Lab 4 Notes One of the most neglected concepts is proper configuration of the microscope with regard to illumination.

. Optimizing illumination is the key to enhanced microscope performance. Light emitted from various locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm is very important. In plant cells, parts of the cytoplasm may show cytoplasmic streaming, which may result in broad cytoplasmic strands that transiently traverse the vacuole. Individual organelles such as the mitochondria, will exhibit short periods of rapid abrupt movements with or against the cytoplasmic flow. There is a layer of cytoplasmic and organelles close to the PM that does not take part in the cytoplasmic streaming of the cytoplasm. The nucleus maintains its position in cell regardless of cytoplasmic movements around it. Fluorescein diacetate (FDA) is an uncharged and non-fluorescent derivate of the fluorescent molecule-fluorescein. Because FDA is uncharged, it passively crosses the PM but once inside the cell, cell esterases convert FDA to fluorescent, polar molecule. Fluorescein cannot easily diffuse back across the plasma membrane escape from the cell. Dead cells that lack esterase activity or have a damaged PM do not retain fluorescein. Therefore, FDA is commonly used as a method to distinguish live or dead cells. Fluorescein fluoresces a green or yellow colour when irradiated with blue light. (green or yellow depends on pH). The use of genetically coded fluroescent proteins (e.g. GFP) has allowed us to tag and track an enormous range of proteins in space and real time in their natural environment without the application of exogenous probes or fixation of cells and tissues that can produce artifacts. Magnification: an apparent increase in size. Resolution: the resolving power being the ability to distinguish between two very closely positioned objects. l.r.= (0.61lamda)/N.A. The limit of resolution (I.r.) is related to the shape of the cone of light entering the objective lens. N.A.= cone or numerical aperture Lamda=wavelength The larger the N.A., the greater the resolving power. The smaller the wavelengths of light, the greater the resolving power. Brownian motion is vibrating particles in cell, this means cell is dead or dying.

Article GFP was first discovered as a companion protein to aequorin, the chemiluminescent protein from A. Victoria. GFP by itself was found to fluoresce under excitation, requiring no substrate or coenzymes. GFP contained all of the information needed for proper synthesis of the fluorophore. S65T= was found to accelerate the speed of fluorophore formation. Some mutations helped the molecule to fold correctly at 37 degrees. The protein expression was improved by converting wild-type GFP condons to form enhanced GFP (EGFP). This produced brighter molecules. EGFP can be seen in low light intensities and little photo-bleaching. Developments of GFP provided variants with differing absorbance and emission spectra, allowing the simultaneous visualization of distinct GFP variants in a cell. BFP= blue. Used for multicolor imaging and FRET ( fluorescence energy transfer) CFP =cyan. B/w BFP and EGFP YEP= red-shifted yellow. Brighter than CFP. CFP YEP has been used to study protein-protein FRET 4D microscopy= time-lapse observations of fluorescent molecules are collected as 3D data rather than 1 image in a single focal plane. FRAP= fluorescence recovering after photobleaching. An area of the cell is photobleached with a high intensity laser and the movement of the unbleached molecules moving into the bleached area is revered. This is done without disrupting protein pathways. FRAP can provide an estimation of the effective diffusion coefficient (Deff) and mobile fraction (Mf) of a protein. Deff= reflects the mean squared displacement that an idealized protein moves by random walk over time and is inversely proportional to the size of the protein. For example, when the diffusion rate is significantly lower that what is predicted based on the proteins size, the protein could be incorporated into a large complex. Mf= mobile fraction. If the mobility of a protein is faster than predicted, the protein might be showing directed transport by motor proteins. A decrease in Mf could mean that the protein could be binding to a fixed molecule. An increase in Mf could indicate that the protein could be released from a fixed scaffold. A FLIP (Fluorescence loss in photobleaching)= Performed by repeatly photobleaching fluorescence in one area of the cell while collecting images of the entire cell. By monitoring the fluorescence in the nonphotobleached regions, the mobility of a

fluorescently tagged protein can readily be observed and the continuity of cellular environment determined. FCS (fluorescence correlation spectroscopy)=fluorescently labeled molecules diffusing in and out of a small defined focal volume are measured over short periods of time. The fluctuations in molecules moving through the focal volume can be analyzed to obtain a proteins diffusion coefficient, binding constants, and concentrations. GFP chimeras are continuously being synthesized and at any particular time, we are observing proteins at different life stages. One way to characterize a proteins expression lifetime is to use destabilized variants of GFP. This way, younger protein chimeras can be distinguished from older ones that have lost their fluorescence due to GFP degradation. We can also use photoactivable fluorescent proteins. These proteins display little initial fluorescence under excitation at the imaging wavelength but increase their fluorescence after activation by irradiation at a different wavelength. Because only photoactivated molecules exhibit noticeable fluorescence, their lifetime and behaviour can be studied independently of other newly synthesized proteins. PA-GFP, Kaede and KFPI show to display 30 times increase in fluorescence after photoactivation. Although Kaede displays the largest contrast b/w pre and post-photoactivation, both it and KFPI self-associated to form tetramer. This leaves PA-GFP to be a reliable protein. However, photoactivation of KFPI required light(532nm) is likely to be less harmful to cells than the near-ultraviolent light required for the other two(400nm).