Research Facilities in high resolution fluorescence microscopy
Figure 1 the Delta Vision microscope. The hood around the microscope allows a constant incubation temperature for the growth of cells during real time imaging.
The Delta Vision Microscope.
A state-of-the-art Delta Vision microscope allows high resolution 3-dimensional restoration microscopy (figure 1). The Delta Vision uses wide-field epifluorescence and a high precision robotic stage to take Z-sections through the specimen. A sophisticated deconvolution algorithm is then used to restore the out-of-focus blur that inevitably arises in such microscopy. The algorithm is restorative rather than subtractive, which means that all the fluorescence emitted from the specimen is used in to produce the final image. This optimises the use of the excitation light resulting in minimal phototoxicity and photobleaching compared to alternative forms of microscopy such as confocal methods. The overall result of the deconvolution is the resolution that actually exceeds the 180nm limit of light microscopy. The low level of photoxicity is ideal for real-time imaging of living cells and the deconvolved Z-stack can be further processed to produce three-dimensional models of the image.
Examples of our Research : morphogenesis in Candida albicans
We use the Delta Vision for research into the molecular and cell biology of fungi. Dr Sudbery's group studies morphogenesis in the human fungal pathogen Candida albicans. This is a common pathogen of vulnerable patients such as those in intensive care, especially those with compromised immune systems. It has a very high mortality, which can be as high as 70%. This organism can grow as yeast (figure 2A) or in a hyphal form (Figure 2B). The ability to grow in these different morphologies is necessary for virulence. The focus of research is molecular and cellular mechanisms which operate when the yeast form switches to hyphal-form growth. Imaging with the Delta Vision revealed that the extreme polarised growth of the hyphal form is driven by a Spitzenkorper (Figure 2C), a collection of secretory vesicles at the tip which acts as a supply centre for the raw materials needed for the new cell wall and membrane incorporated into the growing tip. Computer modelling of high resolution images reveals that the Spitzenkörper is a three-dimensional structure which lies just behind the plasma membrane at the hyphal tip. (Figure 3).
Figure 2. Candida yeast and hyphae: Panels A,B: DIC images of Candida albicans growing as yeast (Panel A, [left]) and hyphae (Panel B, [centre]). Panel C:[right] A strain was genetically engineered so that a fluorescent protein, YFP, is fused to Mlc1 (myosin light chain). This allows the protein to be visualised in living cells. The image shows the tip of a hypha with the cell walls visualised by the binding of concanvaline A conjugated to Alexafluor 388 which fluoresces blue. Mlc1-YFP localises to the Spitzenkörper just behind the hyphal tip.
Figure 3 The Spitzenkorper. The image on the left shows an enlargement of a hyphal tip where the plasma membrane is stained with Filapin and the Spitzenkorper with Mlc1-GFP. The image on the right shows a three dimensional model of the Spitzenkorper constructed from the image on the left using the information in the Z-stack. (The software used was VolocityTM published by Improvision. We are grateful for the assistance of Alan Tilley of Improvision for the production of this model.)
Examples of our Research : endocytosis in yeast
Dr Ayscough’s group studies endocytosis in yeast. This is the process by which the plasmembrane invaginates to recycle membrane proteins and to internalise the extracellular fluid. The process is driven by the actin cyctoskeleton which is assembled in cortical patches at the sites of endocytosis. The recrutiment of actin to the cortical patches is organised by complex of proteins which disassemble once the actin has arrived. Figure 4 shows an example of the Delta Vision being used to follow protein dynamics in real time in living cells.
Figure 4 Real time imaging of proteins in living cells. The animation is large (2 Mbyte) and is available via the right hand menu or below. This figure shows the start (left) and finish (right) of the animation. The white dots show a protein involved in the internalisation (endocytosis) of material from outside the cell. The two dots on the upper right disappear as the internalisation process is completed and the complex of proteins involved in the process begins to disassemble. The images were taken at 1 second intervals and are replayed in real time in the animation.