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.
- *Laboratory schedule with learning outcomes and assignments will be distributed to registered students.
- Instructor: Robert C. Burghardt, Ph.D. | Professor & Image Analysis Laboratory (IAL) Director
| Week | Lecture Topics | Lab Topics |
|---|---|---|
| 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 | Introduction to ZEN software. Acquire and process czi files; Stage micrometer; C-mounts, optivar settings |
| 3 | Applications of wave optics, diffraction, and interference; the diffraction apparatus and image formation in a microscope; brightfield microscopy | Abbe diffraction apparatus; brightfield microscopy/achieving theoretical limit of resolution |
| 4 | Contrast in optical microscopy; compare brightfield, darkfield, phase contrast, and differential interference contrast microscopy; compare contrast generation in optical vs. transmission electron microscopy (TEM) | Darkfield and phase contrast imaging; TEM brightfield and diffraction imaging |
| 5 | Basics of digital images; digital imaging ethics | DIC imaging on Axioplan 2 and inverted live cell workstations |
| 6 | Introduction to widefield fluorescence microscopy | Introduction to Axio Imager.M2 Motorized Upright Microscope. BF, DF, Phase, DIC, z-stacks, tiling |
| 7 | Written Examination Submit project proposal | Laboratory Practical Examination |
| 8 | Fluorescence probes of cellular function; steady state and kinetic analysis for live cell imaging | Training on ZEN software on AxioImager.M2 automated widefield microscope |
| 9 | Confocal microscopy | Intro to ZEN Software for Zeiss 780 and Zeiss CellDiscoverer 7 instruments |
| 10 | Confocal and multiphoton microscopy | Image acquisition of fixed samples on Zeiss 780 |
| 11 | Confocal microscopy & Automated live cell imaging | Image acquisition of live samples on Zeiss 780 and Celldiscoverer 7 |
| 12 | Breaking the diffraction limit: Super-resolution microscopy-Intro to Airyscan superresolution | Introduction to Airyscan imaging on Zeiss 780 |
| 13 | TIRF and Correlation Coefficients | Data acquisition using high-end instruments |
| 14 | Data acquisition | Data acquisition using high-end instruments |
| 15 | Final presentations & written project reports are due | Presentation of research projects |
