Opto-digital image processing tools, such as Computational Clearing , can help to improve the signal-to-background ratio and reduce the out-of-focus blur. The associated contrast enhancement can reveal additional information in microscope images.
Amongst sequencing techniques, bioinformatics, and electron microscopy, Ogando et al.  analyzed infected cells by fluorescence microscopy: Vero E6 cells were grown on glass cover slips, infected with SARS-CoV-2, and fixed with paraformaldehyde. Then, the cells were incubated with antisera from rabbits or mice which were exposed to SARS-CoV beforehand (Figure 2). The SARS-CoV-originated antibodies, which bind to SARS-CoV-2 structures in Vero E6 cells, were then detected by fluorescently labelled secondary antibodies. In addition, nuclei were stained with Hoechst. Fluorescence imaging was done with a DM6 B upright fluorescence microscope.
Immunofluorescence microscopy revealed cross reactivity of many SARS-CoV antisera in SARS-CoV-2 infected cells (viral proteins nsp3, nsp4, nsp5, nsp8, nsp9, nsp13, nsp15, N, M). This fact means that antisera produced against SARS-CoV also lead to characteristic fluorescent staining in SARS-CoV-2-infected cells (Figure 3). Whereas nsps proteins were found in the perinuclear region of infected cells, the N protein was spread throughout the cytosol. The M protein was detected in the Golgi apparatus.
The cross-reacting antisera described in the study of Ogando et al.  is a useful tool for the characterization of the replication cycle of SARS-CoV-2. This tool enables researchers to define potential targets for inhibitors of replication.
A relatively simple experimental setup – immunofluorescence microscopy – is sufficient to draw conclusions of the viral replication cycle. Because the cells are grown on cover slips for this method and mounted on glass slides, an upright fluorescence microscope is a practical solution. Automated versions with a motorized stage in combination with a large field of view (FOV) help users to acquire large overviews quickly. If single snapshot images are enough, a manually operated mechanical stage is the more reasonable choice.
- N.S. Ogando, T.J. Dalebout, J.C. Zevenhoven-Dobbe, R.W.A.L. Limpens, Y. van der Meer, L. Caly, J. Druce, J.J.C. de Vries, M. Kikkert, M. Bárcena, I. Sidorov, E.J. Snijder, SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology, Journal of General Virology (2020) vol. 101, iss. 9, pp. 925-940, DOI: 10.1099/jgv.0.001453.
- L. Felts, V. Kohli, J.M. Marr, J. Schumacher, O. Schlicker, An Introduction to Computational Clearing: A New Method to Remove Out-of-Focus Blur, Science Lab (2020) Leica Microsystems.
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