Some more lung cancer cells, nyquist sampling

[kinase]

Someone from Nikon stopped by to help us install an incubation unit on our confocal scope. Someone from a different lab brought a GFP tagged tubulin cell line with him to come give it a test and we had a look. I watched tubulin moving around in a cell in real time at a very fast frame rate in 3D. Needless to say it was one of the coolest things I've ever seen, unfortunately I don't have any of that video. What I did do though is learn how to make the microscope stitch together images that it had taken of my field of view, using nyquist sampling. The end result is the system is operating at its maximum resolving power because the detector and laser settings are set such that they are able to work perfectly with the maximum resolving power of the lens. It's basically setting the unit of detection to be much smaller than the expected smallest detail (which is NA dependent) to be. I also figured out how to play with the pinhole size, which can increase resolution until it's so small that diffraction around the pinhole degrades it. The system will also calculate optimum pinhole size based on lens and wavelength I can't see much of a difference in this case, except for the larger image but I'm happy that I know how to make the system operate at peak efficiency. This is truly a wonderful instrument.

Blue is DNA, red is markers of DNA damage, green is a protein that's supposed to be mostly nuclear. I'm not sure why so much of it was out in the cytoplasm in this set, I'd like to see if the ER is there but I don't have an ER antibody. The other odd thing about this sample was that the innate levels of DNA damage are very high, seen in the second photo, all the red. Maybe they were mad at me or something. I'm redoing this with a more freshly unfrozen batch of cells because of the cytoplasmic green and too much DNA damage.


I believe the couple of cells that don't have red was because the red was out of the focal plane which is very small on a confocal.
I also realized that I've never used regular transmitted light/DIC on this scope and if someone asked what lung cancer cells look like, I'd only have a picture from my phone through the eyepiece normal microscope to show them, or these glowing things. I'll have to grab a DIC image next time.

[75RR]

Striking images!

[Crater Eddie]

Very interesting indeed! Please continue to post images such as these.
CE

[zzffnn]

Very intertesting.

What is that "green" nuclear protein? Did you play around tag concentration and binding specificity?

Can you swell the nuclear membrane without killing cells quickly, with some treatment or under pathological condition? I have not tread about DNA damage for 8 years, so I am just guessing and being curious.

And maybe change to a specific cytoplasmic marker, if you can manage them together?

[kinase]

I just did an antibody dilution test yesterday, the slide's curing right now. It was a chambered slide with 8 wells where on the top row I held the secondary antibody concentration constant and changed the primary, and the bottom row was the reverse of that. I'll have a chance to image it on a confocal on monday. I kind of refuse to take photos on the other fluorescence microscope because the confocal is just so much better.

Green is a certain transcription factor, made in the ER and sent to the nucleus via a nuclear localization signal. I'm unsure if the staining specificity was bad or if it was actually in the cytoplasm, I suspect that might be the ER because it's not all throughout the cytoplasm but only near the nucleus like that, although if it is I don't know why so much of it is there. I don't have a cytoplasm stain or an ER stain. If I did though, theoretically the microscope can handle 32 different dyes, even if they're very similar in color, so if you were inclined to stain 32 different structures, you could. Both primary antibodies are extremely specific for their target, as verified by western blots. And secondary only controls show that the secondaries aren't binding random stuff.

To fix, I used 37C 4% formaldehyde in PBS. I was reading a paper about the nucleus swelling and shrinking, someone fixed a cell while it was on the microscope and watched it. I was reading some formaldehyde chemistry and I think the warm formaldehyde helps because the warmer it is, the more percentage of the solution is actually formaldehyde instead of the non-crosslinking hydrate. Also the increased temperature makes diffusion and chemistry occur faster, and I figured the longer they're at body temp the better.