WO2010067353A2

WO2010067353A2 - System and method for non-invasive diagnosis of melanoma

Info

Publication number: WO2010067353A2
Application number: PCT/IL2009/001140
Authority: WO
Inventors: Ziad Hammudy; Dan Vager
Original assignee: Viramedics Ltd.
Priority date: 2008-12-08
Filing date: 2009-12-03
Publication date: 2010-06-17
Also published as: WO2010067353A3; IL195795A0

Abstract

The application describes a grating storage unit (50) for light multiplexing comprising a grating imprinted or fabricated on a disc (52) or a tape and a reading head (56). The grating storage unit is characterized in that different areas of the grating have different values of the one or more of the following grating parameters: density of the grooves; groove depth, geometrical shape of the grooves, and refractive index. The grating storage unit can be optically- coupled (74) to an Attenuated Reflection (AR) probe. In embodiments of the application the AR probe is a multi-channel attenuated reflection (AR) sensor head adapted for nevi probing. The sensor head can comprise AR pin probes (82) adapted to be used as Attenuated Total Reflection (ATR) probes or Scanning Near field Optical Microscope (SNOM) pin probes. In the latter case the AR probe is used as a MIR sub cellular sensor. The grating storage unit and AR probe can be used in a system for non-mvasive assessment of the condition of a lesion.

Description

SYSTEM AND METHOD FOR NON INVASIVE DIAGNOSIS OF

MELANOMA

Field of the Invention The present invention relates to cancer diagnostics, and more specifically to a system and a method for classifying early stage malignant melanoma of suspicious nevi in vivo.

Background of the Invention Publications and other reference materials referred to herein, including references cited therein, are incorporated herein by reference in their entirety and are numerically referenced in the following text and respectively grouped in the appended Bibliography, which immediately precedes the claims.

Malignant melanoma is a malignant neoplasm of epidermal melanocytes. It is the third most common skin cancer and is responsible of about 79% of the deaths due to skin cancer. In the United States, malignant melanoma is considered as a real epidemic, as its incidence has increased considerably during these last few years. The American Cancer Society (ACS) estimates that in 2008 there will be about 62,480 new cases of melanoma in the US alone from which, about 8,420 people will die from the disease. Early detection and resection of the melanoma is the only treatment currently available. When the melanoma is diagnosed and treated early, survival rates are very high. In contrast, as the melanoma progresses, it becomes increasingly more devastating and deadly. Five year survival rates for patients treated for early stage melanoma exceed 90-95% whereas these rates drop to less than 50% when treatment begins at later stages of the disease [I]. Therefore, the importance of early detection of melanoma can not be overstated. The success rate of current methods in diagnosing melanomas is quite low. Melanomas are mainly diagnosed by dermatologists and/or primary care physicians using a visual clinical evaluation method. Physicians assess pigmented skin lesions according to the evolution of the "ABCDE" (Asymmetry, Border irregularity, Color variation, Diameter, and Evolving) criteria over time. However, this assessment remains subjective and results in a high number of undiagnosed cases of melanoma and a high number of benign mole resections. The ratio of benign lesions (nevi) diagnosed as melanomas to those confirmed after biopsy is about 40-50 to 1.

Some dermatologists who specialize in the management of pigmented skin lesions use dermoscopy to help distinguish benign from malignant lesions. Dermoscopy, also known as dermatoscopy or epiluminescence microscopy, is the examination of cutaneous lesions with a derrαoscope. The dermoscope comprises a magnifier, a liquid medium between the instrument and the skin, and either cross-polarized or non-polarized light for illuminating the lesion without glare from reflected light. Although dermoscopy provides more information than unassisted visual examination, mastering the technique necessitates many years of training and experience. Proper use of dermoscopy can reduce the number of unnecessary biopsies of benign lesions, but even experts in dermoscopy biopsy 3-10 benign lesions for every melanoma detected.

Several diagnostic instruments are now commercially available, e.g., DermLite® and SolarScan® for the diagnosis of melanomas. These instruments all use spectral imaging, ranging from visible to the near infrared (NIR) region, for gathering morphological information on the skin lesion. However, such instruments do not really offer significant diagnostic enhancement over the methods currently used by dermatologists. Indeed, light in the visible-NIR band is known to be predominantly scattered (elastically) by matter with some absorption in the NIR band by overtone molecular vibrations. Therefore, scattering of visible-NIR light mainly provides structural information of macroscopic objects. In contrast, basic molecular vibrations lie in middle infrared (MIR) portion, making MIR scattering useful in assessing biochemical changes. Whereas the MIR region is not the only region providing such information, methods using other wavelengths such as UV or X-ray would be hardly considered non- intrusive, particularly for patients with a sensitive skin or for those requiring frequent screening. Anomalies corresponding to tumor transformation will induce changes in the biochemical activity, long before their morphological manifestation. Therefore, extraction of MIR absorption spectra offers the potential of an effective method for an early stage diagnosis of abnormal transformation.

Since the MIR band is primarily absorbed in the tissue, absorption spectroscopy performed by shining light directly through the specimen is not possible, unless it is done on very thin samples. It thus seems that the only way to extract in-viυo vibrational spectroscopy is either by means of Raman Spectroscopy or by Evanescent Wave Spectroscopy [EWS], which is also referred to as Attenuated Total Reflection [ATR] spectroscopy. Raman processes are rather noisy spectroscopic channels particularly for in viυo samples. On the other hand, in addition to reduced noise, the EWS method has the following advantages:

1. It is possible to measure very strong IR-absorbing materials, e.g. water, without interference.

2. An ATR element can be used as a sensor for measuring the spectrum in places that are difficult to reach, such as within a deep hole or a working engine and the method allows measurements to be made at locations that are distant from the spectrometer. 3. EWS is a non-invasive technique for medical purposes. For example, for measuring pigmented skin lesions there is no need to prepare a sample.

Broadly speaking, EWS is a method of measuring the absorption spectrum of a sample which is in contact with a denser transparent medium through which light propagates. The light passes through the transparent medium, such that, when there is no absorbent material in the vicinity of the medium's exterior boundary, the light is totally reflected from the boundary, and only an evanescent wave crosses the boundary. The evanescent wave can propagate only along the boundary and decays exponentially perpendicular to it. If an absorbent material Lies in the range of the evanescent beam, it will reduce the reflected intensity of resonant frequencies and hence the absorption spectra of the specimen in the proximity of the transparent medium can be obtained. The portion of the transparent medium in contact with the sample is referred to as an ATR element or probe.

A Scanning Near field Optical Microscope (SNOM), is a tool similar to a tunneling microscope, whereby the image of a thin scanned sample may be resolved below the wavelength of the microscope's source frequency. It incorporates a tip, referred to as a near field probe, with a sub wavelength aperture. The tip may be placed above a thin sample supported by a transmitting material in contact with a detector. In this mode, light may tunnel from the tip's aperture through the sample to the detector. The intensity of light thus measured is exponentially decaying with the depth of the sample and depends upon the sample's optical properties. By moving the probe at a fixed height above sample's support, a 2D image can made providing for the thickness or the refraction index of the sample at various locations. The resolution of the image is essentially the size of the aperture at the tip. The same principle can be applied in a reflection mode, where the light reflected from within the probe is measured.

MIR spectroscopy has a great potential for the diagnosis and the prognosis of in vivo tissue anomalies. While already known and discussed in the prior art [2], such potential has not yet lead to any specialized diagnostic or prognostic medical tools, mainly for the following reasons: 1. In general, the characterization of tissue anomaly requires high spectral resolution of a rather wide band or bands within the MIR portion of the spectrum. Due to current limitations in material engineering technologies, state of the art MIR or Fourier Transform

Infrared (FTIR) spectrometers capable of providing high enough resolution tend to be large, of general purpose, expensive and unfriendly for non-specialists.

2. In many circumstances, in particular in melanoma, the biochemistry corresponding to tumor transformation is characterized by differences in the cell activity (metabolism, biosynthesis, cell to cell transmission and adhesion), rather than the appearance or disappearance of one or more specific biological markers. Therefore, there are no specific peaks appearing in the MIR spectrum and one has to draw the characterization by more detailed structural features of the absorption spectra. Generally such classifications suffer from specificity which does not exceed the diagnostic or prognostic ability of the specialist doctor and hence does not provide a potential diagnostic benefit.

3. Since energy in the MIR band corresponds to the vibrational spectra of organic molecules, it is predominantly absorbed by the tissue. Hence the only realizable method for obtaining an in vivo MIR spectrum is by Attenuated Total Reflection (ATR) techniques, i.e. by means of physical contact with the tissue. Generally this method suffers from limited penetration depth, integration of absorption of non-malignant areas for wide ATR probes, or probing in non- malignant points when needle like probes are used.

The miniaturization of optical spectrometers depends on the advances in engineering technologies, which can provide miniaturized light source, detectors and optical microelements [3-5]. Unfortunately, currently available optical microelements have poor performance in the MIR portion of the spectrum. Therefore, actual wideband high resolution MIR spectrometers tend to be large, non-mobile and are mostly based on the classical free air Michelson interferometer. Recently, ARCoptix [6] proposed "the first hand held near-FTIR spectrometer" using a lamellar grating of variable depth. The bandwidth or spectral resolution is limited by the grating density or length, which is fixed in such a construction, and by the stability extent of the mechanical driving system [4] .

The detection of MIR light is rather complicated due to the thermal radiation of the detector material, which is predominantly in the MIR band at room temperature. Therefore MIR detectors must be cooled. This fact renders detector array based spectrometers unsuitable in the MIR range, at least when miniaturization and cost reduction are considered. Therefore spectrometer designs incorporating only a single or a few detectors must be utilized, e.g. the FTIR design. In such designs, incoming light is parametrically multiplexed to encode the detected intensity, such that measuring the intensity as a function of the encoding parameter permits a decoding of the intensity to the frequency or wavenumber domain. The multiplexed encoded light source is obtained either by using an array of narrow band light sources such as a LED array [3], by narrowing a wideband light source by a filtering method, or by an interferometric method. In the filtering method, the light is filtered through a variable band pass filter array which controls the outgoing intensity at each band pass filter, and then, the resulting light beams are recombined to form a narrow ray of multiplexed light encoded by the narrow band pass intensities [7]. However, in the MIR region of the spectrum, current technologies are unable to provide a high spectrum resolution when using either of the first two of the above methods. Therefore, only interferometric encoding methods are currently suitable for producing high resolution MIR spectrum.

