PMB 185: Techniques in Light Microscopy
F2022/ PMB 185
Lecture (27166) MWF 9-10AM
Lab (27167) F 1-4, 208 GPBB
3 Units
Denise Schichnes Ph.D.
(with help from Steve Ruzin, Ph.D.)
Biological Imaging Facility
381 Koshland Hall
The course will be a detailed overview of the practice of light microscopy as applied to scientific investigation. The emphasis of the course will be on the use of the light microscope by biological scientists, however students of other disciplines are welcome. We will cover optical microscope theory, microscope components and mechanics, and all modern optical techniques from Brightfield through Deconvolution microscopy, Structured Illumination, and others. Instruction will consist of lectures and Demonstrations.
Lab Friday 1–4PM 208 GPBB
Grading: There will be a Midterm written and practical exams as well as a written Final Exam. For labs, students will be required to complete a worksheet for each of the labs. In addition, a project is required for the class. It consists of a written paper describing your forensic analysis of an unknown substance.
The textbook is available at Instant Copying and Laser Printing (2138 University Ave).
| Lecture | Topic in Lecture | Reading pages | Lab topic (Fridays) |
|---|---|---|---|
| 08/24/22 | Introduction | ||
| 26 | Light and Refraction | 12–17 | No Lab |
| 29 | Microscope Optics: Lenses and Conjugate planes | 18–22 | |
| 31 | Microscope optics: Imaging and Resolution | 22–24 | |
| 09/02/22 | Numerical Aperture and Resolution | 24–27 | Optics |
| 5 | Holiday: No Class | ||
| 7 | The compound microscope, Microscope Standard Lengths | 35–39 | |
| 9 | The Condenser: history, optics & Köhler illumination | 40–44 | Brightfield MicroscopyFocus Examples |
| 12 | Lens aberrations (spherical, chromatic) | 28–34 | |
| 14 | Objectives, objective designations, Eyepieces | 45–54 | |
| 16 | Darkfield microscopy, Rheinberg Illumination, Oblique Illumination | 56–60 | Darkfield Microscopy |
| 19 | Polarized light, Anisotropy, Types of birefringence, Polarized light microscope | 61–73 | |
| 21 | Polarized light microscopy, Generation of colors | (ditto) | |
| 23 | Polarized light microscopy: Quantitative PLM | 74–84 | Polarized Light Microscopy |
| 26 | Phase Contrast microscopy I | 85-88 | |
| 28 | Phase Contrast microscopy II | 88–92 | |
| 30 | Inverted Microscope, Stereo Microscope | 54–55 | Phase Contrast |
| 10/03/22 | Interferometry, Differential Interference Contrast Presentation; DIC microscope | 93–102 | |
| 5 | PlasDIC | 103–104 | |
| 7 | Hoffman Modulation Contrast | 104–106 | DIC |
| 10 | Review of transmitted light techniques, Key to determining imaging method Here's a Jeopardy you can use to study. | 107–109 | |
| 12 | Midterm Exam. Example from 2014 | ||
| 14 | TBA | Lab Practical Exam | |
| 17 | Fluorescence Microscopy, Stokes Shift, Types of sample fluorescence | 110–114, 120–121 | |
| 19 | Fluorescence Microscopy II | 114–119 | |
| 21 | Fluorescence Microscopy III | 136–140 | Epifluorescence microscopy |
| 24 | Laser scanning confocal microscopy I | 126–131 | |
| 26 | Laser scanning confocal microscopy II, resolution | 131–135 | |
| 28 | Spinning Disk & Sweep Scanners, Nipkow disk | (no reading assignment) | Confocal Microscopy |
| 31 | Digital Imaging, Nyquist sampling, Digital imaging for microscopy. | 141–145 | |
| 11/02/22 | Two photon microscopy I | 146–153 | |
| 4 | Two photon microscopy II | (ditto) | Fluorescence Probing |
| 7 | Restoration microscopy I, Basis, PSF, Methods, | 162–171 | |
| 9 | Restoration microscopy II, Here's a short tutorial on Filtering | (ditto) | |
| 11 | Holiday: No Class | NA | No Lab |
| 14 | Super Resolution: Structured Illumination Microscopy (SIM) | 172–180 | |
| 16 | Super Resolution: STED, PALM/StORM | 181–194 | |
| 18 | FSPIM (Lightsheet imaging) | 154–161 | Project Explained (Project lab open) |
| 21 | Advanced fluorescence imaging: FRAP, FRET, FLIM | 121–125 | |
| 23 | Holiday: No Class | ||
| 25 | Holiday: No Class | Holiday: No Lab | |
| 28 | Total Internal Reflection microscopy, TIRF microscope | 124 | |
| 30 | Links to review resources • TL review • Fluor Review Then there's Finals Jeopardy | 195–207 | |
| 12/02/22 | The History of the Microscope | 208–218 | ALS Tour during normal lab hours |
| 5 | RRR Week: No Lecture | ||
| 7 | RRR Week: No Lecture. | ||
| 9 | RRR Week: No Lecture. Project Due 5PM. Upload to bCourses | ||
| 12–15 | The final exam will be a Take-Home exam. At midnight Sunday 12/12 you will get a link to the exam. It will be due 12/15 by 11:59PM. |