Announcements
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Transcript of Announcements
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Announcements
• Please pick up syllabus and index card from front desk.– Fold the card lengthwise, write your name big and
bold and use it as a name plate on your table.
• Lecture notes will be available on Blackboard before class.
• TBA this week: continue working with your samples, practicing image collection and file management.– For viewing, sign up on confocal room door and with
either me or Phil Oshel.
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Agenda, Week 1
A. Introduction to Confocal: Syllabus, etc.
B. Lab: Fixation and staining of brine shrimp with fluorescent probes
1. Sytox Green for DNA2. Rhodamine-phalloidin for microfilaments (enriched in muscle)3. Unstained control for background fluorescence
C. Seminar from 4-5, Brooks 176: Heather Wiatrowski, Microbiology job candidate.
D. TBA this week and next: complete staining, view results with Dr. Hertzler or Phil Oshel.
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TBA Availability with Dr. Hertzler:Two 3-student groups, 2 hours each
Time Tuesday Wednesday Thursday
8
9 SEM Cell Biology
10 Office
11 Hours
12
1
2
3 UCC
4 Faculty Meeting Seminar
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Artemia Staining• Artemia at 1, 2, and 3 days (at 30oC) are available.• Fix with 3.7% formaldehyde in artificial seawater (ASW) for 1-2 hours.
After seminar:• Wash 3 X 5 min with ASW.• Each group should label 3 tubes and stain overnight, covered with tin foil,
with rocking:a) Sytox Green: 1:5000 in ASW (gloves)b) Rhodamine-phalloidin: 1:40 in ASW (gloves)c) nothing (control)
Tuesday, in Microscope suite (one locker available per group):• Wash 3 X 5 min with ASW.• Mount with either:
a) 50% glycerol/50% ASW then 90% glycerol (store in fridg until TBA time), orb) EtOH dehydration, 3X in 100% EtOH, then methyl salicylate (store at RT in
dark)
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Introduction to Confocal Imaging
A. Confocal versus conventional fluorescence
B. Optical sectioning
C. Imaging modes and applications
D. Advantages, limitations of confocal
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Laser Scanning Confocal Microscope Components
Laser
Scan Head
Microscope
Controllerbox Computer, display
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Conventional versus confocal fluorescence
Conventional epifluorescence Confocal epifluorescence
Sea urchin eggs (100 μm diameter)stained with antibody to tubulin.
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Human brain slice Rabbit muscle fibers Sunflower pollen grain
Widefield
Confocal