研究人员访谈

Dr. Steve Thomas

Senior Lecturer in Cardiovascular Science, University of Birmingham

Please tell us about your research.

We are interested in platelets and megakaryocytes, which are blood cells. Particularly, we study how they are formed, how they function both in normal healthy people but also in disease. We use a lot of imaging to try to learn what is happening at a cellular level, understand the proteins that are involved in making the cells work, how they interact together and what happens when these things go wrong in terms of human disease.

What imaging techniques are you using and how do these imaging techniques help you answer your research questions?

We are using fluorescence microscopy, predominantly super resolution techniques. These are really important for us because they allow us to see the sub-cellular detail that is normally excluded when you are using diffraction limited techniques. Super-resolution has allowed us to really look at interactions between different protein compartments in the cell. We can see how they are organized spatially which tells us something about how they function and what they are doing in the cell. We can’t do that in normal microscopy which does not have the resolution. Both the N-STORM and the N-SIM techniques have been very useful in understanding how these proteins are organized and how that dictates their function in the cell.

Human platelet spread on fibrinogen, stained with 488-phallodin to label the actin cytoskeleton and imaged using 3D-SIM. The image shows platelets at various stages of spreading indicating how actin organisation changes to drive the spreading process.

You mentioned that you use N-STORM and N-SIM – what did each of these two techniques enable you to see particularly?

N-STORM allows us to look at individual molecules within the platelets surface and to try to understand how they are organized. For example, we may label a receptor on that platelet surface and look at how its distribution changes in an activated vs. a resting cell. That can tell us something about how these molecules are helping this cell to signal because we can get information on cluster size and number of molecules, see how they co-localize with other molecules in the cell. Insight into signaling on a molecular level feeds into drug discovery and understanding of the disease by knowing what the normal situation is vs what happens if these processes go wrong. So N-STORM specifically allows us to check what is happening with these proteins at the molecular level.

N-SIM on the other hand is very nice for giving us high-quality images of the cytoskeleton, which is a protein network in the cell. This allows us to look at how this protein network is organized in a normal cell and what happens when you activate these proteins and to drive some of the cell functions. For example, when the platelets are activated they change shape very rapidly. By imaging the cytoskeleton using N-SIM we can understand how these proteins are responsible for that changing shape.

A549 cells stained with Alexa488-phalloidin to label the actin cytoskeleton and imaged by Structured Illumination. Left hand image shows the widefield image and the right hand side shows the 3D-SIM reconstructed image. The super-resolved image allows the detailed organisation of the cortical cytoskeleton to be visualised.
A549 cells expressing Snap-tagged Adenosine A2 receptor and imaged using dSTORM. The left hand images shows the diffraction limited image whilst the middle image shows the reconstructed STORM super-resolution image. In the right hand image, a clustering algorithm has been applied to the data to identify clusters of receptors for quantitative analysis.

You mentioned that you use N-STORM and N-SIM – what did each of these two techniques enable you to see particularly?

N-STORM allows us to look at individual molecules within the platelets surface and to try to understand how they are organized. For example, we may label a receptor on that platelet surface and look at how its distribution changes in an activated vs. a resting cell. That can tell us something about how these molecules are helping this cell to signal because we can get information on cluster size and number of molecules, see how they co-localize with other molecules in the cell. Insight into signaling on a molecular level feeds into drug discovery and understanding of the disease by knowing what the normal situation is vs what happens if these processes go wrong. So N-STORM specifically allows us to check what is happening with these proteins at the molecular level.

N-SIM on the other hand is very nice for giving us high-quality images of the cytoskeleton, which is a protein network in the cell. This allows us to look at how this protein network is organized in a normal cell and what happens when you activate these proteins and to drive some of the cell functions. For example, when the platelets are activated they change shape very rapidly. By imaging the cytoskeleton using N-SIM we can understand how these proteins are responsible for that changing shape.

What do you think of Nikon microscopes?

The quality of the systems is very good, they are well-built and well-made, but primarily I think the support that we get for using the systems is excellent and really helps us to make the most out of our systems. You have got people who really understand them and know how to use them to solve questions. The support behind the systems is very important.

What do you think of Nikon microscopes?

The quality of the systems is very good, they are well-built and well-made, but primarily I think the support that we get for using the systems is excellent and really helps us to make the most out of our systems. You have got people who really understand them and know how to use them to solve questions. The support behind the systems is very important.

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