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===Hybridization and imaging=== ====First reality-check on the imaging of mammalian nuclei==== *Blue and Alice helped to grow PGP1F fibroblasts in the 35mm imaging dishs. One of the dishes was treated with colcemid overnight to capture metaphase cells. After removing culture medium, I added 1-2ml 1X DAPI (provided by Jeff) in 1X PBS to each dish, then imaged using our own 40x objective as well as the loaner high-NA 60x oil objective from Olympus. *In the bright field with phase contrast, the morphology of the colcemid treated fibroblasts looks very different from typical fibroblasts. It was not possible to collect a nice bright field image. So I only took one DAPI image. Only a very small fraction of cells are in the metaphase. I couldn't see the spindle, but can see the compact metaphase chromosomes with the eye piece. However, the boundaries between chromosomes are quite blurry in the image captured by the CCD. *Unblocked fibroblasts looked much nicer, both in the bright field and DAPI channel. I took a few images using both the 40X and 60X objective. *The largest nucleus is roughly 200 pixels in width, and 150 pixels in height with the 60x objective. Given that the pixel size of our ORCA R2 CCD camera is 6.45uM^2, one pixel corresponds to 6.45/60=107.5nm. So the nucleus is ~20x15 microns in size. 60X Unblocked 40X Unblocked [[Image:PGP1F_60X_composite.png|400px]] [[Image:PGP1F_40X_composite.png|400px]] 60X 1-day coldemid block [[Image:PGP1F_colcemid_1d_color.png|400px]] *Conclusions: **The 60X objective does seem better (obvious). **It's unlikely that we can resolve very dense chromosome barcodes using our microscope. **It doesn't appear that going up to 100x would be of any benefit, since with 60X we are already hitting the diffraction limit. **To get a better resolution we need to try confocal or super-resolution microscopy. **I captured the images using the default setting for exposure time, gain etc. The resulting images only have the dynanmic range of ~100-fold. Need to significantly increase the exposure time and gain next time.
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