Two Photons Are Better Than One

I try to stay away from describing the technical aspects of lab work because a lot of what we do is not very interesting and the rest would be very hard to comprehend without two or three years of college bio classes, in large part due to the terminology (one of my bio professors likes to remind us that studying science is like learning another language).  However, yesterday over lunch some of the research assistants from the Moreland  Lab across the hall told me about one of the microscopy techniques they use, and it sounded cool enough that I am going to try to tell you guys about it!  I will try my best to explain things in a way that everyone can get an idea of what I'm saying.  I also should warn you, my knowledge base is very limited, so I don't completely know what I am talking about. ;-)

The majority of techniques that my lab (the McElroy Lab) uses are fairly standard in the research world - many of which I was exposed to in my biology labs at Juniata - like Western blotting, immunohistochemistry, PCR, and RT-PCR (if you don't know what those are, don't worry about it, it probably wouldn't be interesting to you anyway).  The Moreland Lab, which is researching a type of white blood cell called a neutrophil, operates on a much larger scale; Dr. Moreland has gotten two grants of $1 million from the NIH, that should give you an idea of the scale of the lab.  They use the procedures that we do and many more. 

One of the techniques the Moreland lab is using is called "two-photon" microscopy.  This is a special technique that allows scientists to look at living specimens, at a depth of up to 1mm (in the microscopy world this is very thick!).  Basically, two-photon microscopy is an extension of standard fluorescent microscopy - light energy of a specific wavelength is absorbed by molecules which then release the energy in the form of light of a different wavelength ("fluorescence").  The difference is that two-photon microscopy uses light of half the energy (to reduce damage to the specimen, so it can be living) and consequently two photons are needed (rather than one) to excite each fluorescent molecule.  This is a picture of what I just tried to say, for those of you who are familiar with Jablonski Diagrams:

From: Nikon MicroscopyU

You need a special laser to do this because you need a very strong light beam, so not many labs have this technology.  The Moreland lab is using two-photon microscopy to look at neutrophils moving through the bloodstream of a live mouse!  How cool is that?!  To do this, they isolate neutrophils from a blood sample and tag them with fluorescent proteins.  The mouse is put under anesthesia and its foot is glued to the microscope stage.  Then they inject the mouse with a substance to make its veins glow (under the microscope) so that they can easily find the bloodstream.  They inject the tagged neutrophils into the mouse's abdomen, and lead them to the foot by injecting the foot with a "chemoattractant" which causes neutrophils to come to the foot.  Finally, they attempt to excite the fluorescent molecules and observe the neutrophils!  I can't get over this technology; the fact that you can watch a live cell in a live organism using fluorescence is just so amazing!

Although we can't do two-photon microscopy in the McElroy lab, our new microscope can take some pretty cool pictures!  We can do standard confocal microscopy and fluorescent microscopy, here is an example of each:

Small Intestine, 10x

Neutrophils in NEC Intestine, 20x