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Techniques in Light Microscopy

F2017/ PMB 185
CCN: 24269

MWF 9-10AM
201 Giannini Hall

3 Units

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

Laboratory: The laboratory is in 208 GPBB, and will consist of one session per week:

Section 101 (CCN 24270) Friday 1–4PM, 208 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 describing your forensic analysis of an unknown white powder.

Office hour is Wednesday 4-5PM, or by appointment.

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

Lecture/Lab topic, Fall 2016

Reading*
Lecture

Topic in Lecture

Lab topic (Fridays)
Aug 23

Introduction (Presentation) (DS)

Ch 2: p21-30
25
Light and Refraction (DS)
No Lab
28
Microscope Optics: Image formation, Conjugate planes, Resolution
Ch 4: p53-65
30
Microscope optics: Lenses, Imaging
Sept 1

Lens aberrations (spherical, chromatic),

1: Microscope optics
Ch 1: p1-19
4
Holiday. No Lecture
6
Microscope configurations: The compound microscope , Microscope Standard Lengths
8

The Condenser: history, optics & Köhler illumination

2: Köhler illumination
Focus Examples
Ch 1 9-14
11

Objectives, objective designations, Eyepieces,

Ch 6: p103-105
13

Numerical Aperture

Ch 4: p62-71
15

Darkfield microscopy, Rheinberg Illumination, Oblique Illumination

3: Darkfield
Ch 7: p129-133
18
Polarized light microscopy: Polarized light, Anisotropy, Types of birefringence, Polarized light microscope
Ch 1: p1-5
20

Polarized light microscopy, Generation of colors,

22

Polarized light microscopy: Quantitative PLM

4: Polarized Light Microscopy
Ch 8: p135-146
25
Phase Contrast microscopy, presentation, examples
Ch 9: p153-164
167-171
27

Phase Contrast microscopy

29
Inverted Microscope, Stereo Microscope,
5: Phase Contrast
Ch 7: p115-129
Oct 2

Interferometry, Differential Interference Contrast Presentation

4

PlasDIC

6

Hoffman Modulation Contrast

6: DIC
Ch 10: p173-190
9
Review of transmitted light techniques, Key to determining imaging method,
Ch 10: p190-194
11
Microscope Jeopardy, Previous Exam, Zernike's Nobel Speech
13

Midterm Exam

7: Midterm Lab Exam
16

Digital Imaging, Nyquist sampling, Digital imaging for microscopy

18

Fluorescence Microscopy, Stokes Shift

Ch 11: p199-231
20

Fluorescence Microscopy, presentation

8: Fluorescence
23
Laser scanning confocal microscopy I, presentation
Ch 13: p265-275
25

Laser scanning confocal microscopy II

27

Spinning Disk & Sweep Scanners, Nipkow disks

9: Confocal/SD Microscopy
Ch 13: 289-294
30
Two photon microscopy and resolution, presentation
CH 14: 307-324
Nov 1

Two photon microscopy II

3

Deconvolution microscopy, Basis, PSF, Methods Forensics Project Described (DS)

10: Restoration Microscopy (DS)
CH 13: 298-305
6
Restoration Microscopy presentation. (DS)
8

Total Internal Reflection microscopy, TIRF microscope (DS)

Ch 12: 252-261
10
Holiday. No Lecture.
No Lab
13

SPIM (LightSheet imaging) Introduction to the Project

Lab open for Projects
15
Super Resolution: SIM
Ch 15
17
Super Resolution: STED; PALM, STORM
Super-Resolution (SIM: SR)
Explan'd (DS)
20
Advanced fluorescence imaging: FRAP, FRET, FLIM ; Live-Cell imaging; Presentation
Ch 12: 233-252
22

Holiday. No Lecture

24

Holiday, No lecture

No Lab
27
Final Review; Finals Review Resources,
Ch 16
29
X-Ray microscopy
Dec 1

FINALS JEOPARDY

ALS tour
Take the BLUE LBNL bus on Hearst by Tolman at 1:10 PM Friday. Bring your Photo ID and the bar-code LBNL sheet
4

No Lecture.

The Project lab will be open all this week.
6

No Lecture. BF Review, Fluor review

8

No Lecture. Project Due 5PM

TBA

Final Exam TBA

*Murphy and Davidson, Fundamentals of Light Microscopy and Electronic Imaging. Wiley-Blackwell, 2nd Ed.

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Updated Tue, Jun 20, 2017