28 March, 2009

Pretty Pictures That Toaster Takes (SFW?)

Immunofluoresence microscopy is one of my absolute favorite laboratory techniques. The method I use takes about 4 days and time for the slides to "cure", and during that entire time there is absolutely nothing I can do to tell whether or not the technique is even working at all. So I always seat myself in front of the microscope with bated breath, peering in hoping desperately that I haven't just wasted so much time and effort for no results. But, unlike an ELISA, which also takes lots of little invisible steps, the results of immunofluorescence microscopy are often absolutely stunning. I sit in that darkened room alone with these brilliant snapshots of life itself glowing below me as I feebly try to capture their gorgeousness with a camera. Compared to everything I've done so far in science, this is molecular biology at it's most palpable, it's most real. So much of my job consists of moving tiny amounts of one solution to another suspension and spinning it down and waiting and repeating and counting...all invisible stuff. I feel so often like I'm trying to find truth at the bottom of a dark chest with my eyes blindfolded, ears muffled, nose plugged, and hands encased in bubble wrap. When I do find something there it's like touching a loose vacuum tube* and that victory sings along my nerves electric, reawakening out of poor data's despair and repetitive boredom that fundamental hunger for discovery, a thrill of small but bottomless adventures. I study molecular biology not just because I'm an easily captivated nerd in search of shiny things, but because molecular biology is life itself vivisected out in all of its wonder and glory. It's not just difficult and complex, it's the biggest puzzle humankind has yet found and by doing science I am hewing the pieces of that puzzle out of raw ignorance and curiosity into elegance and grace.

Immunofluorescence microscopy, for me at least, bypasses all the trudgy serial dilutions and quantitative cell culture and patches me directly into that holy place of rare wonder and quintessential awe.

Below are a few of the hundreds of pictures I have taken over the past several months. These are all scientifically useless: control slides and slides where the concentrations weren't quite right or just not good enough for inclusion in practicable data. I've loaded each of these as large .jpegs, so if you'd like to download them I can assure you they make for excellent desktop backgrounds.

This is a frozen section of mouse stomach at 100X. The mouse was a germ-free C57/B6 infected with H. pylori and a unique microbiota that we're studying. The picture is at 100X. This is from a test-run I did on stomach tissue to make sure the same immunofluorescence staining protocol I use for mouse ceca works in this tissue type. We can't use this picture in any kind of data or results because it is, by and large, a mistake (pretty nonetheless, though). The red is an APC-labeled anti-mouse CD11c antibody and the blue is just the DAPI (therefore showing DNA) that came mixed in with the Promega Gold Anti-Fade Reagent.

So why is CD11c showing up so well on stomach parietal and smooth muscle cells when it's primarily a marker for dendritic cells?

There're a couple of reasons. First, according to other labs in our working group, CD11c antibodies have to be at a really high concentration to detect dendritic cells well (e.g., 1:10 instead of the 1:400 shown above). Secondly, Fc receptors bind antibodies. Fc receptors are most commonly associated with innate immune effector cells such as dendritic cells, macrophages, natural killer cells, and neutrophils; however, Fc-gamma receptors are widely expressed by several different cell types in diverse tissues. I didn't use any Fc blockers in this particular slide, so the Fc-gamma receptors likely present on the parietal and smooth muscle cells probably bound up my mouse IgG antibody (reason #1 there's high background). Thirdly, I used a mouse antibody in mouse tissue (reason #2 there's high background, also a stupid move on my part). Fourthly, I pulled out my old IF blocking buffer and used it without remembering that it contains normal goat serum instead of normal mouse serum (reason #3 there's high background). I might as well have not bothered blocking at all. Fifthly, these antibodies have only been tested, in the literature and our lab, for flow cytometry and not immunofluorescence microscopy. This last point is the win for the picture as it demonstrates we can use flow cytometry antibodies for this project, which is really really really convenient as it standardizes our data. That being said, however, I recently found out that rabbit anti-mouse monoclonals are coming onto the market for several CDs and if I hear that they do a better job than mouse anti-mouse CD antibodies I will not hesitate to use them instead (although if i do I'll probably wind up doing a direct comparison of the two by flow cytometry).

This is a section of mouse cecal wall. In this one the red is an AlexaFluor 597 phalloidin that binds to actin. Blue is DAPI again. This is a control slide where I was trying to find the optimal concentration of the phalloidin to label actin in cecal epithelial cells. Here the concentration was too low and only clearly labeled the actin in the smooth muscle that lies under the cecal epithelium. The DAPI to the left of the smooth muscle shows where the epithelial cells should be.

Same thing, different field.

Here I got the phalloidin concentration just about right to stain the epithelium. Also, a different person cut these blocks into slides and got much nicer sections without destroying the tissue architecture (it helps when the person operating the cryostat doesn't melt down the OCT and rearrange the tissues to their liking). When we took this tissue from a mouse infected with E. coli O157:H7, we cut along the inferior curvature of the cecum, spread it flat open, washed with 1X PBS three times to wash away food gunk (because that shit autofluoresces) and unadhered bacteria, and cut a ~2mm wide strip of cecum down the length with a scalpel. Then we took a Q-tip swab and rolled the tissue around the end like a cinnamon bun (my boss calls them "Swiss rolls") before embedding it in OCT (like clear syrup that keeps cells from bursting when they freeze) and slow freezing in a -20C freezer (as opposed to snap freezing in liquid nitrogen). The green in the picture above is from a poly-clonal goat anti-E. coli O157:H7 lipolysaccharide. The little green dots along the thicker actin lines (tight epithelial junctions) are enterohemorrhagic E. coli O157:H7 that have stuck themselves to the cecal epithelium (the mechanics of which are discussed here).

*I've done that, once. I was testing out an old Fender bass amp. It was a beautiful amp, 4 10" speakers with a tweeter, sounded great on a 4-string bass, tight and punchy where you can feel the rumble right in your diaphragm. But I play a 5-string bass and it lacked good low-end definition; also, I prefer to run my rig at settings that place the bass rumble firmly in one's duodenum. The vacuum tubes of the amplifier were placed right next to the on-off switch on the back of the amp head where I could see it, so when I went to turn it off I groped too far to the right and wound up feeling up the vacuum tubes' loose connections. I was knocked flat on my ass.


Science Bear said...

I agree, I like pictures like these!

Hermitage said...

*sniffle* spoken like a true mad scientist. Very hot pics, you should start an immunofluorescence microscopy pr0n website.