Lecture 1--microscopy

Inject into an animal egg to make a transgenic animal

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Unformatted text preview: GFP soluble protein. floats in the cytoplams. just fills the cells but does not show specific s tructutres(proteins and organelles) you have the gene encoding cadheren, and want to see where c adhere is in cell. use molclar bio to add GFPto the end off it remove stop codon from cadheren and 5' UTR from GFP make sure they are in the same reading frame, putkeratin driver. in skin cells this will make transcript that has both proteins. and the two will become attached (cadheran and GFP) Two ways to image the inside of 3-D specimens: GFP can interupt folding of the protein. and the protein can become degraded. take tissue and slice it into s alami 1. Tissue slices 2. Optical sections the bulky GFP protein mught block some of adheren's natural interactions if you put too much GFP, then it might spill over to other areas and highlite places where adheren is acyually not present can do antibody staining on GFP; how to control the disrupting function of c adheren. use a mutant and insert the c adheren+GFP to see if it can rescue the mutant. this will tell us if the protein ifs functioning normally Tissues can be sliced into sections Tissue sections can be imaged with fluorescence: 1. fix specimen 2. embed 3. slice 4. image m S Tissues can be stained either before or after slicing m mNm 25 SS uses lazer as a light source Confocal microscopy allows the visualization of 3-D images with optical sections taking optical slices confocal: using a microscope whose imaging system only c ollects light from a s mall spot on the s pecimen, giving greater resolution Confocal Microscopy - Utilizes lasers as a light source - Specimens can be thicker than in light microscope - Allows 3-D reconstructions - Same level of resolution as the light microscope - Requires fluorophores resolution: the degree of detail v isible in a photographis or television image The key to confocal microscopy is the pinhole, which makes it possible to collect light from a single plane (optical section) key part pinhole filters out the light that it not in focus. Confocal image of 36 hpf zebrafish sensory neurons express GFP, skin cells express RFP Confocal image of 36 hpf zebrafish sensory neurons express GFP, skin cells express RFP Confocal images consist of optical planes Optical planes can be combined into a 3-D image Multiphoton microscopy about 500 microns deep, depends on lazer In mutiphoton excitation microscopy fluorescence is limited to a spot at the focus of the pulsed infrared laser beam, resulting in much less damage. The infrared illumination also twice as deep penetrates more deeply into the specimen than visible light confocal imaging: limitations: li...
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