# Transcript

DR KATJA RIETDORF:
Within this video, we will show you some examples to illustrate just how powerful microscopy is to study cellular structures and processes happening inside cells. The main purpose of this video is to share our fascination of cell biology and the techniques of fluorescence and electron microscopy. Honestly, I never get bored looking at organelles moving in cells or cells themselves moving. You will learn about the methods used to obtain the videos shown here elsewhere. So rather than teaching your detailed knowledge, we would like you to sit back and watch the images and videos we present and, hopefully, come to share our fascination.
So first comes the question, how do we take the multicoloured images of living cells that you will have seen already? Each colour shows the staining of one particular structure of protein inside a cell. Depending on the staining method used, the cells can be alive. Here, you can see an image of nuclei shown in light blue.
In the same cells, a different probe showing us a purple colour was used to stain both the cytosol and the nuclei. If you combine both images, you get this two-coloured image. If you then add a third stain with a different colour, here a green probe specific for mitochondria, you will get an image with three colours.
While these images are static, the following examples show the use of fluorescence in living cells. This image shows a cell in which three organelles can be seen, each expressing a different fluorescent protein. Mitochondria are shown in light blue. And the endoplasmic reticulum, or ER, is shown in pink. Both organelles form a three dimensional network that spreads through the cell. In green, you can see the Golgi apparatus, which is much smaller than the other two organelles.
In theory, you can use probes of many more colours to stain different proteins or organelles. However, to ensure that the signal you capture only comes from one probe, the colours must be sufficiently different. Components of the specific microscope you use will determine how many colours you can separate and use inside a cell. If you rotate the cell, you get an even better idea of the three dimensional arrangement of the organelles. At certain angles, you can see an empty elliptical shape, which is the nucleus.
Let us now observe the process of cell division. During cell division, the cell changes its shape. And the organelles move towards the opposite poles of the cell. The cell then becomes rounded. Both the ER and mitochondria are found near the cell membrane at the cell’s periphery.
Finally, cytokinesis, the process of the physical division of the cell, takes place. You see the cell membrane constrict, and two daughter cells form. This whole process took around two and a half hours.
You will have heard about imaging techniques to visualise tissues inside the body, for example, to detect a cancer. Tissues can also be imaged using fluorescence microscopy. You will now see an example of immune cells being imaged inside a blood vessel.
Cells in this image express a yellow protein in the cell membrane. That’s why the walls of the blood vessel on the right and left are showing in a brighter fluorescence than the lumen of the blood vessel, which you can see here in the centre. The lumen is the hollow part of the blood vessel. It is filled with blood, which contains several cell types, including immune cells.
Within the lumen of the vessel, you can see small blue dots. These are fluorescent particles that have been injected into the blood. Because they are foreign to the body, they will be taken up by immune cells. And we will see this process once I start playing the video.
You can see a cell entering the blood vessel from the bottom of the image. Now, you can see a second cell appearing. This cell has already taken up several of the blue particles. Both cells move within the lumen of the blood vessel and constantly change their shape. They are only clearly visible when they are in focus. And you can see the cell moving through a three dimensional space, leaving the area that is in focus and disappearing at times.
This video was taken over a period of 55 minutes. I feel it is fascinating to see immune cells in their native environment and it is absolutely amazing to see how these cells constantly change their shape whilst moving around. If you use distinct colours to stain different cell types, techniques like this also allow you to observe the interaction of different cell types over time.
After seeing a whole blood vessel, let us zoom in again and look at one individual cell. Here, you see an isolated heart muscle cell. It is loaded with a fluorescent indicator that is sensitive to changes in the intracellular calcium concentration. When the intracellular calcium concentration increases, the indicator increases its brightness. An increase of the intracellular calcium concentration causes muscle cells to contract.
So what you will see in this video are small local increases in the calcium concentration. Approximately every 14 seconds, you see a big increase in the brightness, starting in the bottom right-hand corner and moving to the top left. This indicates a large increase in the intracellular calcium concentration, which causes the cell to contract. Several billion of these cells make up your heart and contract rhythmically throughout your lifetime. Did I mention before that I can spend a long time looking at these cells and being fascinated by how much detail we can observe?
So this is the end of our journey. We hope you enjoyed it and see why we love using microscopy to study cell biology in living cells. One question remains, which is your favourite?