CN1902475A - 荧光计 - Google Patents
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Abstract
在用于在样品表面处产生和检测荧光的设备中,该设备高于该样品表面的高度减小,并且由于反射损耗和光散射而导致的所发射的荧光的损耗被最小化。该设备包括三维弯曲的光反射表面(40),其横向于来自光源(32)的光的原始路径而引导该光,并且将该光聚焦到该样品表面处或该样品表面下的照明区(30)。该反射表面(40)还横向于所发射的荧光的原始路径并朝向检测器(46)收集、引导并至少部分地准直该荧光。
Description
技术领域
本发明涉及荧光计,其是用于产生荧光并使用强度或时间分辨测量来测量荧光的设备。
背景技术
落射荧光显微镜(epifluorescence microscope)传统上具有线性光学装置,其中样品位置、分束器和检测器例如垂直地沿第一方向上的公共轴间隔地设置,且激发光源离置在一侧。此装置规定最小的高度约束,以提供空间使得从样品位置发射的荧光由透镜系统准直、通过分束器、滤波以移除不同于该荧光波长的波长,并最终聚焦到检测器上。这样的传统装置在图1中示出,如下面更详细地讨论的。在荧光应用中,所发射的荧光的强度通常是弱的。另外,如果荧光体位于光散射介质如皮肤,则荧光各向同性地或根据Lambert辐射图案来发射。在这两种情形中,所检测的荧光随光学系统的数值孔径而增大。在通常的落射荧光建立中,大数值孔径通常通过使用许多透镜而获得,该使用即使对涂覆的透镜也引入反射损耗和光散射。
值得期望的是,开发一种具有减小的复杂性的可替换通用光学装置,其可用于减小从样品位置测量的设备的必要高度。
发明内容
因此,本发明现在在第一方面中提供用于在一表面处或下产生和检测荧光的设备,所述设备包括:
光源,用于沿在所述表面上延伸的光路引导荧光激发光;
反射器,具有三维弯曲的、类似壳形的光反射界面,其被定位以接收从光源沿所述光路的一部分在所述表面上通过的光,并且相对于光路的所述部分横向地反射所述光,以便将所述光聚焦在所述表面处或下的照明区上用于在所述区激励荧光,并且用于收集在所述区发射的荧光,并且反射并至少部分地准直所述光以沿光路的所述部分返回;以及
检测器,用于接收在所述界面反射之后的以荧光发射的所述光。
上面所说的‘表面’可以是由两种不同材料之间的界面限定的物理表面,或者可以是相对于该设备可限定的虚拟表面。
该设备可以进一步包括分束器,其反射由所述光源发射的光以将其传到所述反射器,并且从所述反射器接收荧光,并且将所述荧光传到所述检测器。
所述反射器、光源和检测器优选地以一般是共面的方式设置,并且所述分束器优选地具有处于与反射器、光源和检测器的共面正交的平面上的平的反射表面。
该设备可以进一步包括激发滤光器,其从由光源发射的光选择激发波长以传到所述反射器。
该设备可以进一步包括发射滤光器,选择所发射的荧光波长以传到所述检测器。
该设备可以包括一透镜,其将荧光聚焦在所述检测器上。可替换地,可以使用又一个聚焦反射界面。
该或每个所述反射器界面可以基本上具有部分抛物面、非球形、环形或双锥形表面的形式。这样的反射器界面可以是抛物面、非球形、环形或双锥形表面。球形表面和非球形表面如双曲面、抛物面、椭球和扁椭球反射器界面可以由一等式限定:
其中:
c从0.07到0.5并且k从-1.5到-0.7,其中z是沿旋转轴的z坐标的“垂度(sag)”,c是曲率(半径R的倒数),k是圆锥常数且r是径向坐标。其它表面可以通过包括增加的Taylor展开的类似等式来描述。类似地,也可以得到用于环形和双锥形表面的等式,该环形例如有小面的环形,或分段的线性环形。
