One week in the life of a TIRF microscope
The Total Internal Reflection Fluorescence (TIRF) microscope in the Bally lab sees a variety of different samples and applications. The week can be quite busy and the schedule full. What is imaged at the TIRF microscope also gives a good impression on what is going on in our research, since imaging is central to our biophysical approaches which allow us to take a closer look (literally) on individual virus(-like) particles.
The week starts with Konrad, the PhD student in our lab. He is preparing bilayers with lipid extracts from enteroids (“miniguts”) which mimic the surface of a cell. Konrad is interested in norovirus binding kinetics, meaning that he looks at the binding and dissociation of norovirus virus-like particles (VLPs). The proteins of the virus capsid are labelled with a fluorophore that emits green light. To look at virus-membrane interaction dynamics, he takes movies of the fluorescent VLPs as they land on and leave from the bilayer. This experiment is repeated for surfaces of different compositions to compare the effect of membrane composition on the virus binding behaviour. Through such experiments, he hopes to learn more about the factors that make cells susceptible to norovirus infection.
On Tuesday, Fouzia has a long lab day. During the first hours, she is preparing surfaces with various glycoaminoglycans (GAG), sugars that are present on the cell. These surfaces allow her to study virus-GAG interactions under different conditions and in a controlled environment. As with all surface preparations in our lab, she is taking great care in keeping them hydrated and not to damage the surface. Once everything is ready, she moves with the sample to the microscope and adds her virus of choice – human papillomavirus 16 (HPV16). The fluorescent HPV16 is kindly provided by our collaboration partners in Mario Schelhaas´s lab from the University of Muenster, Germany. Fouzia is interested in the diffusion behaviour of HPV16 and in how the different GAGs impact the movement of the particles on the cell surface. This is different from the kinetics experiments as she is looking at the movement of the particle after landing on the surface. Understanding how viruses attach to and move on cell surfaces to efficiently infect the cell can give insights into how to design new anti-viral treatments.
For Wednesday, our Erasmus student Julius has managed to get some microscopy time. He is supervised jointly by Konrad and Dario and he is studying SARS-CoV-2, the coronavirus responsible for the current pandemic. Today he is performing an experiment together with Dario. For his experiment, he is preparing native supported lipid bilayers (nSLBs). nSLBs are formed from membrane material purified from cultivated cells. They thus contain all lipids, proteins and sugars found in the membrane of the source cell and are closer to living cells than the purely artificial surfaces mentioned so far. Julius is using material from lung cells, which are susceptible to SARS-CoV-2 infection. In parallel he is preparing lipid vesicles that contain fluorescent lipids and are decorated with SARS-CoV-2 spikes. The spike protein is the surface protein responsible for virus attachment, thus these vesicles mimic the real virus in Julius’s experiments. When everything is ready, the coverslips with the nSLBs are mounted on the microscope and kept at 37°C. Now Julius is carefully adding a defined concentration of vesicles presenting spikes on their surface. Similar to Konrad, he is interested in the binding kinetics of these vesicles to the nSLBs and is comparing different coronaviruses to each other. In this way we want to investigate if the disease severeness and/or infectivity of some coronaviruses can be predicted by their interaction with the cell surface.
Our TIRF microscope can also be used for normal fluorescence imaging. On Thursday, our virologist Lifeng is using this feature for immunostaining experiments. He is interested in the role of human apolipoproteinE (ApoE), in the life cycle of Herpes simplex virus (HSV). Studies indicate that a HSV infection can play a role in the development of Alzheimer’s disease (AD), which in turn often coincides with the presence of a variant of the protein (ApoE4), which is carried by a percentage of the human population. Furthermore, he would like to understand why people with this variant are more likely to develop AD. To investigate this aspect more closely, Lifeng has created stable cell lines that can express different variants of ApoE. He then infects those cell lines with HSV and compares the progress of the infection at different time points. Both, the HSV and the ApoE, are fluorescently labelled with different colours, allowing Lifeng to see if and where they co-localise within the cells. In contrast to the previous users who took movies to see fast dynamics Lifeng is taking single images. This is necessary as the virus life cycle is orchestrated on a bigger time scale and Lifeng is taking snapshots at time points a few hours apart.
On Friday, Kerstin is using the microscope to test a method to label proteins that she has genetically modified. She has added a SNAP-tag into the surface protein of Ebola Virus by cloning. She then transfected cells with the DNA so that the cells express this mutant on their cell surface. After giving the cells 24 hours to produce many proteins, she is trying today to label them with a fluorophore that binds to the SNAP tag. If everything works well, she will be able to see the fluorescence of those cells that were successfully transfected. In the future, she will use the genetically modified plasmids to produce Ebola glycoprotein-presenting virus particles (pseudotypes). The SNAP-tag will allow to choose the fluorophore on virus particles more freely, and to label the surface protein directly. So far, we label other parts of the virus particles (e.g. lipids or capsid proteins) or we perform immunostaining with specific antibodies. With the direct label on the protein, we will further be able to do step-wise bleaching to count how many glycoproteins are present on an individual viral particle. This will give us new information about the characteristics of the viral particles we use in our studies.
By Kerstin