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Unveiling the invisible: A short journey into fluorescence microscopy

Cell observation through high resolution images

Reading time: 5 min - Words: 1063

Fluorescence microscopy, a subset of light microscopy, leverages the fluorescence effect to visualize structures and processes at the nanometer scale. This technique relies on fluorochromes, which, when excited by specific wavelengths of light, emit light of a different color. The applications of fluorescence microscopy span various scientific fields, including biochemistry, biophysics, and medicine, enabling real-time observation of diverse cultures. The following blog post looks at the importance of fluorescence microscopy in life science applications and describes its general mode of operation.

Fluorescence microscopy - a subset of light microscopy

Fluorescence microscopy stands out as a captivating subset within the realm of light microscopy, offering unparalleled insights into the intricate world of cellular structures. By harnessing the phenomenon of fluorescence, this technique allows scientists and researchers to delve into the microscopic universe with exceptional precision. Unlike traditional light microscopy, fluorescence microscopy employs fluorochromes, molecules that emit light upon excitation by specific wavelengths. This not only enhances contrast and visibility but also enables the visualization of dynamic processes within living cells. In the exploration of cell observation through high-resolution images, fluorescence microscopy emerges as a powerful tool, unraveling the mysteries of cellular function and behavior at a level of detail previously unimaginable.


Optical precision for enhanced results


Advancing the optical precision in fluorescence microscopy involves a meticulous consideration of multiple factors. The optimization of beam paths is imperative, ensuring that the excitation light reaches the sample with the utmost efficiency. Additionally, the selection and integration of precisely fitting filters become paramount, allowing only the desired wavelengths to contribute to the fluorescence signal, thereby enhancing signal-to-noise ratios. The application of high-quality coatings further refines the optical components, minimizing reflections and aberrations that could compromise image clarity. In this intricate interplay of optical elements, each component contributes synergistically to elevate the overall precision of the microscopy system, resulting in enhanced resolution and facilitating intricate measurements at the nanometer scale.

Components of a fluorescence microscope


Figure 1: Working principle of a fluorescence microscope

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:

  • Excitation filters
  • Emission filters
  • Dichroic beam splitter

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.


Figure 1: Working principle of a fluorescence microscope

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 (see Fig. 2). 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.

Figure 2: Measurement sample illuminated with a Gauss profile (left) compared to an illumination using a Top-Hat profile (right). The Top-Hat provides even illumination and thus uniform activation of the molecules as well as borderless stitched imaging with minimal image overlap. © CREOL
Figure 2: Measurement sample illuminated with a Gauss profile (left) compared to an illumination using a Top-Hat profile (right). The Top-Hat provides even illumination and thus uniform activation of the molecules as well as borderless stitched imaging with minimal image overlap. © CREOL

Problems like these can be overcome by using asphericons Top-Hat beam shaper the a|TopShape. This compact system utilizes aspheres for beam shaping. Its use enables the transformation of the Gaussian 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.

Fluorescence microscopy reference

As shown in the previous section, 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 (see Fig. 2).

Figure 3: (a) Beam shaping schematic. (b) Experimental setup. FFI was expanded 1.5x by using the BeamExpander behind the TopShape to provide a full FOV illumination to the sample. © CREOL
Figure 3: (a) Beam shaping schematic. (b) Experimental setup. FFI was expanded 1.5x by using the BeamExpander behind the TopShape to provide a full FOV illumination to the sample. © CREOL

A research team at the College of Optics and Photonics, University of Central Florida in Orlando (CREOL), was able to overcome these problems by integrating aphericon’s beam shaper a|TopShape and the a|BeamExpander into a microscope set-up (see Fig. 3). 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.


About the author

Anna Polinski
As a graduate in communication science with experience in HR marketing and event management, Anna Polinski joined asphericon in 2016.