The interferometric method for producing a multiplexed encoded light source generally uses the Fourier Transform (FT). State of the art FT interferometers, e.g., Mach-Zehnder or Michelson type interferometers, are realized by means of a two fold interference pattern. This interference pattern is obtained by splitting a single input ray of light into two rays, one ray further following a fixed length optical path, the other ray following a length- variable path, and then recombining the two rays into a single output ray. The length difference between the two optical paths generates the interference pattern, the variation of the optical path length of the second ray constituting the encoding parameter.

A typical prior art system (10) comprising a MIR-ATR spectroscopic system for the diagnosis and the prognosis of an in vivo tissue anomaly (12) in a patient (14) is shown schematically in Fig. 1. The main components of system (10) are: an optical unit (18), an ATR probe (16), and a processing unit (20). The type of FTIR spectrometer generally utilized in the art is based on a Michelson interferometer which encodes the source intensity frequency profile by its Fourier transform, the corresponding encoding parameter is the path distance difference between the two interfering rays. Optical unit (18), i.e. the FTIR spectrometer is composed of an IR source (22), an interferometer that is usually a Michelson interferometer (24), and a detector (26). Michelson interferometer (24) comprises a fixed mirror (28) and a moving mirror (30), which is moved backwards and forwards linearly by drive motor (32). The beam from the source (22) is split and recombined by beam-splitter (34) such that the path difference, 2x between the recombined beams can be varied by moving mirror (30) in one of the interferometer arms. The Michelson interferometer is not fiber-optic based, which means that additional optical devices such as collimators and couplers (36) (air to fiber, fiber to probe) must be added to couple FTIR spectrometer (18) to the ATR probe (16).

Formally, varying the path difference realizes the x Fourier component of the Fourier transform of the incoming beam. Generally the specimen (12) for spectra extraction is illuminated by the output of interferometer (24) and the resulting scattering is measured by the detector (26). If the specimen has a linear optical response, then the measured intensity is the x Fourier component of the Fourier transform of the source intensity profile multiplied by the cross-section for the scattering process of each illumination wavelength. By scanning the sample using variable x, the Fourier transform is obtained and the desired spectrum is extracted by computing the inverse transform.

In an effort to reduce the size and complexity of the spectrometer more recent developments in the field replace the interferometer (24) with twofold, i.e. binary, variable diffraction elements such as a lamellar grating [5]. A binary diffraction element splits the wavefront such that the diffracted light is a superposition of two wave fronts with a global phase difference. In a sense, binary gratings act as a beam splitter and re-combiner in one element. The far-field zero order diffraction of such binary diffraction elements realizes the FT. More generally, if a diffraction grating could be temporally manipulated at will, in principal any desired interference pattern could be obtained. Thus, variable diffraction elements with large variability extent would be useful for miniaturization of high performance spectrometers.

Variable diffraction gratings are crucial optical components in modern single detector multiplexed optical spectrometers, and channel multiplexing systems in fiber-optic communication. While state of the art variable gratings employ novel mechanical and material engineering technologies, they all suffer from a rather poor range of variability. As such, the resulting multiplexing will suffer, either from a limited bandwidth or a limited spectral resolution. At the moment, the most sensitive, large range of variable gratings used for encoded multiplexing spectrometers are lamellar gratings.

Fig. 2 schematically shows a portion of a variable lamellar grating (38) having rectangular shaped grooves of equal groove width, having period a and variable depth d. Grating (38) comprises a mechanism (not shown) for varying the depth of the well. When illuminated by an incoming beam superposition between the light reflected from the front facet (40) and rear facet (42) realizes a binary diffraction. Until recently, lamellar grating interferometers were limited to long wave bands (>100μm) because of tolerances related to the mechanism for manipulation of the well depth. In their device referred to herein above, ARCoptix has incorporated MEMS technology to create a mechanism which permits high resolution control of the grating depth. For a fixed spectral resolution, the operational band for such a device is limited due to the fixed grating length and maximal admissible depth.

While more general transformations then the Fourier Transformation are admissible with general variable diffraction elements, it is instructive to describe the lamellar grating in more detail in order to appreciate the essence of the limitations of the only such device that is presently commercially available. The far field reflected beam may be represented as a product of three intensities I ∞ h h h indicating the predominant contributions;

represents a diffraction from a single facet of width a/2,

is the interference pattern from a grid of period a with N line like reflecting facets, where K-ksinalA and a is the angle between AB to BC in Fig. 2. Up to a phase, the product I₁ h corresponds to the wavefront reflected from either the front or back facets. The third product h=coskd accounts for the phase difference of these two wavefronts. For zero order reflection only /3 comes into play and thus the output of the Michelson interferometer is reproduced if one replaces x in Fig. 1 by d. Broadly speaking, for long wavelengths λ ~ a the fringe interference term h dominates, so that the zero order intensity is drastically attenuated. For wavelengths much shorter than the period α, the diffraction from a single facet becomes ray-like hence there is a reduction in facet-facet interference. A crude estimation may be made as follows: for a fixed wave length, the first node in the single diffraction term Ji, defines an opening angle of a ray reflected from a single groove. If there are a fixed number of groves N participating in the interference then wll < λ/a, where w = Na is the effective width of incoming ray, and / is the distance at which the reflected light is collected. Another limitation comes from the depth of the grating. When the grating depth becomes sufficiently large, the properties of the "walls" of the well come into play. This limits the range of applicable depth long before the limitation caused by the mechanism for varying the groove depth. Essentially the walls of the wells constitute a loss channel which reduces the intensity only of the back facets and hence causes a reduction in contrast. To obtain fixed resolution with a wider band of operation more detailed aspects of the grating must be varied, or in other words, a variable diffraction grating is necessary with a range of variability larger then the range available with the current state of the art variable gratings.

Evanescent wave vibrational circular dichroism (EWVCD) is the extraction of vibrational circular dichroism (VCD) information from the Attenuated Total Reflection (ATR) spectrum by means of evanescent wave spectroscopy. Generally, circular dichroism (CD) or VCD are means for extraction of the transmission optical activity dependence on the transmitted wave length. When light is transmitted through a non- isotropic media, the transmission, reflection and absorption amplitudes are different for left and right handed polarizations. Stated otherwise, the scattering amplitude at each wavelength is polarization dependent. Specifically, when linear polarized light is transmitted through a non- isotropic or a chiral material, the effective index of refraction of left handed and right handed polarizations are different. If there is no absorption in the process, then the usual description is that the transmitted light will be a linear combination of left and right polarization with phase coefficients that depend upon the optical path, and the concentration of chiral centers in the medium, resulting in an overall rotation of the incoming linear polarization, the rotation angle per unit length is defined as the optical activity of the sample. CD is the dependence of optical activity on the incident wavelength. There is a subtle point here usually missed in discussing this phenomenon, namely, for a sample with polarization dependent index of refraction, both the magnitude and the phase of the reflection amplitude will generally be polarization dependant. This means that the transmitted left and right handed rays will differ in magnitude, so that the transmitted ray will have an elliptical polarization with non-trivial eccentricity rather then being strictly linear. Nonetheless, optical activity may still, as is done in practice, be assessed as the inclination angle of the principal axes of the transmitted elliptic polarization. This point is stressed for comparison with the proposed evanescent equivalent to be considered hereinbelow. If the incoming wavelength is such that absorption is involved, then the difference between the left and right hand absorptions will result in a further deformation of the outgoing polarization ellipsoid, which in this case tends towards a circular polarization, as well as an over all reduction in intensity, i.e. polarization wise integrated intensity. When the incoming ray is in the IR band, the absorption process is principally due to molecular vibration and the assessment of absorption dependent optical activity as a function of wavelength is referred to as VCD.

The assessment of absorption optical activity through the entire range from UV to IR provides significant information on the geometric conformation of biomolecules. Of these, the most informative for 3D conformation are the UV and MIR bands. Broadly speaking UV is sensitive to electronic orbitals ranging through a large portion of the molecule, and MIR is sensitive to vibrations of molecular bonds which are restricted by secondary structure. For a healthy tissue the 3D conformation of biomolecules uniquely defines their cellular activity and is nearly perfectly determined by the primary conformation and is crucial for all cell activities [8]. Abnormal cell activity is thus manifested principally by abnormal 3D conformation and hence VCD is a crucial element in the malignancy assessment of a tumor. It is an object of the present invention to provide a handheld device for classifying early stage malignant to non-malignant melanoma of suspicious moles.

It is another objective of the invention to provide a method for enabling the extraction of in-vivo MIR absorption spectra of intact lesions, which can be spatially resolved to sub cellular levels. The method will be referred to herein as MIR-AR spectroscopy.

It is another object of the invention to provide a method enabling the miniaturization of high performance MIR-ATR spectrometer with multichanneling capabilities.

It is still another object of the invention to provide a method for extracting Vibrational Circular Dichroism from the ATR spectrum.

It is still another object of the invention to provide new spectral biomarker fingerprints to assess malignancy status of the suspicious anomaly.

Other objectives and advantages of the invention will become apparent from the following description taken in conjunction with the accompanying drawings wherein are set forth, by way of illustration and example, certain embodiments of this invention. Summary of the Invention

In a first aspect the invention is a grating storage unit for light multiplexing. The grating storage unit of the invention comprises:

A. a grating imprinted or fabricated on a disc or a tape; and B. a reading head comprising: i. a broadband light source; ii. a system adapted to illuminate a small area of the grating with light from the broadband light source and to transmit light reflected from the small area of the grating illuminated by the broadband light source to an external device; iii. means for causing relative motion between the grating and the reading head; and iv. a tracking mechanism for positioning a desired area of the grating under the reading head. The grating storage unit is characterized in that different desired areas of the grating have different grating parameters. The grating parameters can be one or more of the following: density of the grooves; groove depth, geometrical shape of the grooves, and refractive index.

