Fluorescence Microscopy

Epifluorescence

Fluorescence describes the emission of electromagnetic radiation from organic molecules, resulting from the excitation of an electron to higher energy levels through the absorption of higher frequency radiation. Due to energy losses, the emitted radiation is always of longer wavelength than the absorbed radiation. This phenomenon is exploited in

fluo-rescence microscopy. Fluofluo-rescence microscopes are designed to study the emission signal from fluorophores, without interference from excitation radiation. Emission brightness can be three to six orders of magnitude lower than illumination. The fundamental chal-lenge is therefore to produce high-efficiency illumination of the specimen, while simulta-neously capturing weak fluorescence emission that is effectively separated from the much more intense illumination band. This is mostly achieved through a set of radiation filters.

A standard set-up (as shown in figure 3.26 A) includes a high power white illumination source, various excitation filters to select a single emission wavelength, a dichromatic mirror that allows the emitted light to pass through while blocking the excitation radi-ation, an emission filter that further filters out only a narrow wavelength range and a detector that records the fluorescence. Traditional widefield epifluorescence microscope objectives focus a wide cone of illumination over a large volume of the specimen, which is uniformly and simultaneously illuminated. A majority of the fluorescence emission directed back towards the microscope is gathered by the objective and projected into the detector. This allows for rapid sample visualisation [?,109].

The fluorescence microscope used for the combined microscope-RIfS set-up, as well as for sample monitoring, was the upright fluorescence microscope BX-51 purchased from Olympus (Shinjuku Monolith, Tokyo, Japan).

Figure 3.26.:Basic illumination and collection pathway of an epifluorescence microscope (A) in comparison to a confocal microscope (B). The schematic drawing was adapted from a graphic found in [110].

Confocal Laser Scanning Microscopy (CLSM)

As the name implies, confocal laser scanning microscopy uses a laser beam as illumination light-source. Apart from that, a pinhole is placed in the illumination path, resulting in a

3.4 Complementary Methods

significantly smaller volume, from which fluorescence is excited compared to epifluores-cence. The laser illumination source is first expanded to fill the objective rear aperture, and then focused by the lens system to a very small spot at the focal plane. Confocal spot size is determined by the design of the microscope, the wavelength of incident laser light, the objective’s characteristics, scanning unit settings, and of course the specimen’s optical properties. The resolution in the xy plane is on the order of the point spread func-tion (PSF) about 200 nm in diameter. Resolufunc-tion is worse in the z-direcfunc-tion because of a more diffuse PSF and is typically about 900 nm for a water-immersion 63×objective (NA

= 1.0) [111,112]. In CLSM, the fluorescence image is generated by scanning the focused beam across a defined area in a raster pattern, controlled by two high-speed oscillating mirrors. Fluorescence emission, as shown in figure 3.26 B, is collected by the objective, passed back through the confocal optical system, focused at the detector pinhole aper-ture and converted into an analog electrical signal by the photomultiplier. The confocal image of a specimen is reconstructed, point by point, from the photomultiplier, thus the image itself never exists as a real image, observable through the microscope eyepieces.

Aside from a higher lateral resolution than epifluorescence, confocal microscopy allows to take multiple single x-y focal plane images in the z-direction from which a composite 3-dimensional projection of the observed specimen may be reconstituted.

The CLSM used for measurements in this thesis was the upright confocal laser scanning microscope LSM710 purchased from Carl Zeiss GmbH.

Fluorescence Recovery After Photobleaching (FRAP)

FRAP is a method developed to measure the diffusion of labeled molecules in a two-dimensional matrix, making it an ideal method to determine the diffusion of labeled proteins or lipids in a lipid membrane [113]. During a FRAP measurement, a circular region of interest (ROI) is chosen, which is exposed to high light intensities for a short time (1 - 2 s) to bleach the fluorophores present, and subsequently the fluorescence intensity is recorded as a function of time. Irreversible photobleaching of organic fluorophores occurs after repeated excitation, where the fluorophores undergo an intersystem-crossing into a triplet state, chemically react with oxygen species in solution and are deactivated because of the loss of the π-conjugated system [114]. For FRAP measurements, the bleaching has to occur rapidly (< 2 s) in comparison to the diffusion time of labeled species within the bleached area (few µm). The exposed fluorophores within the ROI are permanently deactivated. If the system under investigation is fluid, the fluorescent molecules surrounding the photobleached area diffuse within the ROI and the depleted fluorescence recovers with a speed that is characteristic of the diffusion coefficient (D) of the labeled molecule. Finally, if certain parts are mobile and certain parts are immobile, then the fluorescence recovery will only be proportional to the mobile fraction within the sample. The recovery signal is typically normalised to a reference fluorescent area that

only undergoes bleaching because of repeated imaging. When diffusion proceeds in an ordered fashion according to Fick’s diffusion equation, the diffusion coefficient (D) of the labeled molecule is given by [113]

D= ^G

2r

4t_1/2 , (3.49)

where Gr is the Gauss radius of the circular bleached ROI andt_1/2 is the time required for half the fluorescence to recover.

FRAP experiments were carried out using a CLSM equipped with a water immersion objective with 63× magnification (Zeiss, Jena, Germany). For fluorescence excitation and bleaching an Argon laser (λex = 488 nm) was used. The mobile fraction was de-termined with the supplier’s software for data acquisition (ZEN, Carl Zeiss GmbH).

Time-elapsed CLSM images (4 frame/s) were analysed with a program written in Igor Pro (WaveMetrics Inc., Portland, Oregon, USA) to obtain lateral diffusion constants.