Wednesday, April 04, 2012

Fluorescence microscopy is awesome

One of the things I've been doing recently (yes, lots of life happens even when I'm not blogging about it) is optimizing an older Zeiss microscope for fluorescence imaging at the lab where I work. A picture of the microscope appears below:





It's an Axiovert S 100 inverted scope with trinocular head. I didn't even know what an inverted microscope was before I started working with this one, but it's pretty obvious when you see it in person: it's called "inverted" because the objectives point up from the base of the scope. This is very useful when looking at, say, liquid contents of Petri dishes.

That aside, the main reason we got this scope (I mean other than it being a very reasonably-priced Zeiss inverted model) was to do fluorescence imaging. And as the resident wrench wench here, the task fell to me to get things functioning in that department.

The main challenge I faced was the fact that the 75 watt xenon arc lamp (vital for fluorescence) the scope had come with did not include a power supply. Initially I tried to make use of the first cheap Zeiss supply I found on ebay, but that one turned out to be, shall we say, a bit of a dud. On the plus side, I got some nice refresher experience tracing out circuitry:



...but on the minus side, even after determining the connector pinout (with the much-appreciated advice of various members of the Yahoo microscopes mailing list, in particular one Mr. Forster), the darn thing still wouldn't light the lamp.

I'd like to at some point go into greater technical detail about the ins and outs of arc-lamp power supplies, as it really is fascinating, but for the moment hopefully the reader can forgive my making a long story short(er). That is, after a fair bit more research and experimentation, what ended up getting the lamp lit was a power supply we already had in the lab!

It had come with another (cheap) scope that nobody really liked, and was not the ideal thing for the 75 W xenon lamp (being geared toward 100 W mercury bulbs!), but it did at least work. Mind you, I ended up having to put a giant-ass power resistor in series with it in order to get the current to the required 5.4 amps demanded by the bulb we were actually using, and modify the connector to observe proper polarity, and the whole setup is still a bit persnickety, but it's been good enough until now to at least turn the xenon lamp on and start getting some decent imaging data.

[Needless to say, I am VERY pleased to have finally found the "proper" power supply for this lamp (an EBX 75 unit) and with any luck will get to try it out tomorrow (it just arrived this evening!).]

Ultimately we're going to be using this scope to look at cells and parts of animal cells, with the fluorescence being used to identify the presence/absence of particular Items of Import (proteins, etc.) within said cells. But for the purposes of testing out our imaging capabilities, I've ended up collecting a whole bunch of random interesting samples of various things that exhibit autofluorescence (which tends to persist longer than the illumination of fluorophores, making it easier to test photography techniques on).

For instance, below is a cross-section of some sort of flowering plant (found growing near the lab parking lot):


You can clearly see the cell wall structure along the outside, and there also seem to be some little particles inside the plant that fluoresce more than the surrounding cells. I am not sure what they are but I am thinking possibly chlorophyll (though any visiting plant biologists can feel free to speak up if they have a better idea!).

As for how the picture was taken, basically I built an adapter thing out of a camera lens cover and mounted my DSLR (a Canon Rebel T3i) onto the phototube (the third protrusion on the binocular head). Again, I'd like to get into more details about this at some point but for now I am just going to post this because I am terribly rusty at blogging and have found that if I insist on making every post into a novel-length textbook thing I never post anything at all.

3 comments:

Comrade Physioprof said...

FUCKE, YEAH!!!!! We have a really nice Zeiss compound fluorescence microscope in my lab. It has a motorized Z stage, and allows for the collection of z-stack data that is then deconvolved with software. It is like a "poor person's" confocal microscope.

Justthisguy said...

Oooh, you got a JOB! My most earnest and cheerful congratulations to you, Ma'am!

In other, and sadder, news, my kitteh left the planet last month. He was, we think, about 17 years old. The Donovan put up a very nice eulogy for him on his blog. Please see:
http://www.thedonovan.com/archives/2012/05/an_empty_bowl_o_1.html

Anne Corwin said...

JTG: Oh yes indeed. I've been employed full time again since October 2011, which has been good for my brain and bank account but not so good for the blogging. I have been updating my cat blog recently, though, as it seems that no matter how saturated my brain is, I can almost always find something to say about cats.

...and on that note, I am so sorry about your cat's passing. He sounded like a very fine self-respecting feline.