Fluorescence Microscopy
Fluorescence microscopy belongs to the family of light microscopy and is based on the physical effect of fluorescence. The color properties of the so-called fluorochromes are exploited, which are excited by light of a certain wavelength and reflect the absorbed light back again with a different wavelength.
Fluorescence microscopy enables morphological investigations, analyses of measured values in the nanometer range and processes of most diverse cultures that become visible in real time. Whether in the field of biochemistry, biophysics or medicine: fast and detailed detection of bright, colorful fluorescence facilitates the measuring process of fluorescence microscopy and forms the basis for new findings. Optimum measurement results and the best resolution require the most precise optics - whether through the optimization and focusing of beam paths, precisely fitting filters or high-quality coatings.
The visualization of fluorescence under the fluorescence microscope is ensured by special filters that allow the passage of individual wavelengths. Special filters of a fluorescence microscope include:
Individual excitation filters allow the respective wavelength of light to pass which is necessary to excite a specific dye in the sample to be examined. The dichroic mirror reflects the stimulating wavelength to the objective, which concentrates the beam onto the specimen. The light reflected from the specimen is concentrated in the objective and in its excited state usually has a higher wavelength than the incident light. Passing through the dichroic mirror, the reflected light passes through the emission filter and is reduced to the wavelength of the emission. Residues of the stimulating light that have not yet been stopped at the dichroic mirror are filtered out at the emission filter. Ideally, only the emission light hits a detector built into the microscope and becomes visible in the respective color.
Optimum measurement results require uniform illumination, especially when a large field of view of several micrometers or millimeters is required. In the case of inhomogeneous illumination, for example, uneven activation of the molecules to be examined can occur. The result: The molecules in the center fluoresce more strongly than those in the periphery of the incident illumination beam. If the periphery is not illuminated equivalently to the center, shading continues to occur when the individually recorded image grids are later merged. Therefore, measurements such as cell and tissue samples cannot be used for reliable analysis. Problems like these can be overcome by using the a|TopShape and the a|BeamExpander. Elements like these are possible through the use of aspheres in the systems. Our systems convince with their compact design as well as their precision and highest optical quality. The use of the optical components a|TopShape and a|BeamExpander enables the transformation of the Gauss beam into a uniform Flat-Top profile and thus uniform illumination across the entire field of vision. The generated flat field illumination convinces by a high spatial coherence, unbeatable optical performance and a high homogeneity of > 95 %. The even excitation of the molecules and minimal image overlaps (5%) can be guaranteed to your complete satisfaction.
The following graphic shows the working principle and general structure of a fluorescence microscope.
Quantitative analyses within laser-based fluorescence microscopy can be complicated by non-uniform illuminations generated by Gaussian beam profiles. Factors such as light source and illumination optics affect the uniformity. These features are particularly challenging when a large field of view (FOV) is to be examined. Measuring images are generated in fluorescence microscopy by image grids. The individual images are acquired in such a way that the edges overlap and they can be assembled in post-processing. If the illumination is uneven, the final image will have darkened edges around each individual image - measurements of cell and tissue samples become unreliable. Another disadvantage of uneven illumination: uneven activation of molecules. Those closer to the center of the beam fluoresce more than those at the edge.
A research team at the College of Optics and Photonics, University of Central Florida in Orlando, was able to overcome these problems by integrating aphericon's beam shaper a|TopShape and the a|BeamExpander into a microscope set-up. The flat-field illumination (FFI) set-up shapes Gaussian beams into a uniform Flat-Top profile. The a|TopShape is extremely tolerant to variations in the size of incoming laser beams (± 10 %) and operates achromatically. The very good optical performance (homogeneity > 95%) enables uniform illumination and thus activation of the molecules. In addition, the FFI set-up enables borderless stitched imaging with minimal image overlap (5%).
For more information on the project, see our reference story.
With our wide range of products, we can offer you a multitude of optimal solutions, from improved imaging qualities through aspheres to a wide variety of filter layers for the separation of different wavelengths. Our experienced team will also be happy to respond to your individual design wishes - for a custom-fit and functional solution.
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Looking for a custom solution? Discover our customized aspheres with unsurpassed surface quality.
Custom Aspheres at a gance
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Choose between asphericon a|High-NA, a|Low-NA and a|UV-grade fused silica. Thanks to CNC polishing and grinding this aspheric lens meets the highest demands on production quality and tolerance.
a|Aspheres at a gance
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The a|TopShape is an innovative beam shaper that transforms collimated Gaussian beams into collimated Top-Hat beams.
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The a|TopShape LD generates stable Top-Hat beam profiles at working distances of up to 4.5 m. Since the effective working distance decreases with subsequent reduction of the beam size, this a|TopShape is particularly suitable for applications that require smaller beam diameters.
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The a|AiryShape enables in combination with a focusing lens the transformation of collimated Gaussian beams into different focused beam profiles (e.g. Top-Hat, Donut). Thanks to its compact design, the a|AiryShape can be easily integrated into existing set-ups.
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The a|BeamExpander is a monolithic laser accessory with just one aspheric lens for the highest level of precision. Experience nearly endless possibilities with up to 32× beam magnification and optimized performance for different design wavelengths (355, 532, 632, 780, 1064 nm) - individually measured and certified.
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Based on thermally very stable dielectric layers, we realize customer-specific desired splitting ratios, typically 50 % / 50 % (R/T) or 30 % / 70 % (R/T).
asphericon GmbH
Stockholmer Str. 9
07747 Jena
phone +49 (0) 3641 3100 500
fax +49 (0) 3641 3100 501
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