In embodiments of the grating storage unit the system that is adapted to illuminate a small area of the grating comprises two flattened or planner optical fibers that are attached to the reading head very close to each other and at a slight angle. When attached in this manner the optical fibers direct the incident light through a input fiber from the broadband source of the reading head onto the grating storage disc and

Middle Infrared (MIR) light reflected from the grating back to the external device through an output fiber. The input fiber can be replaced with a filament source at the reading head. In embodiments of the grating storage unit of the invention the external device to which the light reflected from the grating is transmitted is an Attenuated Reflection (AR) probe. In embodiments of the invention the AR probe is attached to elongated fibers and passed through a working channel of an endoscope. In embodiments of the grating storage unit the external device can comprise a variety of sensors. In other embodiments the external device comprises either a multiplicity of single element AR heads or a single AR sensor head comprising a multiplicity of AR elements.

The broadband light source does not have to be located on the reading head in which case light from a remote broadband light source can be conducted via fiber optics to the reading head. The grating storage unit of the invention can comprise multiple reading heads or, alternatively, may comprise a reading head having multiple input/output fibers.

In embodiments of the grating storage unit of the invention the parameters of the grating in the grating storage unit allow measurements of the absorption intensity of a specimen at wavelengths between 2.6 microns and 26 microns. In embodiments the parameters of the grating in the grating storage unit allow measurements having high spectral resolution of less than 8cm ¹.

In a second aspect the invention is a multi-channel attenuated reflection (AR) sensor head adapted for nevi probing, the sensor head comprising: A. an external support tray comprising: i. an upper surface; ii. a lower surface that is pierced by an array of holes; iii. rails, which are attached at their upper and lower ends to the upper surface and the lower surface; iv. a locking mechanism on at least one of the rails; and v. a driving mechanism; B. an internal support tray attached to and able to move up and down on the rails and to which is attached a plurality of AR pin probes; wherein, in a non-active state of the sensor head, the internal support tray is locked on the rails by the locking mechanism such that the tips of the pin probes lay within the external support tray and the sensor head enters an active state by releasing the locking mechanism allowing the internal support tray to be propelled downward by the driving mechanism; whereupon the tips of the pin AR probes exit the sensor head through the arrays of holes in the lower surface, thereby puncturing the outermost area of the nevi.

In embodiments of the multi-channel AR sensor head of the invention the driving mechanism comprises springs. In embodiments_the shape as well as the diameters of the top and bottom of the holes in the lower surface of the external support tray and the diameter of the pin probes are selected to allow the tips of the pin probes to penetrate the skin a depth of 40-50μm.

In embodiments of the multi-channel AR sensor head the AR pin probes are made from silver halide alloys and are at least partially coated with silver. The AR pin probes can be Attenuated Total

Reflection (ATR) probes in which case the sensor head comprises at least one flat fiber punctured to create an array of holes similar to the array of holes in the lower surface of the external tray is attached to the lower surface. In embodiments of the invention the multi-channel AR sensor head according to the second aspect of the invention, comprising ATR probes and an array of circular cross section fibers attached to the lower surface of the external tray.

In an embodiment of the invention the multi-channel AR sensor head comprises an input fiber; an output optical fiber; either a two-to-one coupler, a beam splitter, or a Y-coupler; and Scanning Near field Optical Microscope (SNOM) pin probes or fibers leading to the SNOM probes.

In a third aspect the invention is a system for non-invasive assessment of the condition of a lesion. The system comprises:

A. an optical unit comprising: i. a light source; ii. a multiplexer adapted to convert light emitted by the light source into high resolution multiplexed encoded light; iii. an AR sensor head comprising one or more AR probes, one or more input optical fibers connected to the output of the multiplexer, and one or more output optical fibers; iv. a detector connected to the one or more output optical fibers of the AR probes, the detector capable of detecting the output of the

AR probes; and

B. a processing unit comprising software adapted to produce an absorption spectrum of a sample of light detected by the detector and to provide the discrimination of the lesion from the absorption spectrum.

The system of this aspect of the invention is characterized in that the multiplexer is a grating storage device according to the first aspect of the invention. In embodiments of the system of the invention the light source is a MIR light source and the lesion is a pigmented skin lesion or a mole suspected of being a melanoma. Embodiments of the system additionally comprise a polarizer and a polarization modulator, which provide Vibrational Circular Dichroism [VCD] information about a probed specimen.

In embodiments of the invention the grating storage device, the MIR light source, the detector, the AR sensor head or a connector to an AR sensor head, and at least a part of the processing unit of the system are miniaturized to the point where they are contained in a hand held unit.

In embodiments of the system of this aspect the AR sensor head is the multi-channel AR sensor head of aspect two of the invention.

In a fourth aspect the invention is the use of the system of the third aspect for assessment of the condition of a lesion. The use of the system comprises the steps of:

(a) using the optical unit of the system to provide an interferogram containing information relative to the absorption of the material in the area of the lesion probed by the AR probe of the optical unit;

(b) using the processing unit of the system to obtain an absorption spectrum from the interferogram;

(c) supplying to the processing unit a set of spectral biomarkers that represent biochemicals or biochemical activities that might be relevant to the lesion;

(d) determining the level of each of the spectral biomarkers in the set;

(e) using the levels of the biomarkers to make the assessment of the condition of the portion of the lesion probed by the sensor head of the output unit. In embodiments of the fourth aspect of the invention the use is noninvasive, the light source is a MIR light source and the lesion is a pigmented skin lesion or a mole suspected of being a melanoma.

The set of spectral biomarkers supplied to the processor can comprise one or more of the following:

(a) Amide I /Amide II absorption;

(b) Quantification of the RNA/DNA; (c) Tyrosine amino acid;

(d) The absorbance of the symmetric and antisymmetric bending of CH3 vibration per Amide II;

(e) The lipid absorbance at 1725-1740 cm ¹;

(f) The newer peaks (hidden in- vitro) appearing at 1040 cm ¹ and 1145 cm ¹;

(g) Band widths and asymmetry of Amide I and Amide II;

(h) A large variety of bond deformation, particularly of resonant bonds participating in synthesis, leading to wider absorption bands;

(i) The ratio of the intensity at 1030 cm"l to that at 1080 cm^~l which gives an estimate of the Glucose/ phosphate ratio;

(j) The ratio of the intensity at 2924 cm ¹ to that at 1080 cm ¹;

(k) Absorption in the range 2800-3000 cm ¹; and

(1) VCD spectral markers.

(m) The spectral expression of B-RAF, C-kit, E-cadherin, N-cadherin, (n) The spectral expression of ATP/ ADP ratio. In a fifth aspect the invention is a MIR sub cellular sensor. The sensor comprises: a. one of: a tunable light source, an interferometer or a multiplexer; b. one or more SNOM pin probes; c. one of: a two-to-one coupler or a beam splitter, or a Y-coupler that is attached to each of the SNOM pin probes or fibers leading to said SNOM probes; d. input fibers to conduct light from the tunable light source, interferometer, or multiplexer to each of the two-to-one couplers, beam splitters, or Y-couplers that are attached to each of the SNOM pin probes; and e. and output fibers to conduct light from each of the two-to-one couplers, beam splitters, or Y-couplers that are attached to each of the SNOM pin probes towards a detector.

Brief Description of the Drawings

The above and other characteristics and advantages of the invention will be more readily apparent through the following examples, and with reference to the appended drawings, wherein:

- Fig. 1 schematically shows a prior art MIR-ATR spectroscopy system;

- Fig. 2 schematically shows a portion of a variable lamellar grating;

- Fig. 3 is a block diagram schematically showing the principle components of the system of the invention;

- Fig. 4A and Fig. 4B respectively schematically show side and top views the main components of the grating storage unit of the invention;

- Fig. 4C schematically shows a magnified view of a small area on the surface of the storage disk shown in Fig. 4B; - Fig. 5A and Fig. 5B are respectively cross-sectional views along planes B-B and A-A respectively of the reading head of Fig. 4A showing schematically the details of the reading head for a lamellar grating disc embodiment that is specialized for evanescent wave spectroscopy; - Fig. 5C schematically shows a portion of a grating storage disc emerged in a cooling socket;

- Fig. 6 schematically shows a reading head grating storage unit of the invention have multiple input/output fibers;

- Fig. 7A schematically shows a specialized connector for use with multiple input/output fibers to a reading head;

- Fig. 7B schematically shows the fiber distribution geometry of the connector shown in Fig. 7A;

- Fig. 8 schematically shows a MIR-ATR spectroscopy system according to the present invention; - Fig. 9A and Fig. 9B are schematic cross-sectional views showing respectively the non-active and active states of an ATR sensor head that has been designed for the use in the system of the invention specifically for nevi probing;

- Fig. 9C is a schematic front view of the ATR sensor head shown in Fig. 9B;

- Fig. 1OA and Fig. 1OB respectively schematically illustrate in a cross- sectional view and a frontal view a multi-channel ATR sensor head comprising an array of conventional circular aperture fibers;

- Fig. HA shows a section of the skin in the area of the lesion being investigated;

- Fig. HB is a magnified view of a section of Fig. HA showing the penetration of a single pin probe of the ATR sensor; - Fig. 12 is a block diagram showing an outline of the analysis done by the processing unit of the system of the invention;

- Fig. 13 is a block diagram showing the basic computational steps that transfer the output of the optical unit to a standard absorption spectrum;

- Fig. 14 schematically shows the relationships between incident, reflected, and transmitted beams for an ATR probe immersed in vacuum and an absorbing sample respectively;

- Fig. 15 schematically shows a FTIR-FEWS in situ spectra of epidermis and nevus in the region 4000—680 cm-1 after a baseline correction and min-max normalization; and

- Fig.16 schematically shows a SNOM pin probe coupled to an input/output optical fibers via a Y-coupler.

