Techniques in Light Microscopy

F2011/ PMB 185
CCN: 70525

TuTh. 9–10AM
3109 Etcheverry Hall
3 Units

Steven Ruzin Ph.D.
CNR 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, demonstrations, and practice using the light microscopes in the CNR Biological Imaging Facility and the GPBB Teaching facility.

Laboratory: The laboratory is in 209 GPBB, and will consist of one session per week. Each student is responsible for one laboratory session per week. Lab times are:

Section 101 (CCN 70528): Thursday 2–5PM, 209 GPB
Section 102 (CCN 70531): Friday 2–5PM, 209 GPB

Grading: There will be a Midterm written exam and lab practical, plus a written Final Exam. For labs, students will be required to complete at least one worksheet for each of the labs. Worksheet is due at the end of that lab. In addition, a project is required for the class. It consists of a written paper. A description of the project will be given toward the middle of the semester.

Syllabus

Office hour: Wed 4-5PM, 381 Koshland Hall
Date

Lecture/Lab topic

Lecture

Topic in Lecture

Lab topic
Aug 25

Introduction, Light and Refraction

No Lab
30

Microscope optics: Lenses, Imaging, and Resolution

Sept 1

Lens aberrations (spherical, chromatic), The compound microscope , Microscope Standard Lengths

Sample preparation (1,2)
6

The Condenser: history, optics & Köhler illumination

Köhler illumination (8,9)
Focus Examples
8

Objectives, objective designations, Numerical Aperture

13

Contrast derived from Light Scattering: Darkfield microscopy, Rheinberg Illumination, Oblique Illumination

Darkfield (15,16)
15

Eyepieces, Inverted Microscope, Stereo Microscope

20

Polarized light microscopy I: Polarized light, Anisotropy, Types of birefringence, Polarized light microscope

Polarized Light Microscopy (22,23)
22

Polarized light microscopy II, Generation of colors, Quantitative PLM

27

PLM II

Phase Contrast (29,30)
29

Phase Contrast microscopy, presentation, examples

Oct 4

Interferometry, Differential Interference Contrast Presentation

DIC (6,7)
6

PlasDIC, Hoffman Modulation Contrast

11

Review of transmitted light techniques, Key to determining imaging method, Sample Exam Questions

Lab Exam (Oct 13)
13

Midterm Exam

Lab Exam (Oct 14)
18

Fluorescence I, Stokes Shift

20

Fluorescence II

Fluorescence (20,21)
25

Digital Imaging, Nyquist sampling, Digital imaging for microscopy,

27

Laser scanning confocal microscopy I, presentation

Confocal Microscopy I (28,29)
Nov 1

Laser scanning confocal microscopy II

3

Deconvolution microscopy I, Basis, PSF, Methods

Confocal Microscopy II (3,4)
8

Deconvolution microscopy II, presentation

10

Two photon microscopy and resolution, presentation

No Lab (Holiday 10,11)
15

High Speed and Live-Cell imaging: Spinning Disk & Sweep Scanners, Nipkow disks, Slit scanning confocal microscopes

Deconvolution (16,17)
17

Advanced fluorescence imaging: FRAP, FRET, FLIM, FLIP

22

Total Internal Reflection microscopy, TIRF microscope

No lab
24, 25

Holiday

No Lab
29

Super Resolution: STED, PALM, Structured Illumination

Dec 1

Historical Perspectives: The Golub Collection
Project Due

ALS tour (Dec 1)
Take the BLUE LBNL bus on Hearst by Tolman
7

Final Review. Comparison of Advanced Fluorescence Techniques: BF Review, Fluor review, Advanced Review,

ALS tour (Dec 2)
Take the BLUE LBNL bus on Hearst by Tolman
9
No lecture. For you to use in preparation for the exam.
Dec 13,
3–6PM

Final Exam

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Updated Mon, Dec 12, 2011