Microscopy Workshop Summer 2009. Objectives Learn how the microscope works – Trace light paths,...
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Microscopy Workshop Summer 2009. Objectives Learn how the microscope works – Trace light paths, identify major parts of the microscope, and compare microscopy
Objectives Learn how the microscope works Trace light paths,
identify major parts of the microscope, and compare microscopy
technologies: light, fluorescent and electron microscopy. Explore
Microscopy Applications Perform simple stain, use the laser
scanning confocal microscope, and gain experience with microscopy
websites/tutorials
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Microscope
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Antonie von Leeuwenhoek
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Tracing connections in the brain
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Architecture of Brain Regions
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pH Measurements Plant Physiology 4 th Ed Chap 15
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Protein Behavior and Organelle Shape Dnm1-GFP and mito-Red in
WT cells
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Microscopy Applications
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Parts of the Microscope 1 3 2 7 5 6 4 8 10 9 12 11 13
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Objective
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Light Path Figure 9-3a Molecular Biology of the Cell ( Garland
Science 2008)
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Magnification
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Spectrum of Light
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What type of microscope do I need to use?
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STERILIZATION OF A LOOPSTERILIZATION OF A LOOP BY FLAMING
Figure 1. Flaming of loop. Place the loop in flame starting at the
loop and move it through the flame so that the wire becomes red-hot
along 4 to 6 cm of its length. Allow the wire to cool for about 10
seconds. Pick up the sample with the cooled loop and distribute it.
Repeat the flaming/cooling procedure before laying the loop down on
the desk. Always flame the loop immediately prior to using it for
any purpose. PROCEDURE: STEP 1 AND 2
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PREPARATION OF A BACTERIAL SMEAR Figure 2. Preparation of a
microbial smear for STAINING. For a dry sample, use your
sterile/cool loop to first put a small drop of water or sterile
medium on the slide. Then pick up a tiny bit of the microbe sample
from the source (colony, wound) with a sterile/cool loop and mix it
into the liquid on the slide, being careful to not put too much or
too little sample on the slide. Spread the sample-drop for drying
and fixing as illustrated above and as demonstrated by your
instructor. STEP 1 AND 2
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STAINING Figure 3. Adding stain to the fixed bacterial smear.
After the bacterial smear has been heat-fixed to the slide, lay it
over the sink on the slide-support. Carefully drop the appropriate
staining solution onto the smear so as to cover it entirely. Allow
it to sit for 30 to 60 seconds. Then tip the slide so the excess
stain drops into the sink. Then gently run tap-water or a spray of
deionized water over the smear, washing off any remaining stain.
Finally, dry the smear either in the air or by gently patting it
with absorbent paper. Examine under the microscope, first using the
10X to locate areas of stained material, then place a drop of oil
on the dried, stained sample and rotate the oil- immersion lens
into the oil drop. STEPs 5-6
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Fluorescence Property of giving off light at a particular
wavelength (Emission) when illuminated by a light of a different
wavelength (Excitation)
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Applications of Fluorescent Microscopy
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Attach a Fluorophore Figure 9-18 Molecular Biology of the Cell
( Garland Science 2008) Figure 9-24 Molecular Biology of the Cell (
Garland Science 2008)
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Fluorescent Scope Light Path Figure 9-13 Molecular Biology of
the Cell ( Garland Science 2008)
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Optical Sectioning
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Confocal Imaging Figure 9-21 Molecular Biology of the Cell (
Garland Science 2008)
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How a confocal works Figure 9-20 Molecular Biology of the Cell
( Garland Science 2008)
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Results Figure 9-22 Molecular Biology of the Cell ( Garland
Science 2008)
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Mitochondria
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Mitochondrial Structure Budding Yeast Mouse Sperm Mammalian
COS-7 Mouse B lymphocyte Human RBC Fission Yeast