Detailed Description of the Invention

Fig. 3 is a block diagram schematically showing the principle components of the system of the invention. The system (100) is similar to the prior art system shown in Fig. 1 that is used for chemical reaction monitoring in gasses and liquids, analytical chemistry of solid powders, and has been suggested to be used for the malignancy assessments of a tumor or a neoplasm. According to the present invention significant changes have been made in the design of many of the components. These changes lead to significant improvements over the prior art system. In particular the improvements enable suiting the system to the clinical environment in the sense of size, cost and more importantly, they will enable clinically sensible measurement times.

The system (100) comprises an optical unit (10') and a process unit (20). The optical unit (10') is intended to extract data that enables the deduction of the molecular vibration absorption spectra in the proximity of a lesion (12) probed by the evanescent wave (102) produced by an ATR sensor head (16')- In another embodiment the ATR probes in the sensor head are replaced by SNOM probes. The SNOM is applied in a reflection mode, where the light reflected from within the probe is measured; thereby providing means for in-vivo MIR absorption spectra measurements of sub cellular components in intact tissue. This use of SNOM techniques is applicable for MIR spectra extraction of any tissue reachable by a needle or through an endoscope. Since the invention utilizes either the ATR or SNOM probes, the notation AR (attenuated reflection) is used frequently herein where appropriate to describe the system, system components, or method of the invention without reference to a specific type of probe. The output of the optical unit (10') is a molecular absorption spectrum (104) which is the input to the processing unit (20).

Processing unit (20) estimates the malignancy status of the tumor via spectral classification parameters, i.e. spectral fingerprints which enable estimation of molecular biomarker content referred to as spectral biomarkers. Processing unit (20) comprises software for spectral marker assessment (106) and for classification (108). The output of the processing unit (20) is the anomaly class of the lesion and the diagnosis (110).

The optical unit (10') of the invention is a miniature version of a conventional mid FTIR spectrometer (18) specialized for AR spectroscopy as shown in Fig. 1 or for FTIR spectroscopy in the case in which the sample is remotely located and the light the light is conducted by MIR fibers from the spectrometer to the sample and back. The miniature spectrometer of the invention is enabled by replacement of the Michelson interferometer in an ordinary FTIR spectrometer with an innovative device referred to as a grating storage device (50), which will be described in detail herein below. The grating storage device (50) of the invention can be made to mimic the output of the Michelson interferometer without loss of spectral resolution and with the ability of incorporating multi-channel AR sensor heads (16').

The present invention combines means to enable miniaturization of the FTIR spectrometer with means that allow the spectrometer to be tuned to relevant bands and to the spectral resolution required for the given task by the exchange of a single spectrometer element. In an embodiment the AR probe is coupled to a linear polarizer and a polarization modulator (e.g. a photoelastic modulator) which, along with proper analysis, provide vibrational circular dichroism [VCD] information of the probed specimen. Since VCD is much more sensitive to secondary structure then non- polarized-sensitive vibrational spectra, VCD will provide a better determination of the status of conformation disorder. Conformation disorder is common to cancerous environments and hence vital for prognosis and diagnosis of the malignancy status of the tissue anomaly. New spectral biomarker classifiers, to be described herein below, are used which are more detailed and better characterize spectral malignancy features than prior art markers some of which are general for cancerous environments and some are specialized for melanoma. Altogether these features enable the construction of a handheld, user friendly, sensitive tool that overcomes the difficulties of the prior art discussed herein above that have to date prevented the development of specialized diagnostic or prognostic medical tools using MIR spectroscopy for the diagnosis and the prognosis of in vivo tissue anomalies.

For illustrative purposes, the device is described herein as being specifically designed for melanoma screening by designing a multi-channel AR sensor for mole sampling, and the processing unit is designed to respond to spectral biomarkers some of which are predominantly melanoma sensitive. However it will be immediately obvious to persons of the art that other applications are possible. In particular the grating storage device can be used as the principal component of miniature spectrometers for many other applications. For example, MIR ATR spectroscopy is used in a wide range of applications in common industrial practice, e.g. food quality, thin layer quality assessment, and chemical analysis of liquids and gases even in hostile environments such as combustion engines. Medical applications have not yet been realized due to the difficulties indicated in the background section herein above. The present invention changes this situation as far as medical applications are concerned since the AR probe may be attached to elongated fibers and passed through a working channel of an endoscope, or alternatively the probe may be fitted into a needle where the AR probe resides at the needle's tip, thereby enabling the AR spectra at a point, or a small region, of any epithelial tumor. Along with the malignancy spectral biomarkers described herein below the malignancy status of the probed portion of such tumors may be assessed. While the prognostic importance of mapping malignancy regions in a tumor is obvious, the regional malignancy assessment may be used in collaboration with a resection of say a brain tumor, to produce a clear malignancy border and reduce the resection margins dictated by current practice.

The heart of the system of the invention is a variable diffraction grating storage device, which is installed in the optical unit. The grating storage device of the invention performs the function of the Michelson interferometer or of the variable lamellar grating in the prior art spectrometers described herein above. The classification method described herein below for early stage malignant melanoma of suspicious nevi in viυo is based on studying the fingerprint patterns of the IR absorption spectra of the probed nevi and using selected biomarkers to distinguish between melanoma and nevi. To carry out the classification scheme it is necessary to obtain the absorption intensity of the specimen at the desired wavelengths (~2.6-26 micron). This is obtained by coupling a MIR spectrometer to an AR probe. The proposed spectral fingerprints require the spectrometer to be of high spectral resolution (< 8cm ¹) and to cover essentially all the MIR portion of the spectrum. State of the art spectrometers that meet these requirements are general purpose, large, non-mobile and costly. The variable diffraction grating of the invention provides a simple way to reduce the size and cost of the spectrometer. This in turn allows an entire system, which includes the grating storage device, MIR light source, detector, AR/EW/Near Field probe (or probe connector), and at least a part of the processing unit to be miniaturized to the point where it is contained in a hand held unit.

The variable diffraction grating of the current invention is made by printing or fabricating a grating on a disc or a tape such that the grating parameters (density, depth, geometrical shape, refraction indexes, etc.) are continuously changing with disc angle or tape position. The main components of the grating storage unit (50) are schematically shown in Fig. 4A and Fig. 4B in side and top views respectively. In essence, for the case of a grating storage disc, the grating storage unit has the structure of an audio compact disc player. Grating storage disk (52) is rotated about its center by means of a drive motor symbolically represented by shaft (54). A reading head (56) comprising a broadband light source, e.g. a thermal emitter and a lens system, illuminates a small area of the disc and collects the reflected light in the vicinity of, for example, the zero order reflection. Fig. 4C schematically shows a magnified view of area (58) on the surface of the storage disk. At other areas the parameters of the grating are different. It is noted that the light source used by the reading head to illuminate the disc need not be a direct component of the reading head. In some embodiments light from a remote broadband light source may be conducted via fiber optics to the reading head. A tracking mechanism (not shown in the figures) whose function is to position the desired section of the grating disc under the reading head is also a component of the grating storage unit. In an alternate embodiment the grating can be stationary and the reading head can move and or both can move in order to create the relative motion between them that allows different areas of the grating to be illuminated by the reading head at different times.

For a grating made on a tape the grating storage unit is similar with the exception that the drive system is configured to move the tape linearly past the reading head. The method of manufacturing the grating storage device is not a part of the invention; however it is noted that techniques of producing discs and tapes having variable parameters such as depth, width, and period of the grooves are well known in the related art and the grating storage device may be easily fabricated using current technology.

The location of the spot of light on the disc/tape defines the temporal portion of the grating storage to be used and thus acts as a temporal grating, as long as the grating parameter variation is small with respect to spot size. The storage grating device of the invention thus realizes a variable grating with, in principal, an unlimited range of variability in grating geometry.

The grating storage device is not intended to be limited to a specific realization such as a variable lamellar grating. However, when a variable lamellar grating embodiment of the grating storage disc/tape of the present invention is used, the limitations of the presently used lamellar gratings that have been discussed herein above with reference to Fig. 2 may be resolved rather directly. Firstly, it is noted that when imprinting a lamellar grating on the disc/tape, more flexibility is allowed than just depth variation. In particular when the intensity of the beam reflected from the back facets is attenuated by the depth such that the back facet reflected intensity corresponds to grooves of width c^t /2 < a/2 _^ then the width of the front facet may be reduced correspondingly. While this reduces the overall intensity, the contrast will be preserved. The intensity reduction factor is ^a ' ^a and may be trivially factored out before applying the inverse FT. Moreover, for a lamellar grating embodiment of the grating storage device, the back and front facets are rigidly connected, this fact eliminates noise coming from mechanical instabilities such as those that occur in the ARCoptix MEMS device at large facet depth [2.3].

Fig. 5A and Fig. 5B are respectively cross-sectional views along planes B-B and A-A respectively of the reading head (56) of Fig. 4A. showing schematically the details of the reading head for a lamellar grating disc embodiment specialized for evanescent wave spectroscopy. Two flattened or planner optical fibers (68) are attached to reading head (56) very close to each other and at a slight angle to each other in order to direct the incident light through input fiber (64) from the broadband light source of the reading head onto grating storage disc (52) and MIR light reflected from disc (52) back to the AR probe through output fiber (66). Also shown in Fig.δB is a visible light optic probe (70) that is a component of the tracking mechanism of the grating storage unit.

The intersections of the planes corresponding to the planner fibers (68) with the plane of the disc and the intersection of the plane A-A of figure

4B with the plane of the disc (52) form three lines which are all parallel. As seen in Fig. 5B, the perpendicular projections of both planner fibers (68) on plane A-A of figure 4B coincides, thus zero order diffraction is obtained with respect to the grating at the given location. In Fig. 5A the depth of the groove as a function of the angular position of the disc (52) is symbolically shown by the line (60). In Fig.δB the depth of the groove at a given distance (radius) from the center of the disc (52) is symbolically shown by the line (62).

