Methods

Total internal reflection fluorescence (TIRF) microscopy

This technique allows selective visualization of the plasma membrane and sub-plasma membrane regions of adherent cells. It is ideally suited for studying signaling events at the plasma membrane, endocytosis, exocytosis and ion fluxes in living cells. The low intensity light required for TIRF illumination also minimizes fluorophore bleaching and phototoxicity. Current setups allow 2-4 color imaging using either low (10X) or high (100X) magnification objectives at physiological temperatures.

Contact: Olof Idevall-Hagren


Spinning disc confocal microscopy

This technique utilizes spinning discs with microlenses and pinholes to illuminate and block out-of-focus laser light, respectively. It is ideally suited for fast cross-sectional or 3D imaging of living cells. Current setup is equipped with 60X and 100X objectives and enable 2-color (red/green or blue/yellow) imaging at physiological temperatures.

Contact: Olof Idevall-Hagren

Figure 1: A. Drawing of an adherent cell with the nucleus in grey and the endoplasmic reticulum in black. Pictures to the right shows confocal and TIRF images of a cell expressing an ER-resident protein that also binds to the plasma membrane (GFP-E-Syt2). Notice how this protein appears peripheral in the confocal images, whereas it appears punctate in the TIRF images. The punctate structures represent points-of-contact between the ER and the plasma membrane. Images have been inverted so that fluorescence is shown in black. B. Example pictures of cells expressing a fluorescence-tagged lipid-binding protein. The protein is plasma membrane bound under basal conditions, but gets redistributed to the cytosol after receptor-triggered hydrolysis of the lipid. This redistribution is seen as a loss of peripheral fluorescence in the confocal images, and as an overall loss of fluorescence in the TIRF images.


Optogenetics

We have developed a number of optogenetic tools based on blue-light-induced dimerization. This technique allows non-invasive control of protein localization within cells and can be used to e.g. steer a protein of interest to a specific cellular location. Current tools include modules that enable synthesis and degradation of multiple phosphoinositides (e.g. PI[4,5]P2 and PI[3,4,5]P3) in various cellular membranes, including the plasma, ER, mitochondrial and endosomal membranes. The optogenetic modules are easily adaptable to other uses.

Contact: Olof Idevall-Hagren

Figure 2: Principle and examples of light-mediated control over protein localization. A. Light-induced recruitment of a protein to the plasma membrane. In this example, the recruitment of a lipid phosphatase (CRY2-Enzyme) results in rapid loss of the lipid from the plasma membrane. Graph shows the kinetics of plasma membrane binding of the enzyme (green) and the corresponding loss of the lipid (red). B. Light-induced recruitment of a red fluorescent protein to the mitochondria. C. Light-induced recruitment of a red fluorescent protein to the early endosomes. All images have been inverted to show fluorescence in black.


For further information about this research group please contact
Olof Idevall-Hagren