VIBS 627

VIBS 627: Optical Microscopy & Live Cell Imaging

Prerequisites: Graduate status & permission of instructor

Meeting times:
Lecture: Monday 9:00–10:40 a.m.
Labs: Monday 11:00 a.m.–2:00 p.m. or 2:00 p.m.–5:00 p.m.

Instructor: Robert C. Burghardt, Ph.D. | Professor & IAL Director

*Laboratory Schedule with Learning Outcomes and Assignments Distributed to Registered Students

Week Lecture Topics Lab
1 Introduction to optical microscopy: visual perception, resolution of eye and magnification; the magnifying lens; description of optical phenomena; geometrical optics; Koehler illumination Instruments and safety; Axioplan 2 microscope, Axiocam HR and Axiovision software; Koehler illumination on multiple instruments.
2 Applications of geometrical optics; basics of objectives, condensers and light sources Acquire/compare images in different file formats; stage micrometer mag calculation; C-mounts, optivar settings
3 Applications of wave optics, diffraction and interference; the diffraction apparatus and image formation in a microscope; brightfield microscopy. Use of Abbe diffraction apparatus; brightfield microscopy/achieving theoretical limit of resolution.
4 Darkfield and phase contrast microscopy

Comparisons between brightfield imaging, diffraction and contrast generation in optical vs. transmission electron microscopy (TEM).

Darkfield and phase contrast imaging.

TEM brightfield and diffraction imaging

5 Basics of digital images Differential interference contrast microscopy DIC imaging on Axioplan 2 and inverted live cell workstations
6 Introduction to widefield fluorescence microscopy Fluorescence on fixed sections and cell cultures; combination contrast methods
7 Written Examination Laboratory Practical Examination
8 Fluorescence probes of cellular function; steady state and kinetic analysis for live cell imaging Live cell imaging workstation, 3D and 4D imaging; fluorescence deconvolution
9 Confocal and multiphoton microscopy Confocal microscopy, determine lateral and axial resolution, optimal pinhole settings
10 Confocal and multiphoton microscopy, continued Confocal, continued
11 Fluorescence co-localization Pearson’s and Mender’s correlation coefficients; Mander’s overlap coefficient Comparison of widefield, Deconvolution and Confocal
12 Total Internal Reflection Fluorescence (TIRF) Microscopy Capture TIRF image on Zeiss TIRF3 instrument;
13 Breaking the diffraction limit: Super-resolution microscopy Zeiss Elyra S.1 instrument use
14 Written Examination LCM demonstration
15 Presentation of research projects Presentation of research projects