Microscopy Seminar

Last Wednesday, the biology department had another lunch time seminar open to anyone in the department.  This time, it was run by my PI, Dr. Wawersik, who spoke to us about transmitted and epifluoresence microscopy.  Initially I wasn’t sure how much I was going to learn.  I have used transmitted light microscopes numerous times and we use epifluoresence in the lab almost everyday, so I know the basics of both types of microscopy.  However, I have never had a formal introduction to how microscopy actually works, so I learned a ton over the hour session.  One of the things that surprised me the most about compound microscopes is just how complicated each objective lens is (which explains why the lenses are so expensive).  Each lens works to correct various aberrations, such as chromatic aberrations that arise because different colors of light have different frequencies and therefore are refracted at different angles through the lens.  We also talked about resolution and which lenses provide the best resolution for different purposes.  I can now actually understand each part of a label on an objective lens!

In the second part of the session we discussed epifluoresence microscopy.  This technique involves labeling your sample with a fluorochrome or another fluorescent molecule and using a microscope to generate an image.  The fluorochromes are exited by one specific wavelength of light, and then emit a different wavelength.  For example, DAPI, which labels DNA, is exited by purple light at 345 nm and emits blue light at 455 nm.  What I didn’t know, but learned during the seminar, was how the microscope could shine a specific wavelength of light on the sample, but not have that wavelength shine through to the eyepiece, where it would distort the image you were trying to create.  It turns out that this is done by a filter cube, which consists of an excitation filter, a dichromatic beam-splitter, and a barrier.  This allows the emitted light to be one wavelength, but the reflected light to be at another.

The seminar taught me more of the details behind the microscopy that I have been doing all summer.  Now I not only understand what I am doing when I sit down at a confocal microscope, but also how it works.  For my project this summer, I am immunostaining fly larvae with primary antibodies that are specific to my proteins of interest and then secondary antibodies that are tagged with fluorochromes and specific the the primary antibodies.  Then I use fluorescence microscopy to create an image of larval testes.

In a few weeks, there will be a follow up session to this seminar, where hopefully I’ll learn even more!