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Techniques in Light Microscopy

F2016/ PMB 185
CCN: 26116

MWF 9-10AM
138 Morgan 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 208 GPBB, and will consist of one session per week:

Section 101 (CCN 26117) 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 24

Introduction (Presentation)

Ch 2: p21-30
26
Light and Refraction
Sample Prep
29
Image formation, Conjugate planes, Resolution
Ch 4: p53-65
31
Microscope optics: Lenses, Imaging
Sept 2

Lens aberrations (spherical, chromatic),

Microscope optics
Ch 1: p1-19
5
Holiday. No Lecture
7
Microscope configurations: The compound microscope , Microscope Standard Lengths
9

The Condenser: history, optics & Köhler illumination

Köhler illumination
Focus Examples
Ch 1 9-14
12

Numerical Aperture

Ch 6: p103-105
14

Objectives, objective designations, Eyepieces,

Ch 4: p62-71
16

Darkfield microscopy, Rheinberg Illumination,

Darkfield
Ch 7: p129-133
19
Inverted Microscope, Stereo Microscope, Oblique Illumination
Ch 1: p1-5
21

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

23

Polarized light microscopy, Generation of colors,

Polarized Light Microscopy
Ch 8: p135-146
26
Polarized light microscopy: Quantitative PLM
Ch 9: p153-164
167-171
28

Phase Contrast microscopy, presentation, examples

30
Phase Contrast microscopy
Phase Contrast
Ch 7: p115-129
Oct 3

Interferometry, Differential Interference Contrast Presentation

5

PlasDIC

7

Hoffman Modulation Contrast

DIC
Ch 10: p173-190
10
TBA
Ch 10: p190-194
12
Review of transmitted light techniques, Key to determining imaging method, Jeopardy, Previous Exam, Zernike's Nobel Speech
14

Midterm Exam

Midterm Lab Exam
17

Digital Imaging, Nyquist sampling, Digital imaging for microscopy

19

Fluorescence Microscopy, Stokes Shift

Ch 11: p199-231
21

Fluorescence Microscopy, presentation

Fluorescence
24
Laser scanning confocal microscopy I, presentation
Ch 13: p265-275
26

Laser scanning confocal microscopy II

28

Spinning Disk & Sweep Scanners, Nipkow disks

Confocal/SD Microscopy
Ch 13: 289-294
31
Two photon microscopy and resolution, presentation
CH 14: 307-324
Nov 2

Two photon microscopy II

4

Deconvolution microscopy, Basis, PSF, Methods Forensics Project Described

Confocal/SD Microscopy
CH 13: 298-305
7
Restoration Microscopy presentation. Room 203 GPBB OPEN for projects
9

Total Internal Reflection microscopy, TIRF microscope

Ch 12: 252-261
11
Holiday. No Lecture.
No Lab
14

SPIM (LightSheet imaging)

16
Super Resolution: SIM
Ch 15
18
Super Resolution: STED
Restoration/ SIM
21
Super Resolution: PALM, STORM
Ch 12: 233-252
23

Holiday. No Lecture

25

Holiday, No lecture

Holiday
28
Advanced fluorescence imaging: FRAP, FRET, FLIM ; Live-Cell imaging; Presentation
Ch 16
30
FLIM review, X-Ray microscopy
Dec 2

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
5

No Lecture. Finals Review Resources, The Project lab will be open all this week.

7

No Lecture. BF Review, Fluor review

9

No Lecture. Project Due 5PM

Dec 15

Final Exam 7–10PM

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

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Updated Fri, Dec 2, 2016