Fluorescence microscopy is an imaging technique used to examine cells and their internal components. Specific cellular components can be labeled with fluorophores.
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When fluorophores absorb of a specific wavelength of light (called the excitation wavelength), their electrons move to a higher energy level. These electrons then produce a longer wavelength (called the emission wavelength) as they return to their original state.
The Stokes shift defines the difference between these values. These differences can be used in fluorescence microscopy to image multiple fluorophores in one sample.
Steps involved in fluorescence microscopy
Fluorophore labeling
Some samples naturally contain fluorophores, whereas others require fluorophore labeling through other methods, such as immunofluorescence labeling, DNA manipulation to stimulate specific proteins to produce fluorophores or through fluorescent dyes.
Methodology
There are multiple types of fluorescence microscopy, however, the vast majority use a light source to illuminate the sample, exciting electrons within the fluorophores and leading to the production of a specific emission wavelength. This wavelength is captured and quantified to visualize the location of proteins.
What is photobleaching?
Photobleaching is a process whereby fluorophores lose their ability to fluoresce effectively, leading to fading of the fluorescent signal. This occurs when fluorophores react with each other, permanently covalently modifying each other and fading the fluorescent signal.
If the fluorescent signal is low, this can lead to false negative results and skewed data. Therefore, various methods have been suggested to reduce the amount of photobleaching.
How to minimize photobleaching
Use different dyes
Some dyes are more susceptible to bleaching than others, and the number of cycles required to fully bleach a sample varies dramatically. Therefore, by using a different or more resistant dye can dramatically decrease photobleaching in a sample. More resistant dyes include Alexa Fluor or DyLight Fluors.
Reduce bright light
The bleaching process can be reduced by using a light source with fewer photons. There are multiple potential light sources available, including mercury, xenon arc-lamps, and LEDs. The traditional mercury and xenon-arc lamps provide very intense wavelengths of light, leading to high levels photobleaching and phototoxicity.
Modern applications frequently use LEDs which can be highly controlled and photobleaching can be reduced. A lower photon exposure can also be achieved through neutral-density filters which only allow a few photons to reach the sample or through altering the gain setup of the microscope. However, these methods can lead to a dimmer image and reduce the resolution of the image.
Reduce sample exposure
Photobleaching can be minimized by reducing the amount of sample exposure to the light. This can be achieved by focusing the image using transmitted light, focusing on the area next to the area of interest, or by using a sub-optimal exposure time.
The lower intensity of fluorescence can be normalized by producing photobleaching curves, allowing the comparison of different treatment conditions without allowing bleaching to skew the data.
Use antifade
If the sample requires mounting, antifade mounting media can prevent photobleaching of the sample. However, a different medium is required for different fluorophores and therefore a range of optimization experiments are required to identify a suitable medium.
Fluorescence Recovery After Photobleaching (FRAP)
This technique uses information gathered from bleached and non-bleached areas of the sampe to recreate the image.
Fluorescence Recovery After Photobleaching (FRAP)
Further Reading
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