To increase the throughput, the input fiber (64) may be replaced by a filament source at the reading head (68). This however, may result in overheating the facets of the grooves to a point where their thermal radiation become appreciable. To prevent overheating, which could affect the parameters of the grating storage disc, a portion of the disc is emerged into a cooling socket as schematically shown in Fig. 5C. Note that this method of cooling is feasible due to the short exposure time of a given area of the grating storage device to the illumination source. For diffraction elements that are constantly illuminated, the cooling arrangement must be more elaborate.

It is well recognized that the grating storage unit 50 described herein above may be constructed with multiple reading heads or, alternatively, such that the reading head have multiple input/output fibers as shown schematically in Fig. 6. This feature enables the either a multiplicity of single element AR heads or a single AR sensor head comprising a multiplicity of AR elements to be used. Embodiments of the optical unit comprising a grating storage device such as is described in this paragraph are referred to herein as multichannel embodiments.

In a multichannel embodiment it is preferable that each output fiber (66) of the reading head be illuminated independently. This may be achieved by, for example, the following two methods: 1) using a single MIR source that illuminates all of the input fibers (64) of the reading head along with a "blocking" device that permits light to go through selected channels and blocks others at will. Such a blocking device may be realized via liquid crystals. However, in view of the current status of the technology, a mechanical blocking method is more applicable for the MIR band. 2) More directly, each input fiber (64) can be illuminated by its own separate MIR source. The advantage of this independent channel operation is two fold: Firstly, in embodiments where the sensor head comprises multi AR elements in pair wise proximity, "cross talk" may arise depending on the exterior media in contact with the probe. The cross talk may provide vital information at times but only contribute to the background noise at others. In both cases it is best if it is measured. By illuminating one channel and measuring the signals from the rest, the cross talk can be quantified and may be analyzed or treated as part of the background as necessary. The second advantage of independent channel operation is that it enables multichanneling using a detector having fewer channels then those of the grating storage device. If Nd represents the number of detector channels and N the number of reading head channels with an integer ratio N - c Nd, then each detector channel is connected to c fibers. In this construction Nd of the reading head channels are operated at a given time and c sequential operations are required to operate all the heading head channels. Clearly the least time consuming scenario is when N - Nd, however current technology dictates a compromise between the number of detector channels, device size, cooling method, and price.

In an embodiment, the grating storage unit (50) will be such that a variety of sensor elements, i.e. AR sensors and other types of sensors capable of detecting absorbed, reflected, transmitted, or scattered light may be attached to it at will. To this end, the grating storage unit (50) of this embodiment will comprise a reading head (56) having multiple input/output fibers of which the output fibers (66) to the AR sensor element/s and the input fiber/s from the AR sensor element/s to the detector are attached by a specialized connector (74) comprised of two sections, known herein as internal tray (76) and external (78) as schematically shown in Fig. 7A. The ends of the fibers (66) from the reading head are rigidly bundled by the internal tray (76), like wise, the ends of the fibers (66') leading to the probing element are rigidly bundled by the external tray (78). The internal tray (76) and external tray (78) are connected together mechanically by any of the relatively simple arrangements known in the art that will insure not only the required match up of the fibers on the two sides of the connector, but also provide and maintain optical and mechanical continuity. The fibers leading from the probing element to the detector are connected in a similar manner. In this case the output fibers of the probing elements are attached to the external tray (78). The use of two connectors (74) (one for connecting the reading head to the probe and the second for connecting the probe to the detector) is not the only way of making the required connections. Clearly a single connector may be used for which half of the fibers bundled by the internal tray are connected to the reading head and the other half to the detector. Likewise, the input and output fibers of the probes can be attached to a single external tray.

On each tray (76,78), the distribution geometry of the fibers as well as their cross-sections are schematically shown in Fig. 7B. The openings having a flat aperture in the connector trays enable the use of flattened fiber probes with minimal deformation along the path from the reading head all the way to the detector. For a sensor input fiber having an index of refraction different from that of the reading head output fibers, the connecting edge at the exterior tray is graded for impedance matching. This can be done for each exterior channel separately leaving the internal workings of the optical unit unaltered. As shown in Fig. 7B, some of the apertures (shown in black) are filled with a cladding material instead of fibers. This enables the number of probing channels in the sensor head to range from one to the number of reading head outputs. The apertures of the external tray (78) containing the cladding provide a cover for the interior fibers, this is of particular importance when the fibers connected to the internal tray are silver halide alloys which suffer from contamination by sufficiently long exposure to visible light and air. An exterior tray with all openings filled with cladding material can be used as a cover when the optical unit is not in use.

A MIR-AR spectroscopic system (10') for the diagnosis and the prognosis of an in υivo tissue anomaly (12) in a patient (14) employing the diffraction storage unit (50) of the invention is shown schematically in Fig. 8. Spectroscopic system (10') of Fig. 8 is essentially identical to the prior art spectroscopic system (10) shown in Fig. 1 except for the major difference that the optical unit (18') is constructed using a diffraction storage unit (50), which replaces the conventional Michelson interferometer (24) in the prior art optical unit (18). Also shown in Fig. 8 are optional linear polarizer (80') and polarization modulator (80), e.g. a photoelastic modulator, which can provide vibrational circular dichroism [VCD] information of the probed specimen as will be discussed herein below.

For the mid through far IR bands replacing the interferometer with the grating storage unit significantly reduces the size of the spectrometer without spectral resolution loss. The lamellar grating embodiment of the grating storage device with the fiber-optic reading head as described herein above is particularly useful for EWS or for probing methods in which the light is mediated through a optical fibers, since it overcomes the necessity of utilizing non-stable collimation and focusing optical elements as an interface between the conventional free-air Michelson interferometer and the fibers of the AR probe. The output fiber (66) of the reading head (56) is the fiber leading to the AR probe (16). An unclad portion of this fiber may constitute an AR probe or, otherwise, it may be directly connected or fused to the AR probe. Embodiments of the grating storage unit comprising a multi-channel grating storage device enable the usage of a variety of multi-channel AR sensors.

The extent or penetration depth of the evanescent beam or near field is crucial for the application of the AR technique and is very dependent upon the probe geometry. For nevi anomalies a penetration depth of ~100μm is necessary. This depth may be obtained by using needle-like probes.

Fig. 9A and Fig. 9B are schematic cross-sectional views showing respectively the non-active and active states of an ATR sensor head that has been designed for the use in the system of the invention specifically for nevi probing. Fig. 9C is a schematic front view of the ATR sensor head shown in Fig. 9B.

The sensor head (16') comprises a multiplicity of ATR elements (82) to provide a spectral image of the absorption spectra of the mole, at most significant locations i.e. near the boundary between the epidermis (116) and the dermis (118) (see Fig. HA). The sensor head (16') is comprised of two major components: an internal support tray (84) and an external support tray (86).

Internal support tray (84) is a planar rigid surface to which is attached a plurality of pin probes (82). The longitudinal symmetry axis of each of the pins is perpendicular to the planar surface of internal support tray (84). The external support tray (86) provides the rigid backbone of the entire sensor unit. The lower surface (88) of external support tray (86) is a rigid planar surface that is pierced by an array of holes (112). The number and location of the holes (112) are identical to those of the plurality of pin probes (82) attached to the internal support tray (84). Holes (86) preferably have a truncated conical shape as shown in the figures. The shape as well as the diameters of the top and bottom of the hole is selected to allow the tips of the pin probes (82) to penetrate the skin a depth of 40- 50μm. At this penetration depth the evanescent beam of each probe will have a high overlap with internal lesion components all the way down to the basal layer as shown in Fig. 11. A flat fiber (92), is attached to the lower surface (88) of external support tray (86) thereby providing the absorption spectra of the superficial portion of the mole. Flat fiber (92) has been punctured to create an array of holes similar to those in lower surface (88).

The planar rigid structure of the lower surface (88) of external support tray (86) allows the sensor head (16') to be held against and flatten the skin by applying slight pressure. This eases and insures uniform penetration of the skin by the tips of the pin probes (82).

External support tray (86) includes rails (94), which are attached at their upper and lower ends to the upper surface (90) and lower surface (88). The internal support tray (84) can move up and down on rails (94). In the non- active state of the sensor (see Fig. 9A) internal support tray (88) is locked on the rails (94) by locking mechanism (98) such that the tips of the pin probes (82) lay within the external support tray (86) of sensor (16'). Upon release of the locking mechanism (98), the internal support tray (88) is propelled downward by springs (96); thereby puncturing the outermost area of the nevi and enabling the desired penetration. The locking mechanism must release the internal support tray (88) instantaneously such that internal support tray (88) is accelerated rapidly in order to avoid a simple non-penetrating deformation, which would be the case if the pin probes were pushed out of the sensor slowly.

In other embodiments any driving mechanism, known in the art, may be used instead of springs to propel internal support tray (84) toward the surface of the skin as described above. In an embodiment, after the internal support tray (84) reaches its minimal height by the, an additional driving method, e.g. micrometric screws, is activated to slowly lift the tray (84) away from the surface of the skin, thereby enabling spectral image readout at variable depths.

The cone shaped holes drilled in the flattened fiber (92) are coated by a highly conducting material, e.g. silver for a silver halide fiber, to minimize probe to probe coupling. The maximal aperture of the pin probes, with currently available silver halide elements, is the diameter of a conventional silver halide fiber which ranges from 0.25mm to 0.9mm. This dictates that the minimal pin to pin separation be as shown in Fig. 9C for a maximal packing. In Fig. 9C, the dots represent the point of the tips of pin probes (82), the solid circles represent the apertures of the holes in fiber (92) at the interface with the skin, and the dashed circles represent the largest apertures of the pin probes (82). For needle tip penetration of

~50μm, typical diameters of the solid circles are ~50μm and of the dashed circles are ~250μm.

