This past June the MRSEC Center offered a condensed summer course based on the popular graduate course QB120: Quantitative Biology Instrumentation Laboratory.
The course was taught by Zvonimir Dogic of the Physics Department (pictured). Prof. Dogic has extensive experience with several forms of microscopy and his Lab features several home-built or heavily modified optical setups.
The course is designed to offer students hands on experience with building their own optical setups from basic components as well as learning how to optimally acquire imaging data from commercial microscopes. The focus was on understanding the physics behind microscope function and leveraging that knowledge towards improving data acquisition in the lab.
Initially, students used basic lenses, apertures, an objective, a camera and a light source to build the simplest possible light microscope. This initial setup was quickly extended to include Köhler illumination, a core principle in microscopy which allows even illumination of the sample as well as access to the conjugate image plane for image filtering.
The next project required students to build a fluorescence microscope, a highly relevant and ubiquitous technique in biological imaging. To image a slide with fluorescently labeled beads students used a dichroic mirror to separate excitation light at one wavelength from emission light at another wavelength. A schematic diagram, a photo of this setup with the light path superimposed and actual data acquired with one of these microscopes can be seen in the video below.
Next, a more advanced technique in microscopy, total internal reflection microscopy (TIRF), was introduced and an imaging setup using this technique was built. TIRF microscopes excel at imaging small molecules that are immobilized in a small area. A laser beam was pointed to shine through a prism at an angle sufficient to cause total internal reflection and the resulting evanescent wave caused fluorescent excitation of the sample. The video below shows a schematic and imaging data of a TIRF microscope built by students.
Finally, students used commercial microscopes to understand the principles behind phase contrast and difference interference contrast microscopy, both techniques well suited for imaging samples that are nearly transparent.
Overall the Course provided an excellent introduction to the physical principles behind microscope function. I highly recommend it to anyone interested in using microscopes in their research!