引导光到照明区的反射器的反射器界面可以包括抛物面、非球形、环形或双锥形表面的部分,其通过由竖立在该照明区中央的直圆柱体切割抛物面、非球形、环形或双锥形表面而产生。
所述反射器界面可以优选地基本上具有半抛物面的一部分的形式。
该设备还可以进一步包括容纳光源、反射器和检测器并且具有基座表面的机壳,该基座表面包含一窗口,用于将激发光传出机壳并且将荧光接收到机壳中,并且用于在使用中与在其处或其下发生所述荧光的所述表面接合。
优选地,所述光路与由所述基座表面限定的平面成不大于10度的角。
附图说明
将参考附图进一步描述和说明本发明,在附图中:
图1是传统落射荧光测量设备的示意性的侧面正视图;
图2是根据本发明的设备的第一实施例的透视图;
图3是示出当从其上置有镜的表面发射光时,其中收集了大多数能量的抛物面镜的区的图示;
图4是根据本发明的第二实施例的俯视图;
图5是图4的设备的透视图;
图6是图4中标记的线VI-VI′上的横截面;
图7示出图4的设备的修改;
图8示出根据本发明的第三实施例的俯视图;
图9是抛物面镜的形状随着圆锥常数变化的曲线图;以及
图10是示出检测的光功率与照明功率的比随着改变圆锥常数而变化的曲线图。
具体实施方式
用于落射荧光测量的一通常的建立在图1中示出。其设计用于在样品位置10产生和检测荧光,样品位置10可能例如是在例如显微镜载片上的体或物品的表面处或下。典型地,这样的表面将是水平的,并且为了方便,这在该设备的后面描述中被假定。激发光源12如LED定位为离置到样品位置10的一侧并且处于高于样品位置的高度h处。LED发射激发光16,其通过激发滤光器14以选择所需的激发波长。激发光落在成角度的分束器18、如侧面平行的二向色分束器18上。激发光的一部分以直角向下向样品位置反射,并通过聚焦透镜系统20,该透镜系统包括一个或多个简单或复合透镜。
从样品位置发出的荧光和反射的激发光由透镜系统20捕获,并且被准直成平行束,该平行束通过分束器18到理想地移除荧光波长之外所有波长的发射滤光器22,并且从那里到将荧光聚焦到检测器26上的第二透镜系统24。类似透镜系统20,透镜系统24可以包括一个或多个简单或复合透镜。
整个装置具有高于样品的总高度h′,其大约是3×h。
已经提出通过基于荧光的技术监视人或动物体中的化学物质的浓度。这些将涉及将激发光引导到皮肤上并且检测来自皮肤的荧光发射。相关的教示包括WO00/02048和WO02/30275。
我们注意到在这样的环境和其它环境中减小所使用的荧光检测设备的尺度h′是所需要的。
图2示出了根据本发明的设备的一实施例。这里,该设备被描绘为放置在皮肤的表面上或要在其上检测荧光的类似表面上。为了后面描述的方便,皮肤的平面将被视为水平。光源32沿平行于皮肤表面的路径发射光,并且确定近似地等于该装置的总高度h′的用于该设备的高度h。
光通过激发滤光器34到设置在竖直平面上的二向色分束器38,并且被转向朝向取代了传统设备的透镜系统20的半抛物面形镜反射器40。该反射器将光向下聚焦到皮肤表面上的照明区30上或朝向恰好在皮肤表面下的一照明区。该反射器收集并准直来自照明区的荧光发射,并且将发射的光引导回分束器,发射的光通过分束器到达发射滤光器42。从那里,该光由与透镜系统24相同类型的透镜系统44聚焦并且在检测器46被检测。
可以看到,该设备的竖直高度已经从图1中的近似3×h减小到图2中的仅h。
通常,干涉滤光器在荧光计中用作激发和发射滤光器。这些要求:如果要得到预期的波长通过特性,则入射光应该与滤光器的平面正交,因为使用倾斜的入射光,滤光器通带将移向较短的波长。因此要求对入射到这些滤光器上光束进行准直。