At higher pin density, the hole to hole separation will become comparable to the larger MIR wavelengths and Brag reflections will come into play within the flat fiber (92). To avoid the noise produced by the holes in the flat fiber (92), the punctured flat fiber (92) can be replaced by an array of conventional circular cross section fibers (114) as schematically illustrated in a cross-sectional view in Fig. 1OA and a frontal view in Fig. 1OB. This embodiment comes with the benefit of additional superficial special information but at the expense of pin probe density as seen by comparing Fig. 1OB with Fig. 9C. Since one of the functions of the lower surface (88) of external support tray (86) is to flatten the skin surface, the fibers (114) in the fiber bundle that replaces the punctured flat fiber (92) are preferably trimmed as shown in Fig. 1OA so that the surface of the sensor element that is placed on the skin is flat and smooth.

The penetration of a single pin probe (82) of the sensor when applied on epidermis is shown symbolically in Fig. HB. Fig. HA shows a section of the skin in the area of the lesion being investigated. Shown in Fig. HA are the Epidermis (116), Dermis (118), and Subcutis (120) layers of skin. Also shown are sweat glands (122), a lymph vessel (124), hair follicle (126), and blood vessel (128). Fig.llB is a magnified view of the upper left corner of Fig. HA. Shown in Fig. HB are the tip of pin probe (82), basal cells (130), melanocytes (132), and squamous cells (134). As shown in Fig. HB, the penetration depth is smaller than the minimal depth of the epidermis. For acquired moles, the width of the epidermis may serve as a lower bound to the separation from the exterior of the skin to the dermis. Hence, the tips of the sensor, using the above described geometry, will penetrate no more than the epidermis layer of the skin, a layer containing no blood vessels or nerves. This ensures that the sensor is really superficial and may be considered non-intrusive.

A SNOM probe similar to the above described ATR probe is made by replacing ATR elements (82) with needle-like probes that are currently used as near field sub wavelength probes of SNOMs. The utilization of such a probe mutatis mutandis in the AR technique as disclosed herein is an aspect of the current invention. By replacing an ATR probe with a SNOM probe the extraction of MIR absorption spectra from sub cellular components of intact lesions is enabled.

Fig. 16 schematically illustrates a SNOM probe (200), which can be connected such that it can replace the ATR element (82) in Fig. 9. Probe (200) comprises a two-to-one coupler, a beam splitter, or a Y-coupler (202). In embodiments of the invention, for example for endoscopic procedures, optical fibers can be connected directly to the SNOM probes at the distal end of the endoscopicAaparoscopic device and can lead the light to and from the probe to a beam splitter at the proximal end. The method of spectra extraction is similar to the ATR or EW, i.e. light enters the coupler (202) through input fiber (204). A portion of the light is transmitted to the SNOM pin probe (206). The near field in the exterior vicinity of the probe overlaps a portion of the sample, and a portion of the internally reflected light by the probe is transmitted through the output fiber (208) to a detector. Absorption of a given frequency of the near field by the sample will result in an attenuation of the light transmitted by output fiber (208) and hence the absorption spectra may be assessed. The dominant difference between the SNOM method and the ATR method is in the physical mechanisms by which the near field or EW are produced. In contrast to EWS, the near field at the exterior proximity of the SNOM probe is produced via a sub wavelength aperture (210) in the otherwise reflecting coat of the probe. The extent of the near field decay along the probe axes may be modified by the tip angle. In particular with a slight penetration of the pin probe to the epidermis, the near field may have a significant depthwise overlap with the relevant components of a nevus. Corresponding to the wideband application of the invention, by a subwave length aperture, is meant an aperture smaller then the shortest wavelength in the band. However, geometrical details of the aperture or shape of the reflecting coat may be adjusted such that the near field extents of different wavelengths in the band are similar, and optimized. To obtain near field coverage of relevant nevus components in both the horizontal and vertical (depth) directions multi channeling is utilized.

The material chosen for the AR elements must be non-toxic, highly MIR transparent, and capable of being processed to enable formation of an AR element as described herein above. Currently such elements are made from silver halide alloys which answer the above requirements. The silver halide pin probes are coated with silver to form a conducting reflecting surface. The coating ensures that the evanescent beam is predominantly produced at the tip of the pin. Specifically, the SNOM pin probes are coated with silver to form a conducting reflecting surface except at their tips in order to create the required sub wavelength aperture. Since the current invention deals with extraction of MIR AR spectra, it is desirable in embodiments of the apparatus to include a procedure for extraction of VCD information. The procedure is rather direct. The input to the AR probe is set to be linearly polarized by means of polarizer (80') in Fig. 8. By measuring repeatedly with variable modulation parameters of the polarization modulator (80) in Fig. 8, the Stokes parameters or incoherent state parameters, e.g. points in Bloch spheres or a density matrix, of the probes output polarization at each wavenumber are obtained. Noticeably, such an embodiment may be only realized when all transmitting elements (fibers) preserve the polarization coherency to a high extent.

An outline of the analysis done on a single channel of the optical unit (18') by the processing unit (20) of the invention is depicted in the block diagram of Fig. 12. The interferogram, which is the output (104) from the optical unit (18'), or in this case a single channel of it, is first processed in step (136), which is referred to as the basic manipulations step, to appear as an apparatus independent conventional absorption spectrum (138). For a circular dichroism embodiment, in addition to absorbance, the output (138) will contain the polarization density matrix parameters at each wavenumber. The absorption spectrum (138) thus obtained is then estimated for the level of each spectral biomarker. As will be detailed herein below, a spectral biomarker (148) is supplied along with, or is defined by, an algorithm or a function which assigns a number to the output (138) referred to as the level of the spectral biomarker. The level of a spectral marker parameterizes the possible expressions of the biochemical/s, or biochemical activities it represents. From the set of spectral biomarker levels, an assessment is made of the condition of the portion of the lesion probed by the corresponding sensor head's channel. In Fig. 12 This step is referred to as the classification step 144 and the output of the classification is shown in block 146.

A system comprising the optical unit of the invention and a processing unit capable of carrying out the basic manipulations described herein constitutes a general purpose AR MIR spectrometer. The ability of the system to assess the condition of a lesion lies in the structure of the sensor head and the set of spectral biomarkers. Broadly speaking, the sensor head should be specific to the type of lesion whose condition is to be assessed and the spectral markers must be specific to enable discrimination between the different conditions in which the particular lesion may exist. Thus, a system in which the sensor head is the sensor (16') described herein and the spectral biomarkers are the ones to be detailed herein below is a device which is designed to discriminate between a benign mole and melanoma. Basically, the essence of the analysis done on the output (138) is to solve the statistical classification problem, i.e. to assign a lesion condition to the output (138). However, much of the information contained in the spectra (138) is redundant as it is mutual to most cellular environments or is non - robust to the environment in which the lesion is contained. The spectral biomarkers thus provide an intermediate step intended to squeeze out most of the relevant information from the spectra (138) from which a discrimination can be drawn.

Given a set of spectral markers 8^...,S_n, a set of lesion conditions

and an absorbance spectra A, the vector J = (5, ,..., s_n), S₁ = S₁ (A) , is referred to as a feature vector. A function that assigns a feature vector to a member of C is a called a classifier. To distinguish between a method which assigns a condition to the probed lesion and the assignment from the feature space the former will be referred to as a direct classifier, and the latter an intermediate classifier. Presumably, a true classifier exists, referred to as the "gold standard". The performance or power of a classifier is quantified by the agreement with the "gold standard" on a sufficiently large lesion sample on which both methods could be applied. Generally, a classifier may be more sensitive or more likely to agree with the gold standard with respect to a specific condition say d. To this end the agreement with the gold standard is generally quantified for each condition separately as follows: Fixing a condition d, a classification outcome where the classifier assignment is C₁ is said to be positive, otherwise it is said to be negative. The agreement of the assignment with the gold standard is then marked by adding the prefix "True" or "False", thus for example True Positive (TP) refer to an agreement of the two classifiers that the lesion is in condition Ci and so on. Of the lesion samples, the ratio of the TP population to the population which has condition C; according to the gold standard (i.e. TP + FN) is referred to as the sensitivity. The ratio of the TN population to the population which has not the condition Ci according to the gold standard (i.e. TN + FP) is referred to as the specificity. The ratio of the TP population to the P population and TN population to the N population are referred to as the Positive Predictive Value (PPV) and the Negative Predictive Value (NPV) respectively.

In general, two or more lesions with different conditions according to the gold standard may have the same, or approximately the same feature vector. The ratio of the population with condition G and a feature vector having approximately the same feature vector s to the population having approximately the same feature vector s tends to the probability distribution P(c= Ci \ s ) that a lesion is in condition C₁ provided that it has a feature vector in approximately the same as s . This so called posteriori distribution defines the best performance an indirect classifier may have. Moreover it is easily shown, and well known in the art, that an indirect classifier which assigns a feature vector to a condition having that maximal posteriori probability at the feature vector is a classifier with the maximal performance. Thus for a selected set of features or spectral biomarkers the posteriori distribution defines the optimal classification and hence the posteriori distribution quantifies the classification power or quality of the feature space. Having stated this, it is emphasized that to date the choice of features is more an art than a method and a firm theoretical basis for the selection of features is by far more efficient than a statistical search of a parameterized family of features.

In statistical classification, the term "supervised classification" usually refers to an algorithm that determines a classifier by a training set. A training set consist of a feature vector and its gold standard classification. The algorithm is such that an incremental increase of the size of the training set will result in a classifier with improved or non deteriorating classification performance, and such that the resulting classifier tends asymptotically, according to the size of the training set, to an optimal classifier. The classification step (144) carried out by the processing unit of the current invention is a supervised classification where the supervised classification algorithm may be any of the supervised classification algorithms known in the art e.g. artificial neural networks, "Naϊve Bayes classification", linear discriminant, parameterized fit for the posteriori distribution etc.

