Optical instruments like the microscope have made a huge impact on the evolution of science and if you want to know what the best ones are you can find more info here. Even if scientists were able to make a lot of progress using a conventional microscope, the fluorescent microscope made a huge difference.
In this article, we are going to tell you all about how a fluorescent microscope works, what the advantages of using one are, and in what areas you will be able to use one. So, let’s begin your journey in finding out all about the fluorescent microscope.
Contents
What is a fluorescent microscope?
If you are not familiar with how a conventional microscope works, you should know that it uses visible light between 400 and 700 nanometers in order to illuminate an image and then to magnify it so that the person examining the image will be able to see it clearly. This method has been used for a long time, but it is a little bit limiting.
So in order to overcome these limitations, scientists have come up with another type of microscope, the fluorescent microscope. This microscope, in addition to the magnification of the image, uses phosphorescence and fluorescence.
Fluorescence is a term used to name the light produced by a substance when another light source is stimulating it. Practically, a fluorescent microscope uses a laser along with reflected light in order to offer a clear image. In this case, the light does not simply travel through a sample to make the image visible.
Phosphorescence is similar to fluorescence, but the light is not reflected right away by the substance. There are many types of fluorescence microscopes. In essence, any type of microscope that uses fluorescence is a fluorescent microscope, like the confocal microscope, for example.
How does a fluorescent microscope work?
The principles behind fluorescent microscopy are very simple and after reading them, you will clearly understand why a fluorescent microscope is superior to a conventional one.
- Principle no.1: You have to use a dye in order to make some components of the cell visible because most parts of the cell have no color and are not easily distinguished.
- Principle no.2: Dyes that are fluorescent have the ability to reflect light when they are excited by an external light source. These dyes are called fluorophores.
- Principle no.3: The light emitted by these substances can be separated from the light source that excites the fluorophores in order to be able to have a clear image.
Reading about these principles has made it all very clear. The simple way that this microscope uses light in order to illuminate certain parts of a cell is no longer a mystery. However, what dyes do you have to use?
Fluorescent dyes
There are a lot of dyes (stains) specially designed for different types of molecules. The way they work is that some of them are made of molecules that are fluorescent by themselves and, when applied to a sample, they will quickly bind to certain types of molecules in order to make them visible.
The most common stain is DAPI, which is a fluorescent stain that can bind to DNA. DAPI is a very interesting stain that can pass through the membrane of a cell. Live cells’ membranes are not that easy to penetrate, but DAPI can do it, allowing scientists to examine a lot of interesting things that happen inside a cell.
What are the advantages of using a fluorescent microscope?
Fluorescent microscopy helps scientists study the internal parts of cells and helps them observe structures and behaviors that conventional microscopy is not able to identify. This is why the study of different bacteria types is possible. Therefore, fluorescent microscopy comes with a lot of advantages compared to conventional microscopy.
- People who want to observe different types of molecules at the same time are able to do so by using different stains for each of them in order to identify them inside cells.
- The precision of fluorescent microscopy is very high, this is why while observing a cell using a fluorescent microscope, someone can determine as many as 50 molecules in an area measuring 1 cubic micrometer.
- Using this special technique, fluorescent microscopy allows you to observe cells both in vitro as well as in vivo, which is actually pretty amazing.
- A fluorescent microscope can isolate a single protein among other parts of the cell that are not illuminated.
- A fluorescent microscope will help you observe the behavior inside living cells and also the dynamics and is one of the most popular methods to do that.
What are the uses of a fluorescent microscope?
- Observing the structure of a cell
As we have said before, using a conventional microscope makes it hard to observe certain things that would help researchers in their quest for solving health issues or for identifying and solving mysteries that have been present in the minds of humans for a long time. A cell is practically without color and all its components are the same.
A lot of things happen in a cell because most cells have different components and each has a role, so in order to be able to observe the dynamics inside cells or to examine the structure of one of its components, it is best to use a fluorescent microscope.
- Studying cell populations
Cell populations are usually a group of cells that have similar characteristics. You can observe cell populations in order to see how they interact with one another and how the internal structures of these cells interact. You can also determine if the cell population is alive or dead.
- Observing DNA and RNA within a cell
Fluorescence microscopy is very often used to observe DNA and RNA present in cells and scientists are able to measure the DNA present in certain cells. You can also see how DNA and RNA molecules interact with other components of the cell using DAPI, the fluorescent substance that we have talked about earlier in this article.
- Spinning disk confocal imaging
This technique uses a confocal microscope, which is also a fluorescence microscope, in order to use 2D images to construct 3D images. This is amazing because you will be able to create a 3D image of a certain cell or molecule. Confocal microscopes are very complex and use very precise techniques to do that.
- Creating an image of a single molecule
A fluorescent microscope can help you localize and observe the activity of just one molecule inside a cell. This is such a big deal especially because you can observe this without damaging its natural environment and you can actually analyze its activity.
- Electrophysiology imaging used in neuroscience
Many new technologies have helped neuroscientists make astonishing discoveries in the last century. Fluorescent microscopes are optical instruments that have been found very helpful in this field. Using such a microscope to observe brain tissue has led scientists to even be able to find out what the function of such a cell is.
Cellular activity is examined here by analyzing the cells’ response to electrical stimulation, so you can imagine that the possibilities here are infinite. This is how scientists have been able to discover a lot about how our bodies work and about the role that our brains have in everything that is happening to us.
Are there any limitations to fluorescence microscopy?
One limitation would be the fact that when you use such an instrument to examine a sample, you will be able to analyze only the certain structures that have been previously labeled to be examined. Also, some cells might have toxic reactions to certain light wavelengths, so this is also a limitation that a fluorescent microscope has.
Final thoughts
As technology has evolved, so has the ability of humans to observe and develop new ways of making the world a better place. To do so, we must first understand what is going on around us, and fluorescent microscopes can help us do that. Due to the technology they are using, they can help scientists observe life at its deepest levels.
There are a lot of benefits that come along with using this type of microscope even though there are some limitations as well. However, the images provided by such a microscope and the scientific discoveries that it has made possible make it indispensable if we want to move forward with making the world better.
