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Techniques in Light Microscopy

S2019/ PMB 185
CCN: 30536
MWF 12-1
110 Barker Hall

Lab F 1-4
CCN: 30995
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 30995) 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 Tuesday 2PM, or by appointment.

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

Lecture/Lab topic, Spring 2019

Reading*
Lecture

Topic in Lecture

Lab topic (Fridays)
Jan 23

Introduction

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

Lens aberrations (spherical, chromatic),

1: Microscope optics
Ch 1: p1-19
4
Microscope configurations: The compound microscope , Microscope Standard Lengths
6
The Condenser: history, optics & Köhler illumination
8

Objectives, objective designations, Eyepieces,

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

Numerical Aperture and Resolution

Ch 6: p103-105
13

Darkfield microscopy, Rheinberg Illumination, Oblique Illumination

Ch 4: p62-71
15

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

3: Darkfield
Ch 7: p129-133
18
Academic Holiday, No Lecture
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, Zernike's Nobel Speech
Ch 9: p153-164
167-171
27

Phase Contrast microscopy

Mar 1
Inverted Microscope, Stereo Microscope,
5: Phase Contrast
Ch 7: p115-129
4

Interferometry, Differential Interference Contrast Presentation; DIC microscope

6

PlasDIC

8

Hoffman Modulation Contrast

6: DIC
Ch 10: p173-190
11
Review of transmitted light techniques, Key to determining imaging method, example questions
Ch 10: p190-194
13
Microscope Jeopardy
15

Midterm Exam (Answers to 2014 exam)

Midterm Lab Exam
18

Digital Imaging, Nyquist sampling, Digital imaging for microscopy

20

Fluorescence Microscopy, Stokes Shift, Types of sample fluorescence

Ch 11: p199-231
22

Fluorescence Microscope, presentation

7: Fluorescence
25
Spring Recess
Ch 13: p265-275
27

Spring Recess

29

Spring Recess

No Lab
Ch 13: 289-294
April 1
Laser scanning confocal microscopy I, presentation
CH 14: 307-324
3

Laser scanning confocal microscopy II, resolution

5

Spinning Disk & Sweep Scanners, Nipkow disks

8A: Confocal/SD Microscopy
CH 13: 298-305
8
Two photon microscopy and resolution, presentation
10

Two photon microscopy II

Ch 12: 252-261
12
Deconvolution microscopy, Basis, PSF, Methods,
Here's a short tutorial on Filtering.
8B: Confocal/SD plus Advanced fluorescenceprobing (DS)
15

Live cell imaging

17
Total Internal Reflection microscopy, TIRF microscope, Implications (HW)
Ch 15
19
Super Resolution: SIM, Description of the Forensics Project.
9: Super-Resolution (SIM), Imaging,
Project Explan'd
Lab 9 Imaging Assignment
22
Super Resolution: STED; PALM/STORM
Ch 12: 233-252
24

SPIM (LightSheet imaging) Introduction to the Project,

26

Advanced fluorescence imaging: FRAP, FRET, FLIM;
Project Samples distributed during Lecture.

9: Super-Resolution (SIM), Imaging,
Project Explan'd
Lab open for Projects
29
Final Review; Finals Review Resources
Ch 16
May 1
X-Ray microscopy Safety Selfies due 5/1
3

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
6

No Lecture. BF Review, Fluor review

The Project lab will be open all this week.
8

No Lecture.

10

No Lecture. Project Due 5PM

May 15

Final Exam Group 11, 3-6PM

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

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Updated Wed, Apr 17, 2019