Announcements

1
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.

2
Agenda, Week 1

• Introduction to Confocal Syllabus, etc.
• Lab Fixation and staining of brine shrimp with
  fluorescent probes
• Sytox Green for DNA
• Rhodamine-phalloidin for microfilaments (enriched
  in muscle)
• Unstained control for background fluorescence
• Seminar from 4-5, Brooks 176 Heather Wiatrowski,
  Microbiology job candidate.
• TBA this week and next complete staining, view
  results with Dr. Hertzler or Phil Oshel.

3
TBA Availability with Dr. HertzlerTwo 3-student
groups, 2 hours each
4
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
• Sytox Green 15000 in ASW (gloves)
• Rhodamine-phalloidin 140 in ASW (gloves)
• nothing (control)
• Tuesday, in Microscope suite (one locker
  available per group)
• Wash 3 X 5 min with ASW.
• Mount with either
• 50 glycerol/50 ASW then 90 glycerol (store in
  fridg until TBA time), or
• EtOH dehydration, 3X in 100 EtOH, then methyl
  salicylate (store at RT in dark)

5
Introduction to Confocal Imaging

• Confocal versus conventional fluorescence
• Optical sectioning
• Imaging modes and applications
• Advantages, limitations of confocal

6
Laser Scanning Confocal Microscope Components
Scan Head
Microscope
Controller box
Laser
Computer, display
7
Conventional versus confocal fluorescence
Conventional epifluorescence
Confocal epifluorescence
Sea urchin eggs (100 µm diameter) stained with
antibody to tubulin.
8
Widefield
Confocal
Sunflower pollen grain
Human brain slice
Rabbit muscle fibers