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.

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.

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

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)

Introduction to Confocal Imaging

A. Confocal versus conventional fluorescence

B. Optical sectioning

C. Imaging modes and applications

D. Advantages, limitations of confocal

Laser Scanning Confocal Microscope Components

Laser

Scan Head

Microscope

Controllerbox Computer, display

Conventional versus confocal fluorescence

Conventional epifluorescence Confocal epifluorescence

Sea urchin eggs (100 μm diameter)stained with antibody to tubulin.

Human brain slice Rabbit muscle fibers Sunflower pollen grain

Widefield

Confocal