Department Pathology and Clinical Bioinformatics
Principal investigator Johan Slotman
E-mail address j.slotman@erasmusmc.nl
Website www.erasmusoic.nl
Expansion microscopy to reach electron microscopic resolution with light
Supervisor: Johan Slotman, j.slotman@erasmusmc.nl
The resolution of optical microscopy, including fluorescence microscopy is limited to approximately 200 nm, due to the properties of light. However, new techniques have been developed that can circumvent these hard limits. These techniques are collectively known as super-resolution microscopy and require advanced and specific microscopes. An alternative method to visualise details in cells that are too small to resolve is to physically increase the size of sample, known as expansion microscopy (ExM). In this technique, samples are embedded in a gel, the cell content, including fluorescently labeled proteins or other biomolecules is then cross-linked to the gel and digested. Finally the gel is allowed to expand 4x to 10x. Microscopic imaging such an expanded sample then reveals details that initially were too small even when they are too small to visualise by super-resolution microscopy techniques. One potentially very promising application is to combine ExM with a so called pan-staining, which aims to label every protein in a cell with a fluorophore. In regular microscopy such staining will yield no discernable features since they lack resolution. However, using ExM several research groups have recently successfully applied pan-staining and claimed to achieve electron microscope (EM) resolution. This project involves setting up ExM and pan-staining in our lab to investigate focal adhesions. Focal adhesions are structures by which cells attach to a surfaces and move on it. We have already observed distinctive features of focal adhesions in EM and aim to observe the same using ExM and pan-staining. Additionally we want to combine this technique with regular staining of proteins to go beyond the capabilities of EM in which specific labeling of proteins is difficult. The aim of this project is to bridge the resolution gap between EM and light microscopy.
Techniques
- Super-Resolution Microscopy
- Expansion Microscopy
- Cell Culture
- Fluorescence staining
Further reading
Light microscopy of proteins in their ultrastructural context
Tracking of androgen receptors using lightsheet microscopy
Supervisor: Johan Slotman, j.slotman@erasmusmc.nl
The androgen receptor (AR) is a nuclear receptor that plays a role in development and maintenance of male phenotype. Malfunctioning of AR is closely associated with prostate cancer, the most prominent form of cancer in males. Upon activation by testosterone, AR translocates to the nucleus, where it binds to DNA and induces transcription of specific genes. During this project you will study the dynamics and location of the AR. For this you will make use of cells expressing fluorescently tagged AR proteins and a Luxendo lightsheet microscope (InviSPIM). In lightsheet microscopy the sample is illuminated through an extra lens perpendicular to the lens used for image acquisition. Using this microscope we can selectively illuminate a sheet in the sample, and image the complete field of view very fast using a sensitive sCMOS camera. This allows us to monitor dynamic proteins within the 3D environment of the cell.
In this project you will learn how to setup and optimize the complete imaging experiment, from sample preparation to live cell imaging and subsequent image analysis. The InviSPIM is equipped with a spatial light modulator (SMD) to create different shapes of the illuminating sheet. Optimizing the beam shape for different kind of imaging experiments will be useful for the future users of this microscope. This study will lead to better understanding of the functioning of AR in 3-D cultured cells, including AR receptor dimerization and its DNA binding properties.
Techniques
- Confocal Microscopy
- TIRF Microscopy
- Lightsheet Microscopy
- Single Particle Tracking
- Cell Culture
- Fluorescence staining
Further reading
Compartmentalization of androgen receptors at endogenous genes in living cells
FRET/FLIM microscopy to analyze androgen receptor dimerization and protein-protein interactions
Supervisor: Johan Slotman, j.slotman@erasmusmc.nl
Fluorescence microscopy is a cornerstone technique in cell biology. Fluorescent resonant energy transfer (FRET) microscopy is an advanced imaging tool to study conformational changes in proteins and protein-protein interactions. FRET is a phenomenon that occurs when two different fluorophores are in close proximity (<10 nm) to each other, where the emission spectrum of one (the donor) overlaps with the excitation spectrum of the other (the acceptor). The efficiency of FRET is highly dependent on the distance between the donor and acceptor fluorophore, and therefore can be used to precisely measure intra- and intermolecular interactions. Using fluorescence lifetime microscopy (FLIM) we can measure FRET by measuring a decrease in fluorescence lifetime of the donor fluorophore.
In this project you will use FRET/FLIM to investigate protein-protein interactions and the role of dimerisation of the androgen receptor (AR). This nuclear receptor is present in the cell in different conformations and dimerizes upon activation by binding to testosterone. Using fluorescently labeled androgen receptors that are fluorescently tagged at both the N- and the C- terminus by a donor and acceptor fluorophore respectively, we monitor the conformation using FRET/FLIM. We hypothesize that AR is in a closed conformation (where the N- and C-terminus are close together and FRET will occur) when it is not bound to DNA and opening up when it is binding to DNA and activating transcription (no FRET, because N- and C-termini are far away from each other. This project is focused on advanced microscopy but will also involve cell culture, as well as the design and cloning of fusion proteins. The results of this study will help us to better understand the function of the androgen receptor. This will lead to further insight about AR and its role in prostate cancer, the most prominent form of cancer in males.
Techniques
- Fluorescence resonant energy transfer (FRET) microscopy
- Fluorescent Lifetime Imaging Microscopy (FLIM)
- Confocal Microscopy
- Cell Culture
- DNA Cloning