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SIRF the Evanescent Waves |
Many of the key events in the cell occur in close proximity to membrane surfaces or at the surface of the cell. Naturally, any optical technique that can visualise these events without interference from the underlying regions within the cell or cellular structure will increase the amount and quality of information collected.
TIRF (total internal reflection fluorescence) microscopy has played a key role in helping us to understand the myriad of cellular processes occurring at the cell's surface. Often referred to as evanescent wave microscopy, it can enable the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction. It is not a new technique but has surged in popularity with the advent of green-fluorescent protein (GFP) as a fluorescent marker and technical developments in laser scanning microscopes.
Now, with the launch of the SIRF (Simple Internal Reflection Fluorescence) system, we are offering individual scientists a total reflection microscope system based exclusively on standard HBO/XBO light sources rather than lasers.
No compromise
SIRF is a revolutionary development that dramatically simplifies the microscope set-up required to undertake cell surface and membrane studies. This will significantly increase the number of scientists and laboratories able to take advantage of total reflection microscopy to selectively excite cellular fluorophores adsorbed, adhered, or bound to the surface and combine it with conventional epi-fluorescence to relate surface effects to internal cellular structures.
 However, the reduction in complexity achieved by our scientists and engineers doesn't mean a reduction in sensitivity or image quality. SIRF boasts optical sectioning performance of the order of 100 nm, compared to the 250 nm of the best confocal systems. And, since there are no interfering fluorescence signals from other layers of the specimen, the SIRF image is very rich in contrast.
So, how does it work?
When light travelling in a high refractive index medium, such as glass, strikes an interface with a material of lower refractive index, such as water or culture medium, above a critical angle of incidence, it is' bounced' back. This is known as total internal reflection. However, a fraction of the energy penetrates the medium as an evanescent wave that can excite fluorescent molecules. Since the light intensity falls off exponentially with distance, the propagation distance may be less than 100 nm and any fluorescing molecules beyond this will not be excited. The effect is demonstrated here.
In both SIRF and TIRF, the emitted fluorescent signal is detected through the same objective used to launch the evanescent wave. For the Carl Zeiss TIRF and SIRF systems, a special high power objective is available - the alpha Plan Fluar 100x/1.45 oil objective.
Sliding into position
 Zeiss already has a TIRF microscopy system based on the highly successful ‘slider’ approach pioneered by the award winning ApoTome contrast enhancement system. In adopting the same principle, the SIRF slider guarantees reproducible alignment, compatibility with existing options and modules, and successful observations.
The SIRF slider is simply inserted into the microscope and an annular diaphragm that can be adjusted in all three spatial directions manoeuvred so that only a ring-shaped area of the exit pupil of the alpha Plan Fluar 100x/1.45 oil objective is illuminated. This ensures that the light leaving the objective strikes the specimen at the critical angle of total reflection.
SIRF will be useful for the selective visualisation of cell/substrate contact regions, tracking secretion in living cells, measuring the binding rates of cell surface receptors, the examination of submicroscopic morphology, and single molecule experiments on molecular motors.
This unique technique continues our strategy of providing complete systems for microscopists of all levels. What’s more, the SIRF slider is retrofittable to existing Axio Imager and Axiovert 200 microscopes and has a free aperture for conventional fluorescence illumination. Changing between SIRF and normal epi-florescence is as simple as moving the slider.
The SIRF system comprises an Axio Imager or Axiovert 200 with epi-fluorescence, the SIRF slider and an alpha Plan Fluar 100x/1.45 oil immersion objective.
For further details, visit the special SIRF section on our website, call us on 08-459 25 00, or email.
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