Optimization of fluorescent labelling and imaging protocols for actin characterisation

Abstract

Since the invention of the microscope in the 17th century by pioneers such as Antonie van Leeuwenhoek and Robert Hooke, the study of cells has played a fundamental role in science. Cell labelling is a technique in which different cell structures or components are marked to ensure optimal visualization under the microscope. To carry out this labelling process, markers are used, such as fluorescent dyes, antibodies conjugated with fluorophores, and genetically encoded proteins. These markers specifically bind to their target, depending on the intrinsic characteristics of the marker. Researchers are not only able to observe the morphology and cellular composition, but also the biological processes taking place within the cell. This has led to advancements in the study of diseases caused by cellular dysfunction, such as, for example, aiding in diagnosis with the specific labelling of biomarkers in tumour cells.4 This project will focus on the study of actin, the protein that makes up the majority of the cellular cytoskeleton, in fixed HeLa cells. Since fixed cells will be used, the dynamics of actin will not be observed. To carry out the study, different actin markers will be tested, including fluorescent dyes and antibodies conjugated with fluorophores. Additionally, the effects that various cellular fixation processes may have on the preservation and labelling of actin will be studied. To observe and study cellular components, imaging techniques are necessary to obtain detailed images and precise information about the elements of interest, such as in this case, actin. Because of the use of fluorescent markers, the technique of fluorescence spectroscopy is combined with confocal microscopy. The latter, due to its technical features, allows for high-resolution images, minimizing fluorescence interference from other cellular planes. In this study, three-dimensional (xyz) images will be obtained to determine the optimal cellular labelling conditions. Moreover, hyperspectral images (xyλ), with an emission spectrum per pixel, will be employed to compare the affinity and specificity of different markers with actin. When obtaining hyperspectral images with several fluorophores present, it is necessary to resort to chemometric tools to effectively separate the fluorescence signal from each fluorophore. In this study, the tool known as Multivariate Curve Resolution by Alternating Least Squares (MCR-ALS) was used to perform the chemometric analysis. Based on the images obtained in xyz acquisition mode, it was determined that the most suitable fixation method for the fluorescent dyes (phalloidin, SiR-actin, SPY-555) was paraformaldehyde (PFA). Conversely, for the antibodies (anti-beta and anti-pan actin Ab), the fixation method combining PFA and methanol was selected. Using hyperspectral images and subsequent chemometric analysis, it was possible to separate the fluorescent signals and compare two pairs of fluorophores. The pair comprising SPY-555 and phalloidin-568 exhibited regions of colocalization as well as areas where each fluorophore showed a preference for the different actin structures. On the other hand, the pair formed by phalloidin-568 and anti-pan actin Ab (ASTAR Orange) demonstrated very low colocalization. The actin filaments were clearly marked by phalloidin-568, while the antibody produced a more diffuse signal, predominantly localised in the cell's interior. Nevertheless, to obtain more consolidated results, a more extensive study would be needed, acquiring a greater number of images and ensuring that the studied cells are in the same physiological state.