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).

Date

Lecture/Lab topic, Fall 2022

 
Lecture

Topic in Lecture

Lab topic (Fridays)
Aug 24

Introduction

 
26
Light and Refraction
No Lab
29
Microscope Optics: Lenses annd Conjugate planes 
31
Optics: Imaging, diffraction, and its consequences for resolution 
Sept 2

Numerical Aperture and Resolution revisited

1: Microscope optics
5
No Class 
7
Lens aberrations (spherical, chromatic) 
9

Microscope configurations: The compound microscope , Microscope Standard Lengths

2: Köhler illumination
12

The Condenser: history, optics & Köhler illumination

 
14

Objectives, objective designations, Eyepieces

 
16

Darkfield microscopy, Rheinberg Illumination, Oblique Illumination

3: Darkfield
19
Polarized light, Anisotropy, Types of birefringence, Polarized light microscope 
21

Polarized light microscopy, Generation of colors,

 
23

Polarized light microscopy: Quantitative PLM

4: Polarized Light Microscopy
26
Phase Contrast microscopy, presentation, examples, Zernike’s Nobel Speech 
28

Phase Contrast microscopy

 
30
Inverted Microscope, Stereo Microscope,
5: Phase Contrast
Oct 3

Interferometry, Differential Interference Contrast Presentation; DIC microscope

 
5

PlasDIC

 
7

Hoffman Modulation Contrast

6: DIC
10
Review of transmitted light techniques, Key to determining imaging method 
12
Microscope Jeopardy 
14

Midterm Exam (Example)

7: Lab Practical Exam
17

Fluorescence Microscopy, Stokes Shift, Types of sample fluorescence

 
19

Fluorescence Microscope, presentation

 
21

Digital Imaging, Nyquist sampling, Digital imaging for microscopy

8: Epifluorescence microscopy
24
Laser scanning confocal microscopy I 
26

Laser scanning confocal microscopy II

 
28

Laser scanning confocal microscopy III

9: Fluorescence Probing
31
Two photon microscopy and resolution, presentation 
Nov 2

Two photon microscopy II

 
4

Spinning Disk & Sweep Scanners, Nipkow disks

10: Confocal microscopy (Zoom)
7
Restoration microscopy I, Basis, PSF, Methods, 
9

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

 
11
No Class
11: Project Explained
14

Super Resolution: Structured Illumination Microscopy (SIM)

 
16
Super Resolution: STED 
18
Super Resolution: PALM/StORM
12: SIM (Zoom)
21
Advanced fluorescence imaging: FRAP, FRET, FLIM 
23

Holiday: No Class

 
25

Holiday: No Class

Holiday: No Lab
28

Total Internal Reflection microscopy, TIRF microscope, Implications;

 
30
Finals Review Resources; Finals Jeopardy; BF Review, Fluor review 
Dec 2

The History of the Microscope

13: Tour of the Golub Collection
5

RRR Week: No Lecture

 
7

RRR Week: No Lecture.

 
9

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

 

12–16

Final exam TBA