Palette of life: flow cytometry and confocal microscopy

confocal microscopySometimes you can see arguments about whether science should be considered an art, or is it a craft. And, I must say, recently in science there is less and less room for inspiration, now the work of a scientist is more like a mixture of an accountant's routine and literary criticism. Ordering a huge array of numbers, and interpreting them according to given rules.

And yet, there are elements in scientific work that resemble the work of an artist. They are mainly related to obtaining those numbers that you will have to work with later. Sometimes you have to get creative to get exactly the data you need and weed out the rest. Sometimes hypotheses and theories and truth are born under the influence of inspiration. And there are whole methods in science that are somewhat similar to fine art.

About a couple of such methods that use paint and light and shadow are important, today we will talk. We will talk about flow cytometry and confocal microscopy.

 

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Body cells and flow cytometry

Our body is not a single monolithic system. It is a collection of billions of cells of hundreds of different types. And all these cells constantly interact, self-organize, form jointly working populations. But in order for the cells to understand with whom and how to interact, each cell is labeled with many different markers. Most often, these are surface proteins, or complex structures, such as glycoproteins, if the protein is associated with a carbohydrate residue, or lipoprotein, if the non-protein part of the molecule is of a lipid nature.

Typically, such molecules are either immersed in the cell membrane or penetrate it through and through. The cell produces them when it is in a certain state. A cell that has just appeared will not be similar in a set of markers to a differentiated cell, that is, an adult cell that has not yet begun to work. And both of them will be different from the activated cell, which has already begun to perform its function. Different populations of the same cell type will also differ. For example, among leukocytes, the cells of the immune system most often studied using flow cytometry, there are T- and B-lymphocytes, killers and helpers, central and effector memory cells. And each of these populations will have its own set of surface markers.

Signaling molecules, as they are also called, not only mark the cell according to its functional group, but also ensure its functions. As soon as the part of the molecule sticking out of the cell membrane binds to the appropriate substance - often these are also surface markers of other cells - the entire signal molecule changes its configuration. As a result, either the signaling molecule itself somehow changes, or its new conformation serves as a catalyst for chemical reactions inside the cell.

What is the connection of all these nuances of cellular life with flow cytometry? The fact is that scientists have learned to divide cells in accordance with surface markers, components of intracellular reactions, or the size or shape of the cell.

This is done quite simply. First, the researcher needs special paints. They are derived from one of the most versatile tools in modern biology, monoclonal antibodies. These are substances that can bind to a well-defined set of atoms. For example, with the functional part of the surface marker. A substance is chemically attached to the desired monoclonal antibody, which, when illuminated by a laser, glows in a certain color. Then, with such a paint, or rather, with a whole palette of such paints, a population of cells is processed. And then these cells are processed in a cytometer.

And here the most technological part of this analysis begins. The cells enter a very thin fluid stream that only fits one cell. Each cell that floats in this way is illuminated by several lasers at once. And photodetectors read the light with which the cells begin to glow. In this way, not only surface markers can be determined. The shape of the cells can be recognized by assessing how much the light passing through it deviates. The composition of the cells will be clear if you look at how the cell scatters light. In addition, by perforating the cell wall of the cell, even its internal contents can be stained.

Today, the most frequent object that is analyzed using flow cytometry is the immune system. By tracking changes in the ratio of different types of cells, one can understand what the general state of immunity of a particular organism is, how it reacts to certain influences, and whether there are any anomalies in the system. Cytometry is also used in oncological, toxicological and hematological studies. In addition, in addition to medical applications, flow cytometry is useful in many other areas. The study of microorganisms, both grown in the laboratory and obtained from the natural environment, tracking cell growth, determining the phases of the cell cycle - all this is possible with the help of flow cytometry.

Features of confocal microscopy

Another method, often using the same stains on monoclonal antibodies, is confocal microscopy. This method differs from conventional microscopy in that we look only at a tiny point in the sample under study at any given time. Rather, we are not looking, but the computer, people already see the final result of the microscope. And this result looks like a three-dimensional model of the studied sample. In order to obtain its image, the microscope sequentially removes each tiny section of the object under study. Moreover, if the sample is transparent, not only its surface is scanned, but also all the internal contents.

Usually, either a cell or some kind of cellular organelle, or even a molecule, acts as a sample. Yes, with the help of confocal microscopy it is possible to see a single molecule. The point is that we are looking at the image through a tiny hole. Thanks to this, it is possible to cut off the illuminating radiation, and greatly increase the sharpness of the focus. Of course, a molecule that can be seen in this way must be very large, consisting of thousands of atoms. So only huge molecules of viruses, or densely twisted gigantic DNA molecules, can be seen in this way.

And what about paints? The point is that in this method, too, the light source is a laser, which is very convenient for inducing fluorescence in special substances. Of course, in this case we are no longer talking about individual molecules, in this case the paint molecule would be comparable in size to the object being painted. However, for the study of whole cells, this method is very convenient. Thanks to staining, you can very clearly see where exactly in the cell the reactions take place, and which ones. Considering that even living cells can be examined using confocal microscopy, it is no exaggeration to say that this method allows us to see life itself.

However, even in individual molecules, it is sometimes possible to highlight certain parts. True, for this, the atoms that make up the substance themselves must be capable of excitation when illuminated by a laser.

These are the modern scientific arts. Researchers around the world paint nature in different colors to better understand it. Progress brings us ever new ways to look at seemingly familiar phenomena. And it’s hard to even predict what other miracles the future has in store for us.

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