可以以许多方式构造抛物面反射器40以产生所需形状的反射界面。例如,该界面可以是在抛物面内部的空气与一体的反射凹内部表面之间。可替换地,该界面可以形成在立体半抛物面元件的凸外部表面处,例如玻璃或塑料的半抛物面块的镀银的外部。
整个所示出的设备将受机壳或壳体(如图6中所示)的限制,该机壳或壳体具有底表面,该底表面包含孔,该孔可被填充以一透明窗口(适当地是玻璃、石英(silica)或者在一些应用中是塑料),用于保护光学元件不受灰尘和湿气影响,并且用于允许将激发光和荧光传到和传自照明区中的样品。所述设备因此可以是壳体中的简单开口或者光学窗口。
来自检测器的信号可以被馈送到适当的电子线路,用于以已知方式分析。LED可以如已知的由适当的电子线路来供给功率,并且LED供给线路和检测信号处理线路一般将形成一集成线路的一部分,该集成线路用于例如在基于FRET(荧光谐振能量转移)的化验中产生荧光的基于强度的或时间分辨(频域)的测量。
所示出的设备提供了用于捕获来自照明区的荧光的高的数值孔径,同时实现紧凑的光学几何,适合用于空间极其宝贵的地方,例如在将穿在体上的装置中。
不需要所有示出的抛物面表面以获得良好的结果。抛物面不需要如所示地设置为其轴与其上待使用该装置的表面一致,而是可以向上成角度以得到用于检测器光学元件的表面上的某个附加高度。荧光束可不被反射器完全准直,但是可由辅助的透镜系统部分地准直。该设备的物理照明区不需要位于反射器的焦点处。反射器界面不需要是真正抛物面的形式。这些概念在后续的图中进一步示出。
如图3中所示,将接收从照明区发射的荧光的大部分并且将其准直输出到检测器的抛物面表面M的区域,将是由位于照明区中央上的直圆柱体C与抛物面表面自身的相交所限定的区域。抛物面表面的剩余部分因此不需要存在。清楚地,圆柱体越小,捕获的光就越少,所以优选地至少存在与由与半径r的圆柱体的相交所限定的一样多的抛物面,其中该半径r不少于从抛物面的原点到焦点的标记为f的距离的50%、优选地不少于f的75%、更优选地不少于f的90%。圆柱体的半径r不需要小于抛物面镜的焦距,如图3中所示的情形,但是可以更大。
同时在图2中,示出透镜系统将光聚焦到检测器上,应当理解,这也可以替代为反射器系统,其可适当地相似于用于将激发光聚焦在照明区上的系统。这种系统在图4到6中示出。
在所示设备中,除了透镜系统44被相似于镜40但被设置成将荧光聚焦到现在定位了检测器46的侧的部分抛物面镜45所取代之外,所示出的部件与图2中的相同。如图6中所示,该设备包括机壳50,其具有位于皮肤上的基座板52并且包含如前面描述的限定照明区30的窗口54。机壳包括上和下半壳,其具有内部构成(formation)以包容(cradle)并支持光学部件,壳如所示地由螺钉固定在一起。
如图7中所示,机壳的基座表面可以与抛物面表面的轴成角α,其优选地不超过10°,更优选地不超过5°。这将给予一些附加的用于光学和电子部件的空间,但是也将导致设备的检测器端的某个附加的高度。此修改可以相关于如所示的在每个端使用镜的实施例并相关于图2的实施例而采用。另外,镜40的焦点可以与样品的位置不同,如图7上由距离δ所示出的,以补偿如检测皮肤下的荧光的情形中的样品或周围介质的辐射模式和散射特性。
如图8上所示,定位在镜40前方的包括一个或多个简单或复合透镜41的辅助透镜系统可以用于产生进一步的准直。
类似地,反射器的形状可以偏离抛物面的形状,以适应于如检测皮肤下的荧光的情形中的样品或周围介质的辐射模式和散射特性。如果这导致荧光发射的不完全准直,可以使用如所示的辅助透镜系统41执行进一步的准直。
可以使用的可替换的弯曲表面包括环形、非球形和双锥形表面。
对于由下面的等式限定的非球形表面