For the system, i.e. optical and computational units, to act as a classifier that discriminates a benign mole from a melanoma, the "gold standard" is the pathology result of a biopsy test. The system is ready to perform as a diagnostic tool once a sufficiently large training set is recorded to provide a classifier having a reasonable classification performance. A mole diagnosed as melanoma either by the device, by the physician, or both will be resected. According to the standard of care such resections will always undergo pathology examinations. The pathology report can then serve to increase the training set even after the system is operational thereby to increase the classification performance with time.

With the sensor head (16') for mole probing of the current invention, the output of the single channel analysis depicted herein above consists of the classification benign (B) or malignant (M) and the approximation for the difference in the posteriori probabilities:

D(J) = P(M I ?) - P(B | ? ) = 2 P(M I J) - I

Since a FN classification has far more critical consequences than a FP, the classification procedure is endowed with a penalty or a threshold d such that the assignment J to M is given provided D + d is positive. The penalty d constitutes a tradeoff between the sensitivity and specificity. When one of the channels assigns M to the probed environment the mole will be classified as Melanoma. Otherwise, from the special distribution of the pin probes, a fit is made for the special distribution of the posteriori distribution from which the expected maximal value of the posteriori distribution or D is evaluated from which the malignancy assessment is drawn.

Fig. 13 is a block diagram showing the basic computational steps (136) that are performed to transfer the output (104) of a single channel of the optical unit (18') to a standard absorption spectrum (138) having the form AQz), i.e. a graph showing the absorbance as a function of wavenumber k. The output of a single channel of the optical unit (18'), i.e. the transformed intensity transmitted through the AR probe as a function of grating parameter is referred to as an interferogram (104), in analogy to common FTIR nomenclature. In the case of embodiments utilizing VCD, the output of a single channel at a parameter θ of the polarization modulator (80') will also be referred to as an interferogram.

The inverse transform (156) of the interferogram (104) is calculated to produce the intensity transmitted through the output fiber of the probe I_r ik) as a function of wavenumber.

Fig. 14 schematically shows the relationships between the incident, reflected, and transmitted beams for an AR probe immersed in vacuum

(top) and an absorbing sample (bottom). When an AR probe is "emerged in vacuum", a portion I_r of incident beam I_1n is reflected from the body of the probe, another portion I_vac is lost due to non-totally reflected components of the internal beam of the probe, and the rest I _B of the incident beam I_1n is transmitted. I_n is referred to as the background and is predominantly a feature of the source and geometry of the probe. The background is ideally measured in vacuum, although pre-sampling background measurements in air provide a good estimate of its value. Since the idealized background may be recorded when the probe is manufactured, the departure of pre- sampling background measurements from the idealized background may serve as an indicator of device malfunctions, poor connectivity, contaminated probes, etc. Upon sampling, the transmitted spectra is approximately given by I_T = I_B - I_ola = I_B - l_mA

The absorption spectra is thus given by

The incident intensity is measured at the stage of manufacture of the optical unit (18') simply by "shorting out" the probe, i.e. by using a clad fiber which directly connects the grating storage devise to the detector. The spectrum produced by subtracting the background is smoothed using standard smoothing techniques (160). Finally, to clearly show the peak features, a base line correction (162) is applied which reduce linear trends between dominant minima. The outcome of the overall procedure is referred to as the standard form of the absorption spectra (138).

For a VCD embodiment the step (158) of Fig.13 is replaced by the following procedure: After calculating IBQI, θn), Iτ(k,θn) by the inverse transform (156) for each value of the selected polarization modulating parameter θn, the polarization state parameters are approximated. The modulating parameter θn-0, refers to a non active state of modulator (80') where it transmits light isotropically and hence provides a measurement of the total transmitted intensity IBQI), Iτ{k) from which the absorbance AQi) is then calculated in step (158) and then undergoes the remaining steps of the basic manipulations. The state of polarization S(k) may be represented as a point r(fe) in the Bloch sphere:

2S(Jk) = 1 + f(fc) ^■ σ

The projection of the point to the x-z plane, represents the extent and direction of the linear polarization of the beam (i.e. the major axis of the polarization ellipse) with the north and south poles (±5) corresponding to the linear polarization direction of polarizer (80') and a perpendicular linear polarization respectively. The projection to the y direction represents the extent of circular polarization. The length llr|l represents the purity of the polarization state with 1 as a pure state and 0 as a non polarized state. After the state of polarization is approximated, the step (158) follows adjusted as A(Ic) = ^Fs^s-^h^ _an(j ^₈ ^₈ f_ou_owe(j by smoothing step (160).

Fig. 15 schematically shows FTIR-FEWS (Fourier Evanescent Wave Spectroscopy) in situ spectra of epidermis (solid line) and nevus (dashed line) in the region 4000-680 cm-1 after a baseline correction and normalization by fixing the maximal absorbance peak laying in the amide I band to 2. This is a conventional normalization in the art and does not constitute a stage of the basic manipulations (136). The AR probe used to produce the spectra in Fig. 15 was an unclad portion of a circular aperture (0.9mm diameter) silver halide fiber, and the spectrometer was a commercial FTIR (TENSOR™ 27 model produced by Bruker Optics).

The spectra contain the main absorbance bands that correspond to known molecular groups in biological tissues. The spectra are dominated by two absorbance bands at 1645 and 1545 cm ¹ known as Amide I and II, respectively. Amide I arises from the C=O hydrogen bonded stretching vibrations and Amide II from the C-N stretching and CNH bending vibrations. The peaks in the spectral range 1300-1000 cm ¹ are due to phosphate stretching modes P=O, C-O, C-C stretching modes, and C-O-C deformation modes of carbohydrates. The bands in the range 1400—1460 cm ¹ are related to the C-O and C-H deformation vibrations of lipids and proteins. The region at 3600-2800 cnr¹ corresponds to the O— H, N-H, and C-H stretching modes. The band in the region 1720-1745 cm ¹ (1730 in the figure) is due to the absorbance of the hydrogen bond ester carbonyl groups of lipids.

It is evident by comparison with the prior art, e.g. [2], that the in-vivo AR spectrum shown in Fig. 15 very closely resembles those extracted by FTIR transmission spectra on in-vitro samples. As such, the guidelines for selection of spectral features for the benign/malignant lesion classification, is essentially the same. Using these guidelines the inventors propose the following set of predominant, theoretically feasible, spectral biomarkers that can be employed in the invention: 1. Amide I /Amide II absorption: This ratio sheds light on the change in DNA content corresponding to tumor/neoplasm transformation.

2. Quantification of the RNA/DNA: Nucleic acids are vital as they are altered during cancerous conditions where cell division and growth are affected. The processes of transcription and DNA duplication during carcinogenesis lead to increased signals from unwound DNA.

Similarly, increased signals can arise from RNA during transcription and translation in cancer cells/tissues compared to normal cells/tissues. This is the scientific basis of selecting absorbance of these biochemicals for analysis. Moreover, changes in the nuclei to cytoplasm ratio would make it probable that a larger quantity of nuclei is measured in a given area. The importance of nucleic acids as characteristic benign/malignant classifiers was readily recognized in [2]. It is thus crucial to find the most relevant spectral features that provide sensitive nucleic acid assessment: The phosphates of nucleic acids absorb predominantly in the region

900-1300 cm ¹: RNA at 1244, 1121, and 996 cm ¹, DNA at 1230, 1020, and 966 cm ¹. To improve the distinction of melanoma tissues from nevus the inventors quantify the bases of nucleic acids (denine (A), guanine (G), thymine (T), and cytosine (C)) absorbance at specific wavenumbers in the measured spectra.

3. Tyrosine amino acid: Tyrosine absorbs at wavenumbers 1512-1515 cnr¹ and has been reported [2] to be an important biochemical that changes during malignancy or development of malignant melanoma. 4. The absorbance of the symmetric and antisymmetric bending of CH3 vibration per Amide II, which is reported [2] to be due to membrane protein bands contribution. While this is a general feature of cancer, it is well known that melanocytes undergo crucial alterations in membrane structure as well as membrane to membrane adhesion upon transformation. This feature is thus expected to be particularly sensitive in melanoma.

5. The lipid absorbance at 1725-1740 cm ¹.

6. Peaks visible in the AR spectrum of in-vivo samples but hidden in- vitro appearing at 1040 and 1145 cnr¹ are expected to contribute collagen or other proteins with glycosylation.

7. Band widths and asymmetry of Amide I and Amide II. Rapid proliferation is expected to increase imperfection of bonds from their idealized covalent structure. Additional bond deformation may arise from secondary structure that is expected to be disordered upon transformation from that of the usual molecular conformation in cellular environments. All in all a large variety of bond deformation will lead to wider absorption bands. This is particularly expected for resonant bonds participating in synthesis, the carbonyl group is an important example of these. 8. The ratio of the intensity at 1030 cm"¹ to that at 1080 cm"¹ which gives an estimate of the Glucose/ phosphate ratio, an index of proliferation rate.

9. The ratio of the intensity at 2924 cm ¹ to that at 1080 cm ¹ which gives an estimate of glucose/phospholipids. 10. Absorption in the range 2800-3000 cm ¹, which is due to strong absorption of CH2, CH3 stretching vibrations of phospholipids, cholesterol and creatine.

11. The spectral expression of known melanoma related biomarkers e.g. B-RAF, C-kit, E-cadherin, N-cadherin; 12. The spectral expression of ATP/ADP ratio.

