PMB 185: Techniques in Light Microscopy

F2023/ PMB 185
Lecture (27166)
MWF 9-10AM
201 Gianinni

Lab (27167) F 1-4, 208 GPBB

3 Units

Denise Schichnes Ph.D. OH: Tu 10-11AM
Steve Ruzin, Ph.D. Emeritus OH: F 10-11AM

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). It’s $40 and will be available Aug 22.

LectureTopic in LectureReading pagesLab topic (Fridays)
25Light and Refraction13-18No Lab
28Microscope Optics: Lenses and Conjugate planes19-22 
30Microscope optics: Imaging and Resolution22-29 
09/01/23Numerical Aperture and Resolution44-46Optics
4Holiday: No Class  
6The compound microscope37-41 
8The Condenser: history, optics & Köhler illumination 42-44Brightfield MicroscopyFocus Examples
11Lens aberrations (spherical, chromatic)30-36 
13Objectives, objective designations, Eyepieces47-57 
15Darkfield microscopy, Rheinberg Illumination, Oblique Illumination59-62Köhler Illumination
Darkfield Microscopy
18Polarized light, Anisotropy, Types of birefringence, Polarized light microscope64-79 
20Polarized light microscopy, Generation of colors(ditto) 
22Polarized light microscopy: Quantitative PLM80-90Polarized Light Microscopy
25Phase Contrast microscopy I91-103 
27Phase Contrast microscopy II(ditto) 
29Inverted Microscope, Stereo Microscope 57-58Phase Contrast
10/02/23Interferometry, Differential Interference Contrast Presentation; DIC microscope104-114 
6Hoffman Modulation Contrast116-119DIC
9Review of transmitted light techniques, Key to determining imaging method
Here's a Jeopardy you can use to study.
11Midterm Exam. Example from 2014  
13TBA Lab Practical Exam
16Fluorescence Microscopy, Stokes Shift, Types of sample fluorescence123-129 
18Fluorescence Microscopy II129-135 
20Fluorescence Microscopy III135-138Epifluorescence microscopy
23Laser scanning confocal microscopy I1157-167 
25Laser scanning confocal microscopy II, resolution(ditto) 
27Spinning Disk & Sweep Scanners, Nipkow disk168-1711) Confocal Microscopy
2) Project Explained
30Digital Imaging, Nyquist sampling, Digital imaging for microscopy.142-148 
11/01/23Two photon microscopy I1172-174 
3Two photon microscopy II174-1801) Confocal Microscopy
2) Project Explained
6Restoration microscopy I, Basis, PSF, Methods,181-191 
8Restoration microscopy II, Here's a short tutorial on Filtering(ditto) 
10Holiday: No Class
NANo Lab
13Super Resolution: Structured Illumination Microscopy (SIM)198-205 
15Super Resolution: STED, PALM/StORM208-219 
17FSPIM (Lightsheet imaging)159-156Super-resolution: SIM
20Advanced fluorescence imaging: FRAP, FRET, FLIM135-138 
24Holiday: No Class Holiday: No Lab
27Total Internal Reflection microscopy, TIRF microscope138-140 
29Links to review resources

TL review
Fluor Review
Then there's Finals Jeopardy
12/01/23The History of the Microscope237-255ALS Tour during normal lab hours
6RRR Week: No Lecture  
8RRR Week: No Lecture.  
10RRR Week: No Lecture. Project Due 5PM. Upload to bCourses