Fluorescence Microscopy

Fluorescence microscopy is a technique where fluorescent substances can be examined and analyzed with high sensitivity, as well as specificity, over traditional light microscopy. In principle, a specimen is illuminated, or excited, with light of a relatively short wavelength, like blue light or ultraviolet (UV). Here, light energy (or photons) are absorbed by an indicator then emitted after a short period of time. Inherently, some energy is lost in this step so the emitted photon will produce less energy than the absorbed photon. Since light with a short wavelength has higher energy than light with a longer wavelength, light emitted from the indicator has less energy than that of the excited light; this phenomenon is also known as Stokes shift. Ultimately, the known excitation/emission (ex/em) spectra of a specific fluorophore can be used for targeted visualization.

Once a known ex/em is identified, the fluorescent microscope can be set to capture images at those specific settings. The specimen, containing at least one fluorescent component, can then be examined through a barrier filter that will absorb light used for illumination and transmit the produced fluorescence. Simple forms of fluorescence microscopy utilize standard luminosity techniques to allow fluorescent components to stand out starkly against a dark background. In this way, fluorescent constituents can be seen even in extremely small amounts.

Currently, many modern fluorescence microscopes also employ the use of epi-illumination. In epi-illumination, light used for excitation is reflected onto the specimen through the objective, which acts as a condenser. This technique allows for the visualization and examination of opaque samples, very thick objects, or even the skin of living people.

Due to the efficacy and variability of fluorescence microscopy, multiple subtypes and specialized versions exist. Common examples include:

• Wide field (WF) fluorescent microscopy
• Confocal Microscopy
• Multi-Photon Microscopy
• Total Internal Refraction Microscopy (TIRFM) and variations (prism- and objective-based)
• Super-Resolution Microscopy and variations (PALM and STORM)
• Fluorescence lifetime imaging microscopy (FLIM)

Fluorescence microscopy can be adaptable and diverse. Conventional absorption-based microscopy utilizes regular transmitted light, with simple instrumentation, and is appropriate for colored objects of resolvable size. Colorless, transparent samples can also be studied through retardation techniques by incorporating polarization, phase-contrast, or interference elements into the microscopy. A dark ground illumination technique may also be preferred, where transparent objects can be revealed by reflection and/or refraction at interfaces of different refractive indices. Such microscopy is highly suitable for extremely tiny particulates, which may be too small to be resolved by other methods.

The dynamic ability of fluorescent microscopy provides many advantages in research. It is not only highly sensitive for the detection and quantification of small amounts of fluorescent components, but can also allow opaque objects to be adequately visualized. Since fluorescence microscopy involves two wavelengths (ex/em), optical specificity can be increased by the selection of filters that favor the fluorophore. Furthermore, fluorescence microscopy allows specific cellular components to be observed through molecule-specific labeling, and structures can be observed inside a live sample in real time.

Assaywise Letters:	FAQs:	Tool:
Fluorescence Live Cell Imaging Application Note: iFluor® Dye Selection Guide	Why is fluorescence more sensitive than UV-VIS absorption spectroscopy? What is the difference between absorption and excitation spectra? How does fluorescent microscopy work?	Fluorescence Spectrum Viewer

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Resources

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Fluorescence Microscopy
Types of Imaging, Part 2: An Overview of Fluorescence Microscopy
Fluorescence Microscopy
Three basic types of fluorescence microscopy and recent improvement
Super resolution fluorescence microscopy
Comparison of frequency-domain and time-domain fluorescence lifetime tomography