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).

LectureTopic in LectureReading pagesLab topic (Fridays)
26Light and Refraction12–17No Lab
29Microscope Optics: Lenses and Conjugate planes18–22 
31Microscope optics: Imaging and Resolution22–24 
09/02/22Numerical Aperture and Resolution24–27Optics
5Holiday: No Class  
7The compound microscope, Microscope Standard Lengths35–39 
9The Condenser: history, optics & Köhler illumination 40–44Brightfield MicroscopyFocus Examples
12Lens aberrations (spherical, chromatic)28–34 
14Objectives, objective designations, Eyepieces45–54 
16Darkfield microscopy, Rheinberg Illumination, Oblique Illumination56–60Darkfield Microscopy
19Polarized light, Anisotropy, Types of birefringence, Polarized light microscope61–73 
21Polarized light microscopy, Generation of colors(ditto) 
23Polarized light microscopy: Quantitative PLM74–84Polarized Light Microscopy
26Phase Contrast microscopy I85-88 
28Phase Contrast microscopy II88–92 
30Inverted Microscope, Stereo Microscope 54–55Phase Contrast
10/03/22Interferometry, Differential Interference Contrast Presentation; DIC microscope93–102 
7Hoffman Modulation Contrast104–106DIC
10Review of transmitted light techniques, Key to determining imaging method
Here's a Jeopardy you can use to study.
12Midterm Exam. Example from 2014  
14TBA Lab Practical Exam
17Fluorescence Microscopy, Stokes Shift, Types of sample fluorescence110–114, 120–121 
19Fluorescence Microscopy II114–119 
21Fluorescence Microscopy III136–140Epifluorescence microscopy
24Laser scanning confocal microscopy I126–131 
26Laser scanning confocal microscopy II, resolution131–135 
28Spinning Disk & Sweep Scanners, Nipkow disk(no reading assignment)Confocal Microscopy
31Digital Imaging, Nyquist sampling, Digital imaging for microscopy.141–145 
11/02/22Two photon microscopy I146–153 
4Two photon microscopy II(ditto)Fluorescence Probing
7Restoration microscopy I, Basis, PSF, Methods,162–171 
9Restoration microscopy II, Here's a short tutorial on Filtering(ditto) 
11Holiday: No Class
NANo Lab
14Super Resolution: Structured Illumination Microscopy (SIM)172–180 
16Super Resolution: STED, PALM/StORM181–194 
18FSPIM (Lightsheet imaging)154–161Project Explained (Project lab open)
21Advanced fluorescence imaging: FRAP, FRET, FLIM121–125 
23Holiday: No Class  
25Holiday: No Class Holiday: No Lab
28Total Internal Reflection microscopy, TIRF microscope124 
30Links to review resources

TL review
Fluor Review
Then there's Finals Jeopardy
12/02/22The History of the Microscope208–218ALS Tour during normal lab hours
5RRR Week: No Lecture  
7RRR Week: No Lecture.  
9RRR Week: No Lecture. Project Due 5PM. Upload to bCourses  
12–15The 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.