VIBS 627: Optical Microscopy & Live Cell Imaging
Prerequisites: Graduate status & permission of instructor
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
|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|