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ILM - Microscopy

There are many different light microscopy methods for dealing with biological and medical issues on the cellular level. In many cases, this also involves the use of lasers either for the manipulation of cells or for the laser-assisted microscopic investigation of cells. A number of different confocal laser microscopy methods have proved to be particularly successful in such investigations to the extent that they have become virtually indispensable.

Fluorescence microscopy

The principle of confocal microscopy: confocal microscopes use point illumination and a pinhole to eliminate out-of-focus light. Only light produced by fluorescence close to the focal plane is detected; out-of-focus light is suppressed. © ILM

Besides standard microscopic methods using reflected or transmitted light, or variants thereof, such as phase contrast microscopy, different fluorescence microscopy methods play a hugely important role in cell biology research. It has recently been shown that fluorescent fusion proteins (e.g., green fluorescent protein, GFP, and derivatives thereof, coupled to the protein to be investigated) are very effective in the investigation of living cells, since these proteins can be transiently and stably expressed in cells.

The clarification of photobiological processes in living cells is one of the major reasons why the ILM applies these modern microscopy methods. In addition, the ILM also focuses on elucidating issues related to other cellular mechanisms, often in cooperation with external academic or industrial partners. These investigations focus, amongst other things, on cellular differentiation in tumour biology and tissue engineering.

Confocal laser scanning microscopy

The confocal laser scanning microscope is a tool which adds value to microscopy applications, particularly in the field of biology. Methods that use laser-induced fluorescence are particularly important.

When ultrashort-pulsed lasers, for example Ti:sapphire lasers, are used for the excitation of fluorescence, the range of methods can be expanded by non-linear excitation processes such as the two- or multiple-photon excitation.

The ILM possesses two confocal microscopes for the investigation of living cells. One of these, the LSM 510 Meta (Zeiss), is equipped with spectrally resolving detectors, which enable the acquisition of complete spectres for any image point. The subsequent analysis (online fingerprinting) enables the separation of individual fluorophores, even though their emission spectres partially overlap.

The second confocal microscope used by the ILM, an LSM 410 (Zeiss), is mainly used for producing an image based on the differences in the fluorescence decay rates. Both microscopes are able to acquire images from selected depths (z) (optical sectioning) and images of planes at various depths can be obtained. A deconvolution algorithm is then used to create 3D models. The ILM also uses numerous special imaging software programmes that enable a broad range of image analyses to be carried out, including the optimal analysis of Förster resonance energy transfer (FRET) and fluorescence-recovery-after-photobleaching (FRAP) experiments.

Time-resolved confocal laser scanning microscopy (FLIM)

Fluorescence images of endothelial cells (incubated with calcein AM): total reflection microscopy (AS) vs. reflected light microscopy (B). © ILM

Fluorescence microscopy enables researchers to gain insights into cell biological processes in a contactless way. In such cases, the fluorescence intensity is spectrally resolved or integrally registered. Besides the fluorescence spectrum, another major characteristic of fluorescence microscopy is the fluorescence decay time. This is largely insensitive to fluorophore concentrations, but is very sensitive to changes in the local environment, in particular to the proximity of other fluorophores. The imaging of the fluorescence decay times (fluorescence lifetime imaging, FLIM) enables the selective representation of molecules in different biological environments, i.e. in different cell compartments.

Spectral and time-resolved confocal laser scanning microscopy (SLIM)

The combination of spectral and time-resolved detection is well suited to improving the specificity of different fluorophores and hence is able to considerably expand the application spectrum. We have implemented spectral lifetime imaging (SLIM) methods on the LSM 410 laser-scanning microscope and tested its suitability for different cell biological issues. We have adapted a newly developed module, which consists of a line detector with 16 photomultipliers that enables the time-correlated counting of individual photons, and an upstream spectrometer to the microscope. The individual FLIM images can be assigned to the 16 spectrally different channels, a dimension which enables the even more specific analysis of processes in living cells.

Total reflection microscopy

Total reflection microscopy (TIRFM) involves the illumination of a sample under total reflection conditions through the light of the evanescent electromagnetic field at the interface of two optical media with different refraction indices. The electromagnetic field decreases exponentially with its distance to the interface; its penetration depth depends on the wavelength and the incidence angle of the laser beam. Variations of between 50 nm and 300 nm can be achieved.

In microscopic applications, the different media are created by the cell carrier (glass) and the attached cytoplasmic membrane of the cells. Due to the lesser penetration depth of the light, suitable fluorophores (exogenous markers or fluorescent fusion proteins) can be used to characterise membrane properties or transport processes. In addition, changes in the cell topography can also be quantified. The measurement set-up and the applications were carried out in close collaboration with Professor Dr. Schneckenburger (University of Aalen).

Contact - Fluorescence microscopy and confocal laser scanning microscopy:
Wolfgang Strauß

Contact - FLIM, SLIM, TIRFM:
Dr. Angelika Rück
E-mail: angelika.rueck(at)ilm.uni-ulm.de
Website address: https://www.gesundheitsindustrie-bw.de/en/article/press-release/ilm-microscopy-1