c优选地在0.07到0.5的范围内,且k优选地在-1.5到-0.7的范围内,其中z是沿旋转轴的z坐标的“垂度(sag)”,c是曲率(半径R的倒数),且k是圆锥常数,r是径向坐标。图9中示出对于圆锥常数的各个值的镜的形状。
图10中示出圆锥常数对所检测的光功率的影响。
照明区不需要是一个点或一个圆形区域,但是通过非抛物面反射器形状的使用的效果,可以横向于或纵向于反射器的轴延长照明区。
在此说明书中,除非明确地另外指示,词‘或(or)’以算符的意义使用,该算符在所陈述的条件的任一个或两个都满足时返回真值,与需要仅条件之一满足的算符‘异或(exclusive or)’相对。词‘包括(comprising)’以‘包括(including)’的意义使用,而不是意指‘由…构成(consisting of)’。
Claims (12)
1.用于在一表面处或下产生和检测荧光的设备,所述设备包括:
光源,用于沿在所述表面上延伸的光路引导荧光激发光;
反射器,具有三维弯曲的、类似壳的光反射界面,其被定位以接收从该光源沿所述光路的一部分在所述表面上通过的光,并相对于该光路的所述部分横向地反射所述光,以便将所述光聚焦在所述表面处或下的照明区上用于在所述区激励荧光,并且用于收集在所述区发射的荧光,并且反射并至少部分地准直所述光以沿该光路的所述部分返回;以及
检测器,用于接收在所述界面反射之后的以荧光发射的所述光。
2.如权利要求1的设备,进一步包括分束器,其反射由所述光源发射的光以将其传到所述反射器,并且从所述反射器接收荧光,并且将所述荧光传到所述检测器。
3.如权利要求2的设备,其中所述反射器、光源和检测器以一般是共面的方式设置,并且所述分束器具有处于与该反射器、光源和检测器的所述共面正交的平面上的平的反射界面。
4.如前面任何一项权利要求的设备,进一步包括激发滤光器,其从由该光源发射的光选择激发波长以传到所述反射器。
5.如前面任何一项权利要求的设备,进一步包括发射滤光器,其选择所发射的荧光波长以传到所述检测器。
6.如前面任何一项权利要求的设备,包括透镜或第二反射器,其将荧光聚焦到所述检测器上。
7.如前面任何一项权利要求的设备,其中所述反射器界面基本上具有部分抛物面、非球形、环形或双锥形表面的形式。
8.如权利要求7的设备,其中所述反射器界面由等式
所限定,其中
c从0.07到0.5且k从-1.5到-0.7,其中z是沿旋转轴的z坐标的“垂度”,c是曲率(半径R的倒数),k是圆锥常数且r是径向坐标。
9.如权利要求7或8所述的设备,其中所述反射器界面包括抛物面、非球形、环形或双锥形表面的部分,其通过由竖立在该照明区中央的直圆柱体切割抛物面、非球形、环形或双锥形表面而产生。
10.如权利要求7或8所述的设备,其中所述反射器界面基本上具有半抛物面的一部分的形式。
11.如前面任何一项权利要求的设备,进一步包括容纳该光源、反射器和检测器并且具有基座表面的机壳,该基座表面包含一窗口,用于将激发光传出该机壳并且将荧光接收到该机壳中,并且用于在使用中与在其处或其下发生所述荧光的所述表面接合。
12.如前面任何一项权利要求的设备,其中所述光路与由所述基座表面限定的平面成不大于10度的角。
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PCT/EP2004/014484 WO2005064318A1 (en) | 2003-12-23 | 2004-12-20 | Fluorometers |
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NO338185B1 (no) | 2016-08-01 |
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