Microscopy

The experimental work in this thesis relies on the use of microscopy and as the examined effects are on small length scales, it is required to use high resolution microscopy techniques like Confocal Laser Scanning Microscopy (CLSM). CLSM is a standard microscopy techniques with a high resolution. As described in Abbe’s Law, the minimal resolutiondof a microscope is

d= λ

2NA, (3.1)

whereNAis the numerical apertureA=n·sinα. The wavelengthλ, the refrective indexnand the half opening angle of the cone of lightαare the important quantities in this equation and all have are bound in the small interval. It is possible to overcome this limit with special techniques, as described in (9, 36, 37, 64). But even to approach this limit with traditional techniques is far from trivial. As for widefield epi-fluorescence microscopy, the whole specimen is illuminated, and thus

fluorophores of different layers, the images will contain light from all layers, which will decrease the resolution. The CLSM used in this thesis is a method that overcomes this problem and is presented in the following chapter. An enhanced CLSM, namely the spinnning disc Confocal Laser Scanning Microscopy (sdCLSM) will be explained, because, due to its high temporal resolution, we performed experiments with this setup.

3.2.2.1 Confocal Laser Scanning Microscopy

The main idea of Confocal Laser Scanning Microscopy is the introduction of a pinhole in the light path of the excitation laser. In this way, the excited volume is reduced to, in principal, the minimal spot to which light is able to be focused. Its size is around 200 nm in x- and y-direction and 500 nm in z-direction. The image quality is enhanced as no fluorescence is excited and collected from other spots.

The set-up of this microscope type is shown in Figure 3.2 that can be found at the homepage www.microscopyu.com.

The light path starts with the laser that is restricted to a very small focus by the light source pinhole aperture. Through a dichromatic mirror, the laser light is reflected to the objective that focuses the light to a spot on the specimen. There it will excite fluorophores, that will emit photons of a different wavelength. These photons will be focused by the objective on the photomultiplier in such a way that out-of-focus fluorescence will be blocked by the detector pinhole aperture. Hence one gets a fluorescent signal of a single point of the specimen. By scanning the probe you are able to image everything or just a part, for instance, single confocal planes of it.

Figure 3.2:This sketch shows the lightpath of a laser scanning confocal microscope.

The laser excitation source is shown on the left hand side. After passing a small pinhole the light is reflected by a dichromatic mirror through the objective onto the specimen. The fluorescent light, which is emitted by the specimen will be detected by a photomultiplier detector after having passed through the objective and the dichromatic mirror only if it matches with the detector pinhole aperture.

That is if and only if it was emitted in the confocal plane that is visualized.

Taken from Nathan S. Claxton, Thomas J. Fellers, and Michael W. Davidson:

http://www.microscopyu.com/articles/confocal/confocalintrobasics.html on July 21st, 2015.

3.2.2.2 Spinning Disc Confocal Laser Scanning Microscopy

The sdCLSM is one special form of the confocal microscopy aimed at optimizing temporal resolution. The main difference between this technique and conventional CLSM is that a highly efficient camera is used to create the micrograph instead of the photomultiplier detector. To retain the confocality due to the precisely placed pinholes, the sdCLSM possesses two rapidly rotating discs, one incorporated with microlenses and the other one with pinholes (72, 76). In Figure 3.3 a sketch of the sdCLSM is shown which is taken from the web page of Zeiss-Campuss.de. The illumination of the specimen is done by focussing the laser with the microlenses of the first rotating disc to the pinholes of the second rotating disc and afterwards through the objective. Thus the confocality is obtained similarly as for the conventional CLSM. The emitted fluorescence will be focused through the objective and the pinholes, but in contrast to the excitation light reflected by the dichromatic mirror onto the CCD camera.

3.2.2.3 Di ff erential Interference Contrast Microcopy

Within the variety of different microscopy techniques, differntial interference contrast microscopy (DIC), is a brightfield microscopy technique. The advantage of the DIC microscopy is that objects with volume-like cells can be imaged in a way that the height of the object can be visualized with a grey scale. On a DIC micrograph, one gets a pseudo three-dimensional image.

To accomplish this impression of three-dimensional imaging, the technique uses the interference of light due to a path differences while passing the probe, see Figure 3.4. Therefore the light has to go through a polarizer in the beginning. The polarized

Figure 3.3:Sketch of Spinning Disc operation mode. The laser light is focused by the microlenses of the rotating lens disc through the pinholes of the rotating pinhole disc. Via the objective the specimen is illuminated. The emitted fluorescence of the sample takes the light path through the objective and pinhole disc before being reflected by a beamsplitter onto a CCD camera.

Taken from http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html on July 21st, 2015.

light will be split by a Wollaston (Nomarsky) prism into two light paths. Both light paths will illuminate the specimen after a condenser. Due to the objective the two light paths will be merged into a second Wollaston (Nomarsky) prism and there they will interfere with each other. Dependent on the difference of the optical path which the light had to take, it will be constructive or destructive interference. Hence the image will be brighter or darker. As last step before the eze piece or the camera detector there is a analyzer that assures only correctly polarized will be detected.

Figure 3.4: Sketch of DIC light path. Light is polarized and then splitted by a Wollaston(Nomarsky) prism. After the actual imaging of the probe with a condensor and an objective with a second Wollaston(Nomarsky) prism the two light paths are merged again and can interfere. Due to the analyzer only the correctly polarized light can pass to the ezepieces or the camera.

Taken from http://www.olympusmicro.com/primer/techniques/dic/dicoverview.html on July 21st, 2015