13. VCD spectral markers: In general it is expected that at least near the absorption bands the larger the secondary conformation disorder, the smaller the inclination and the closer the eccentricity will be to unity. The opposite is expected from non-chiral absorbers; water molecules are expected to have polarization independent absorption amplitudes, however, with high synthesis rate, normally water rejecting sites of enzymes and other hydrophobic molecules will increase their overlap with water. Water in the close vicinity of helical molecules will "inherit" the tendency to vibrate in preferred directions. Stated otherwise, chiral molecules induce chiral environment in their vicinity, the larger their overlap with the environment the larger is the extent of the induction. It is noted that in free air transmission spectra such an effect cannot be seen since a major portion of water absorption comes from outside of the sample. The above spectral biomarkers are drawn from a single channel output. The epidermis structure implies a differentiation in the lipid absorbance - spectral marker (5) when measured at to topmost layer of the skin from that measured at the basal layer. In the embodiment of the mole sensor head (16'), the superficial probe (92) or probes (114) provide an averaged absorbance over the contact area of these probes with the skin. The difference in spectral marker (5) by a pin probe channel to that of the superficial probe (92) or probes (114) provide an additional structural marker which will be accessed to the classifier

(144) for each pin probe.

Although embodiments of the invention have been described by way of illustration, it will be understood that the invention may be carried out with many variations, modifications, and adaptations, without exceeding the scope of the claims.

Bibliography

[1] http://www.melanomacenter.org/detection/index.html

[2] Z. Hammody, S. Argov, R. K. Sahu, E. Cagnano, R. Moreh, S.

Mordechai, "Distinction of malignant melanoma and epidermis using IR micro-spectroscopy and statistical methods" Analyst. 133 (3), 372-378,

2008.

[3] US Patent US006031609A "Fourier Transform Spectrometer Using a

Multielement Liquid Crystal Display" Feb. 29, 2000

[4] US Patent US005475221A "Optical Spectrometer Using Light Emitting Diode Array" Dec. 12, 1995.

[5] O. Manzardo, R. Michaely, F. Schadelin, W. Noell, T. Overstolz, N. De Rooij, and H. P. Herzig, "Miniature lamellar grating interferometer based on silicon technology," Opt. Lett, 29, 1437 (2004).

[6] (http:// www.arcoptix.com)

[7] US Patent US007126682B2 "Encoded Variable Filter Spectrometer" Oct. 24, 2006.

[8] http://en.wikipedia.org/wiki/Structural_biology

Claims

1. A grating storage unit for light multiplexing, said grating storage unit comprising:

A. a grating imprinted or fabricated on a disc or a tape; and B. a reading head comprising: i. a broadband light source; ii. a system adapted to illuminate a small area of said grating with light from said broadband light source and to transmit light reflected from said small area of said grating illuminated by said broadband light source to an external device; iii. means for causing relative motion between said grating and said reading head; and iv. a tracking mechanism for positioning a desired area of said grating under said reading head; characterized in that different desired areas of said grating have different grating parameters.

2. A grating storage unit according to claim 1, wherein the grating parameters are one or more of the following: density of the grooves; groove depth, geometrical shape of the grooves, and refractive index.

3. A grating storage unit according to claim 1, wherein the system that is adapted to illuminate a small area of the grating comprises two flattened or planner optical fibers that are attached to the reading head very close to each other and at a slight angle to each other in order to direct the incident light through a input fiber from the broadband source of the reading head onto the grating storage disc and Middle Infrared (MIR) light reflected from said grating back to the external device through an output fiber.

4. A grating storage unit according to claim 3, wherein the input fiber is replaced with a filament source at the reading head.

5.A grating storage unit according to claim 1, wherein the external device is an Attenuated Reflection (AR) probe.

6. A grating storage unit according to claim 5, wherein the AR probe is attached to elongated fibers and passed through a working channel of an endoscope.

7. A grating storage unit according to claim 1, wherein a broadband light source is not located on the reading head and light from a remote broadband light source is conducted via fiber optics to said reading head.

8. A grating storage unit according to claim 1, comprising multiple reading heads or, alternatively, may comprise a reading head having multiple input/output fibers.

9. A grating storage unit according to claim, wherein the external device comprises a variety of sensors.

10. A grating storage unit according to claim 8, wherein the external device comprises either a multiplicity of single element AR heads or a single AR sensor head comprising a multiplicity of AR elements.

11. A grating storage unit according to claim 1, wherein the parameters of the grating in said grating storage unit allow measurements of the absorption intensity of a specimen at wavelengths between 2.6 microns and 26 microns.

12. A grating storage unit according to claim 1, wherein the parameters of the grating in said grating storage unit allow measurements having high spectral resolution of less than 8cm ¹.

13. A multi-channel attenuated reflection (AR) sensor head adapted for nevi probing, said sensor head comprising:

A. an external support tray comprising: i. an upper surface; ii. a lower surface that is pierced by an array of holes; iii. rails, which are attached at their upper and lower ends to said upper surface and said lower surface; iv. a locking mechanism on at least one of said rails; and v. a driving mechanism; B. an internal support tray attached to and able to move up and down on said rails and to which is attached a plurality of AR pin probes; wherein, in a non-active state of said sensor head, said internal support tray is locked on said rails by said locking mechanism such that the tips of said pin probes lay within said external support tray and said sensor head enters an active state by releasing said locking mechanism allowing said internal support tray to be propelled downward by said driving mechanism; whereupon the tips of said pin AR probes exit said sensor head through said arrays of holes in said lower surface, thereby puncturing the outermost area of the nevi.

14. A multi-channel AR sensor head according to claim 13, wherein the driving mechanism comprises springs.

15. A multi-channel AR sensor head according to claim 13, wherein the AR pin probes are made from silver halide alloys and are at least partially coated with silver.

16. A multi-channel AR sensor head according to claim 13, wherein the shape as well as the diameters of the top and bottom of the holes in the lower surface of the external support tray and the diameter of the pin probes are selected to allow the tips of said pin probes to penetrate the skin a depth of 40-50μm.

17. A multi-channel AR sensor head according to claim 13, comprising Attenuated Total Reflection (ATR) probes and a flat fiber punctured to create an array of holes similar to the array of holes in the lower surface of the external tray is attached to said lower surface.

18. A multi-channel AR sensor head according to claim 13, comprising ATR probes and an array of circular cross section fibers attached to the lower surface of the external tray.

19. A multi-channel AR sensor head according to claim 13, comprising an input fiber; an output optical fiber; either a two-to-one coupler, a beam splitter, or a Y-coupler; and Scanning Near field Optical Microscope (SNOM) pin probes.

20. A system for non-invasive assessment of the condition of a lesion, said system comprising: A. an optical unit comprising: i. a light source; ii. a multiplexer adapted to convert light emitted by said light source into high resolution multiplexed encoded light; iii. an AR sensor head comprising one or more AR probes, one or more input optical fibers connected to the output of said multiplexer, and one or more output optical fibers; iv. a detector connected to said one or more output optical fibers of said AR probes, said detector capable of detecting the output of said AR probes; and

B. a processing unit comprising software adapted to produce an absorption spectrum of a sample of light detected by said detector and to provide said discrimination of said lesion from said absorption spectrum; characterized in that said multiplexer is a grating storage device according to claim 1.

21. A system according to claim 20, wherein the light source is a MIR light source and the lesion is a pigmented skin lesion or a mole suspected of being a melanoma.

22. A system according to claim 20, additionally comprising a polarizer and a polarization modulator, which provide Vibrational Circular Dichroism [VCD] information about a probed specimen.

23. A system according to claim 20, wherein the grating storage device, the MIR light source, the detector, the AR sensor head or a connector to an AR sensor head, and at least a part of the processing unit are miniaturized to the point where they are contained in a hand held unit.

24. A system according to claim 20, wherein the AR sensor head is the multi-channel AR sensor head of claiml3.

25. The use of the system of claim 20 for assessment of the condition of a lesion, wherein said use comprises:

(a) using the optical unit of said system to provide an interferogram containing information relative to the absorption of the material in the area of said lesion probed by the AR probe of said optical unit;

(b) using the processing unit of said system to obtain an absorption spectrum from said interferogram;

(c) supplying to said processing unit a set of spectral biomarkers that represent biochemicals or biochemical activities that might be relevant to said lesion;

(d) determining the level of each of the spectral biomarkers in said set;

(e) using said levels of said biomarkers to make said assessment of the condition of the portion of the lesion probed by the sensor head of the output unit.

26. The use of claim 25, wherein, said use is non-invasive, the light source is a MIR light source and the lesion is a pigmented skin lesion or a mole suspected of being a melanoma.

27. The use of claim 26, wherein the set of spectral biomarkers comprises one or more of the following:

(a) Amide I /Amide II absorption;

(b) Quantification of the RNA/DNA;

(c) Tyrosine amino acid; (d) The absorbance of the symmetric and antisymmetric bending of CH3 vibration per Amide II;

(e) The lipid absorbance at 1725-1740 cm ¹;

(f) The newer peaks (hidden in-vitro) appearing at 1040 cm ¹ and 1145 cm ^1; (g) Band widths and asymmetry of Amide I and Amide II; (h) A large variety of bond deformation, particularly of resonant bonds participating in synthesis, leading to wider absorption bands;

(i) The ratio of the intensity at 1030 cm"l to that at 1080 cm"l which gives an estimate of the Glucose/ phosphate ratio; (j) ^{The ra}tio of the intensity at 2924 cm ¹ to that at 1080 cm ¹;

(k) Absorption in the range 2800-3000 cm ¹; and

(1) VCD spectral markers.

(m) The spectral expression of B-RAF, C-kit, E-cadherin, N-cadherin,

(n) The spectral expression of ATP/ADP ratio.

28. A MIR sub cellular sensor comprising: a. one of: a tunable light source, an interferometer or a multiplexer; b. one or more SNOM pin probes; c. one of: a two-to-one coupler or a beam splitter, or a Y-coupler that is attached to each of said SNOM pin probes or to fibers leading to said SNOM probes; d. input fibers to conduct light from said tunable light source, interferometer, or multiplexer to each of said two-to-one couplers, beam splitters, or Y-couplers that are attached to each of said SNOM pin probes; and

Θ. output fibers to conduct light from each of said two-to-one couplers, beam splitters, or Y-couplers that are attached to each of said SNOM pin probes towards a detector.