PRINCIPLES OF MICROSCOPY S. WOLNIAK
Dates Topic Readings*
Aug. 30 Introduction to the light microscope M (ch. 1, 2, 4), R (1-6, 23-24)
Kohler illumination, bright field S (1-31), D (ch. 1,3), Ob
Sept. 1,8 Image formation; diffraction M (ch. 5), R (7-11), S (1-31)
D (ch. 1)
Sept. 13 Resolution, numerical aperture M (ch. 4, 6), R (7-22)
objective lens design S (1-31,63-69)
D (ch. 1)
Sept. 15-22 (Digital) Photography and photomicrography, M (ch. 3,13) R (113-120)
Filters, light temp., chromatic S (70-72),
Sept. 27,29 Phase contrast; darkfield microscopy M (ch. 7),R (25-27,30-31,
Oct. 4,6 Fluorescence microscopy, microfluoro- M (ch. 11), R (27-30,88-90)
metry, microspectrophotometry; S (40-45)
light detectors Z or L or Of
Oct. 11 Polarized Light Microscopy M (ch. 8,9), R (32-36,94)
Birefringence, Compensation, BR S (46-53), Bennett
Oct. 13 Differential Interference Contrast M (ch. 10), R(36-44,92-94)
Hoffman Modulation Contrast S (46-53),
Interference Reflection Microscopy Hoffman
Interference Microscopy S (54-61)
Oct. 18 NO CLASS
Oct. 20 Digital microscopy and computer image M (ch. 13-15), R (121-125), P
Oct. 25-Nov. 3 (Confocal) Scanning optical microscopy M (ch. 12), R (44-49,96-98)
Deconvolution Microscopy Handbook, Wright, Stelzer,
for microbeam surgery,
fluorescence, optical traps Block
Acoustic microscopy M (137-158)
Nov. 10 Exam
Nov. 1- Dec. 11 Training with the 510 and DV microscopes
0107 Microbiology (sign up for microscope use)
Dec. 13 10:30-12:30 Oral Presentations/Picture Analysis
* Readings: M = Murphy, D.B. 2001. Fundamentals of Light Microscopy and
Electronic Imaging, Wiley
R = Rawlins, D.J. 1992. Light Microscopy. Bios Scientific Publishers
S = Spencer, M. 1982. Fundamentals of Light Microscopy
D = Delly, J.G. 1988. Photography through the Microscope.
L = Leitz booklet on fluorescence microscopy
Z = Zeiss booklet on fluorescence microscopy
Of = Olympus booklet on fluorescence microscopy
Ob = Olympus booklet on brightfield microscopy
P = Photometrics booklet on CCD cameras
= article on
Bennett = article on polarized light microscopy
Stelzer = article on confocal scanning light microscopy
Wright = article on confocal scanning light microscopy
Petroll = article on confocal scanning
427 (2 credits) FALL,
PRINCIPLES OF MICROSCOPY S. WOLNIAK
Office hours: By appointment - call 5-1605 or email at firstname.lastname@example.org
November 10th This hourly exam will cover the theoretical aspects of imaging through the light microscope.
In room 0107 of the Microbiology Building, we have two new microscopes that represent the best in confocal instrumentation and digital image processing/deconvolution. This course provides you with an unusual opportunity to learn how to use these microscopes, and I plan the lecture cycle of this course so that you can spend some time using these instruments.
Step 1: training with a simple specimen. Dr. Bob Brown will train you in the basics of confocal imaging with the Zeiss 510 and with the DeltaVision Image Reconstruction Microscopes. You will start with a standard, stable specimen to generate a set of color images that will enable you to see what is possible. Save and transfer these files to the workstations, because you can use them in the presentation (step 3, below), especially if your cells prove to be troublesome.
Step 2: imaging some of your own material. After you have been checked out on the microscopes, you need to speak with me about strategies to get good confocal or deconvolution images with your specimens. Then, you need to able to sign up for some time with one or both of the microscopes in room 0107 Microbiology. Take several micrographs of representative cells and a few sets of image stacks. Try to obtain multi-color images, and/or superimposed fluorescence and transmitted light images (DIC or phase contrast). Transfer the files to the workstations, so that you can manipulate the images without having to sign up for more microscope time. Translate the files to TIFF, so that they can be viewed with Adobe Photoshop. We can burn CDs from the workstations so that you have permanent records of your work, and so that you can show the others in the class what you have been able to generate in a few short hours.
Step 3: presentation time. We will meet at class time on Monday, December 13th, in room 0107 Microbiology to look at everyone’s images. At this stage, it is more important to produce images of cells than to generate masterpieces that will be preserved in museums. If images have flaws, it is actually better, because we can use this presentation class meeting to discuss ways to reduce problems and improve the likelihood that you can get cover pictures and color plates that will convey structural information accurately and beautifully.
From November 1st onward, Bob Brown and I will be available to help you in getting trained to generate images of your cells.