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Techniques in Light Microscopy

F2021/ PMB 185
Lecture (27166)
MWF 9-10AM 106 Wheeler

Lab (27167) F 1-4, 208 GPBB


3 Units

Steven Ruzin Ph.D.

Denise Schichnes 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 for $26 at Instant Copying and Laser Printing (2138 University Ave).

Ruzin Office hour:
Monday 2–3PM,
381 Koshland Hall

or anytime you want, just ask.

Date

Lecture/Lab topic, Fall 2021

Lecture

Topic in Lecture

Lab topic (Fridays)
Aug 25

Introduction

27
Light and Refraction
No Lab
30
Microscope Optics: Lenses annd Conjugate planes
Sept 1
Optics: Imaging, diffraction, and its consequences for resolution
3

Numerical Aperture and Resolution revisited

1: Microscope optics
6
No Class
8
Lens aberrations (spherical, chromatic)
10

Microscope configurations: The compound microscope , Microscope Standard Lengths

2: Köhler illumination
Focus Examples
13

The Condenser: history, optics & Köhler illumination

15

Objectives, objective designations, Eyepieces

17

Darkfield microscopy, Rheinberg Illumination, Oblique Illumination

3: Darkfield
20
Polarized light, Anisotropy, Types of birefringence, Polarized light microscope
22

Polarized light microscopy, Generation of colors,

24

Polarized light microscopy: Quantitative PLM

4: Polarized Light Microscopy
27
Phase Contrast microscopy, presentation, examples, Zernike's Nobel Speech
29

Phase Contrast microscopy

Oct 1
Inverted Microscope, Stereo Microscope,
5: Phase Contrast
4

Interferometry, Differential Interference Contrast Presentation; DIC microscope

6

PlasDIC

8

Hoffman Modulation Contrast

6: DIC
11
Review of transmitted light techniques, Key to determining imaging method
13
Microscope Jeopardy
15

Midterm Exam (Example)

7: Lab Practical Exam
18

Fluorescence Microscopy, Stokes Shift, Types of sample fluorescence

20

Fluorescence Microscope, presentation

22

Digital Imaging, Nyquist sampling, Digital imaging for microscopy

8: Epifluorescence microscopy
25
Laser scanning confocal microscopy I
27

Laser scanning confocal microscopy II

29

Laser scanning confocal microscopy III

9: Fluorescence Probing
Nov 1
Two photon microscopy and resolution, presentation
3

Two photon microscopy II

5

Spinning Disk & Sweep Scanners, Nipkow disks

10: Confocal microscopy (Zoom)
8
Restoration microscopy I, Basis, PSF, Methods,
10

Restoration microscopy II, Here's a short tutorial on Filtering

12
SPIM (Lightsheet imaging)
11: Project Explained
15

Super Resolution: Structured Illumination Microscopy (SIM)

17
Super Resolution: STED
19
Super Resolution: PALM/StORM
12: SIM (Zoom)
22
Advanced fluorescence imaging: FRAP, FRET, FLIM
24

Holiday: No Class

26

Holiday: No Class

Holiday: No Lab
29

Total Internal Reflection microscopy, TIRF microscope, Implications;

Dec 1
Finals Review Resources; Finals Jeopardy; BF Review, Fluor review
3

The History of the Microscope

13: Tour of the Golub Collection
6

RRR Week: No Lecture

8

RRR Week: No Lecture.

10

RRR Week: No Lecture. Project Due 5PM. Upload to bCourses

13–16

The final exam will be take-home

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Updated Mon, Dec 6, 2021