What is the Best Way to Normalize Fluorescent Images?

Fluorescence microscopy has become an essential procedure for understanding cellular structures, physiology, and medical diagnoses. It provides clear and quite colorful images of the sample under study. This makes identification and understanding of the biological processes so much easier. However, there are some challenges with fluorescence microscopy when it comes to obtaining good quality and reproducible images.

fluorescent photo

To counter these issues, normalization of the images is done.

Normalization of images in fluorescence microscopy can be done by (a) calibration using a sample of reproducible fluorescence field, and (b) using 10-50% w/v of concentrated Fluorescein or rhodamine between the cover glass and the slide (c) using the same microscope settings which show a good image across all samples.  Anti-quenching agents (eg. AuNP) are also helpful.

Fluorescence microscopy has many applications in the field of biology and medical science. However, it’s quite often difficult to get identical images of the same slide in different microscopes and even in the same microscope if the images are being taken over some time (say, to study a biological process that might complete over a few days).

This is a major drawback of fluorescence microscopy, and it’s often the major reason why it can’t be used under various circumstances (ex. to make certain medical diagnoses), even though it’s an excellent tool to study various structures, proteins, DNA, and biological processes.

The quenching (or photobleaching) of the sample can be prevented by using anti-quenching agents like AuNP (gold nanoparticles). This safeguards the sample’s fluorescence for a longer duration of time and therefore will provide a slightly longer time for the examination of the sample with minimal changes in the fluorescence quality.

Normalization Of Images

Some of the major challenges with the fluorescence microscopy include:

(A) various instrumental factors, like different – microscopes may use different optics and different light detectors, and so the emitted light may have slight variations in different microscopes, 

(B) shading. Because of shading, some areas of the field may appear brighter while some may appear darker, despite having the same amount of fluorophore (the dye), and

(C) Photobleaching. This can be controlled to some extent by reducing the duration of light exposure, and by using anti-quenching agents like AuNP.

The variation between different instruments is managed by calibration using a sample of a reproducible fluorescence field. Thin and uniform fluorescing reference layers are used here for the comparison of the image for image intensity correction and reproducing imaging conditions and quality control. The characteristics of the imaging situation can be summarized in a SIP (sectioned imaging property) chart for future use and comparison.

Shading is managed by applying a concentrated solution of fluorescence dye. Doing this brightens the image uniformly, and also prevents photobleaching of the sample to some extent. This is a simple, pocket-friendly, and reproducible method— making it a convenient method for routine fluorescence microscopy.

Photobleaching can be prevented by using anti-quenching agents (AuNP). Additionally, as mentioned above, the use of concentrated fluorescein dye also provides a certain degree of protection from photobleaching.

The normalization can also be done using certain imaging programs like metamorph, Isee, and Slidebook.

When performing measurements of intensity on a microscope such as a confocal to determine the amount of a protein for which you stained using an antibody, normalization is required because you will not be able to reproduce conditions from one day to another which would result in erroneous intensity values.  In addition, picking an intensity value that is too high would result in washed out images that could not be used for measurements.

In this case you would want to go through your samples and set your laser amount, gain, etc to a value where all the images can be readable for measurements and not blown out.

Some Background on Fluorescence Microscopy

Fluorescence microscopy is the type of microscopy which uses the fluorescence produced by fluorophore stained specific structures/proteins in the specimen to visualize these structures. 

In a regular light microscope, white light is used to illuminate the background. But in fluorescence microscopy, instead of using white light, a specific color of light is used— depending upon what structure is being studied. More on how it works is discussed below.

The sample is stained with a fluorescent dye (flours chrome/fluorophore) and these emit fluorescent light which helps the structure being studied visualized. Alternatively, the gene under study can also be GFP tagged. A GFP tagged DNA will produce a GFP tagged protein, and thus this protein can be visualized easily as well. 

Principle Of Fluorescence Microscopy

Fluorescence microscopy works on the principle that when a certain wavelength of light is directed on the sample, the electrons in the fluorophore (dye) absorb the energy, and then emit light to get stabilized. 

After absorbing light, the electrons in the atoms of the dye jump from the innermost shell to the outer shell due to excitation. But electrons are unstable in the outer shell. To get stable again, they emit the energy out and jump back to the innermost shell again. 

The energy/light/photon thus emitted by the electrons in the dye is the fluorescence light that is observed in fluorescence microscopy.


How Fluorescence Microscopy Works


  1. The light source in the fluorescence microscopy generates the white light, which then goes through the excitation filter.

  1. The excitation filter allows the light of a certain wavelength to pass only (say, blue light or UV light).

  1. This light is then reflected by the dichroic mirror to the sample.

  1. When the light reaches the sample, the dye absorbs and then emits the energy.

  1. The released energy from the sample goes via the objective lens, passes through the dichroic mirror, then to the emission filter, and lastly to the ocular lens where the observer can view the result.

  1. Fluorescence microscopes usually also have a camera attached to them at the top, which transfers the image to a computer attached.


The light given to the sample is of high energy, short wavelength, and the energy emitted by the electrons is low energy and long wavelength. This is the reason that the light given and the light emitted are of different colors (ex. if blue light is given, green light is emitted). 

The following video briefly explains how a fluorescence microscope works:


Applications Of Fluorescence Microscopy


Some of the advantages of using fluorescence microscopy include:


1. The biggest advantage of fluorescence microscopy is that different structures/organelles/proteins within a cell emit different colors, so it’s easier to see these structures and their locations distinctly.


2. Since this process does not kill the cells under study, various biological processes can be studied using fluorescence microscopy. Similarly, various pathological phenomena can also be studied.


3. Images of the subcellular structures formed by fluorescence microscopy are more magnified and clear when compared to other light microscopes. So, we can obtain much clearer images of these microscopic structures.


Disadvantages Of Fluorescence Microscopy


1.The major disadvantage of fluorescence microscopy is photobleaching. The fluorophores on repeated exposure to light lose their ability to fluoresce. So, the shelf life of the slides may not be very long (varies from dye to dye).


2. Quenching can also occur due to other factors. Quenching refers to the loss of fluorescence due to various factors, like pressure and temperature. This can be dealt with by using anti-quenching agents to some extent.


3. Only those structures that fluoresce can be visualized using this microscopy. 


Points To Remember About Normalization In Fluorescence Microscopy


Due to various microscope-related factors, photobleaching, and shading, obtaining reproducible images in different and even the same microscopes is usually quite difficult. To combat these issues, the following measures can come in handy:


1.To deal with the instrument to instrument variation– uniform, and thin fluorescing reference layers can be used. These can be used for comparison and reproducing imaging control.


2. The best way to tackle shading is by using a concentrated solution (10-50% w/v) of fluorescein or rhodamine dye. Out of the two, fluorescein dye gives the best result. Using these concentrated dyes also prevents photobleaching to a certain extent.


3. Photobleaching can be reduced by limiting the exposure time of the sample with the light, or by focusing the light on an area next to the sample. Anti-quenching agents like AuNP (Gold Nanoparticles) can also help prevent rapid photobleaching.


Read the following article to learn the maximum magnification of a confocal microscope.

Sneha Sunny

Dr. Sneha is a medical doctor (MBBS) based in India. She has always been fascinated with biology. She enjoys exploring and learning about new biological advancements, especially in the field of medical sciences. She finds writing to be a great way for sharing and connecting with others with similar interests.

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