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A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. Source: https://en.wikipedia.org/wiki/Total_internal_reflection_fluorescence_microscope

Background
In cell and molecular biology, a large number of molecular events in cellular surfaces such as cell adhesion, binding of cells by hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often overwhelmed by the background fluorescence due to the much larger population of non-bound molecules. TIRFM allows for selective excitation of the surface-bound fluorophores, while non-bound molecules are not excited and do not fluoresce. Due to the fact of sub-micron surface selectivity, TIRFM has become a method of choice for single molecule detection.

Overview
The idea of using total internal reflection to illuminate cells contacting the surface of glass was first described by E.J. Ambrose in 1956.[1] This idea was then extended by Daniel Axelrod[2] at the University of Michigan, Ann Arbor in the early 1980s as TIRFM. A TIRFM uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. The evanescent wave is generated only when the incident light is totally internally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells as shown in the figure above. Note, however, that the region visualized is at least a few hundred nanometers wide, so the cytoplasmic zone immediately beneath the plasma membrane is necessarily visualized in addition to the plasma membrane during TIRF microscopy. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution.

TIRF can also be used to observe the fluorescence of a single molecule,[3][4] making it an important tool of biophysics and quantitative biology.

Cis-geometry (through-objective TIRFM) and trans-geometry (prism- and lightguide based TIRFM) have been shown to provide different quality of the effect of total internal reflection. In the case of trans-geometry, the excitation lightpath and the emission channel are separated, while in the case of objective-type TIRFM they share the objective and other optical elements of the microscope. Prism-based geometry was shown to generate clean evanescent wave, which exponential decay is close to theoretically predicted function.[5] In the case of objective-based TIRFM, however, the evanescent wave is contaminated with intense stray light. The intensity of stray light was shown to amount 10-15% of the evanescent wave, which makes it difficult to interpret data obtained by objective-type TIRFM [6][7]

Source: https://en.wikipedia.org/wiki/Total_internal_reflection_fluorescence_microscope

前面介绍显微物镜的时候,我们有提到显微镜的分辨率主要由数值孔径(NA)来决定,大家熟悉的XY轴分辨率公式如下。另外,显微镜Z轴的分辨率是和数值孔径(NA)的平方相关,对于一个60倍油镜NA=1.35来说,XY轴分辨率在200nm左右,Z轴分辨率在600nm左右。一般来说显微镜上Z轴的分辨率相对于XY轴来说是比较差的,包括共聚焦成像技术对于Z轴分辨率的提高也比较有限。有一种显微成像技术,Z轴分辨率可以达到100nm甚至更高,比XY轴分辨率还高。这种在细胞生物学领域也比较流行的技术叫全内反射荧光成像(Total Internal Reflection Fluorescence, TIRF),常见于倒置显微镜上。



从上图(左下)我们可以看到,激光通过一个大角度斜入射到倒置显微镜的物镜里,在玻璃(折射率1.518)和水(折射率1.33)的界面,发生了全反射现象,即激光不会穿过玻璃进入细胞培养基里面,而是完全反射回到了物镜里。但是,在玻璃和水的界面(水的那侧),会产生激光的消逝波现象(evanescent wave),激光能量随距离呈指数型衰减,但是也能激发荧光信号。在100nm深度,激光能量衰减到低于50%。前几期介绍光学显微镜发展史上的里程碑(中),我们有提到TIRF技术特别适合观察细胞膜上膜蛋白的运动,极大地提高了荧光图像的信噪比。相比较于传统的宽场荧光或者共聚焦,TIRF技术最大的区别是只有靠近玻璃和水界面的100nm左右的样品才能被激发出荧光,正好是贴壁细胞的细胞膜的位置。其他细胞器的荧光不会被激发,所以荧光图像的背景就非常干净,如下图。


要实现TIRF成像,有几个关键要素需要满足:
1)高数值孔径物镜,一般是60倍油镜NA=1.49
2)高功率激光器,一般40mW以上固体激光器
3)可以使激光斜入射物镜的TIRF照明器,可调角度
4)高灵敏度荧光相机,EMCCD或背照式sCMOS相机

