As you may know, HCS Pharma makes use of Fluorescence Microscopy to perform biological assays and to test customers’ compounds. We often have questions about this technology, whether it is different or not from optical microscopy, and what it offers in terms of analysis. In a series of short articles, we will try to answer. Let’s begin by the basics: what is Fluorescence Microscopy?
Fluorescence Microscopy is based on a physical phenomenon called fluorescence, i.e. the emission of light just after absorption of a photon from an “excitation light”. Rare biological tissues have this property. Therefore, to visualize cellular or subcellular structures, we need to use fluorescent molecules (called fluorophores or fluorochromes) which are able to attach themselves specificly to biological molecules of interest. For example, DAPI fluorochrome is adapted to observe cell nuclei, since it can go through the cell membrane and binds strongly to adenine–thymine-rich regions in DNA. The selection of adapted fluorescence molecules is an important part of the preparation of any observation, and it depends on assay conditions (live cell imaging, cell types, assay duration, etc.) and which cell part is looked. To be able to distinguish them, fluorochromes must have different emission wavelengths.
The observation principle is quite simple: with our microscope, we illuminate the biological sample with the excitation wavelengths of chosen fluorochromes, and we finally observe the emission light! In HCS Pharma, we use the very powerful ImageXpress Micro Confocal High-Content Imaging System. To understand how the excitation and emission lights are excitated/received, look at the picture below and find more information in cited articles at the end.
As you see, for a fluorochrome we get a picture from a very narrow wavelength range. This is why we do not obtain a color picture but a monochrome picture for each specific wavelength. Our microscopes are very sensitive to light, they can see more than 60000 values of light intensity, 65536 to be precise. It’s why we talk about “16 bits pictures” (16 bits = 2^16 = 65536).
Below you can see a picture made from DAPI emission light with Hela cells in our BIOMIMESYS® hydroscafhold. The emission wavelength is close to 447 nanometers.
The emission wavelength for a DAPI picture is close to 447 nanometer, so we are used to colorize this kind of picture in blue… But keep in mind it is only for convenience!