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VIBS 627, Optical Microscopy and Live Cell Imaging (Cr =3)

Prerequisites: Graduate status & permission of instructor

Meeting times: Lecture Monday 9-10:40 am; Labs Monday 11:00 am - 2:00 pm or 2:00 pm – 5:00 pm

Robert C. Burghardt, Ph.D. Professor and Director

Rola Barhoumi Mouneimne, Ph.D. Research Professor, Associate 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