物镜的数值孔径越大,激光斜入射物镜的角度可以更大,更容易实现全反射。对于目前大多数TIRF实验,一般都是NA1.49甚至更高。高灵敏度荧光相机,以往TIRF系统采用电子倍增EMCCD相机较多,主要的原因是其量子效率QE可以达到90%以上。但是价格较为昂贵,市场上最新的背照式sCMOS相机,量子效率也达到了90%,并且总像素达到1600x1200,图像视野更大分辨率更高,更好地兼顾了宽场荧光和TIRF成像的综合需求。从应用角度来说,TIRF成像技术经常用于细胞膜蛋白的活细胞实验,还包括了更为复杂的多色多通道荧光TIRF实验。由于TIRF技术的高信噪比,越来越多的单分子荧光实验也是通过TIRF来实现的,包括单分子FRET实验(single-molecule FRET, smFRET)。还有一个更为知名的TIRF应用,是拿到诺贝尔奖的超分辨成像,PALM和STORM这两个技术就是基于TIRF架构来实现的。

前面介绍显微物镜的时候,我们有提到显微镜的分辨率主要由数值孔径(NA)来决定,大家熟悉的XY轴分辨率公式如下。另外,显微镜Z轴的分辨率是和数值孔径(NA)的平方相关,对于一个60倍油镜NA=1.35来说,XY轴分辨率在200nm左右,Z轴分辨率在600nm左右。一般来说显微镜上Z轴的分辨率相对于XY轴来说是比较差的,包括共聚焦成像技术对于Z轴分辨率的提高也比较有限。有一种显微成像技术,Z轴分辨率可以达到100nm甚至更高,比XY轴分辨率还高。这种在细胞生物学领域也比较流行的技术叫全内反射荧光成像(Total Internal Reflection Fluorescence, TIRF),常见于倒置显微镜上。



从上图(左下)我们可以看到,激光通过一个大角度斜入射到倒置显微镜的物镜里,在玻璃(折射率1.518)和水(折射率1.33)的界面,发生了全反射现象,即激光不会穿过玻璃进入细胞培养基里面,而是完全反射回到了物镜里。但是,在玻璃和水的界面(水的那侧),会产生激光的消逝波现象(evanescent wave),激光能量随距离呈指数型衰减,但是也能激发荧光信号。在100nm深度,激光能量衰减到低于50%。前几期介绍光学显微镜发展史上的里程碑(中),我们有提到TIRF技术特别适合观察细胞膜上膜蛋白的运动,极大地提高了荧光图像的信噪比。相比较于传统的宽场荧光或者共聚焦,TIRF技术最大的区别是只有靠近玻璃和水界面的100nm左右的样品才能被激发出荧光,正好是贴壁细胞的细胞膜的位置。其他细胞器的荧光不会被激发,所以荧光图像的背景就非常干净,如下图。


要实现TIRF成像,有几个关键要素需要满足:
1)高数值孔径物镜,一般是60倍油镜NA=1.49
2)高功率激光器,一般40mW以上固体激光器
3)可以使激光斜入射物镜的TIRF照明器,可调角度
4)高灵敏度荧光相机,EMCCD或背照式sCMOS相机

物镜的数值孔径越大,激光斜入射物镜的角度可以更大,更容易实现全反射。对于目前大多数TIRF实验,一般都是NA1.49甚至更高。高灵敏度荧光相机,以往TIRF系统采用电子倍增EMCCD相机较多,主要的原因是其量子效率QE可以达到90%以上。但是价格较为昂贵,市场上最新的背照式sCMOS相机,量子效率也达到了90%,并且总像素达到1600x1200,图像视野更大分辨率更高,更好地兼顾了宽场荧光和TIRF成像的综合需求。从应用角度来说,TIRF成像技术经常用于细胞膜蛋白的活细胞实验,还包括了更为复杂的多色多通道荧光TIRF实验。由于TIRF技术的高信噪比,越来越多的单分子荧光实验也是通过TIRF来实现的,包括单分子FRET实验(single-molecule FRET, smFRET)。还有一个更为知名的TIRF应用,是拿到诺贝尔奖的超分辨成像,PALM和STORM这两个技术就是基于TIRF架构来实现的。


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