Microscopy Workshop Summer 2009

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

Microscope

Antonie von Leeuwenhoek

Tracing connections in the brain

Architecture of Brain Regions

pH Measurements Plant Physiology 4 th Ed Chap 15

Protein Behavior and Organelle Shape Dnm1-GFP and mito-Red in WT cells

Microscopy Applications

Parts of the Microscope

Objective

Light Path Figure 9-3a Molecular Biology of the Cell (© Garland Science 2008)

Magnification

Spectrum of Light

What type of microscope do I need to use?

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

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

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

Fluorescence Property of giving off light at a particular wavelength (Emission) when illuminated by a light of a different wavelength (Excitation)

Applications of Fluorescent Microscopy

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)

Fluorescent Scope Light Path Figure 9-13 Molecular Biology of the Cell (© Garland Science 2008)

Optical Sectioning

Confocal Imaging Figure 9-21 Molecular Biology of the Cell (© Garland Science 2008)

How a confocal works Figure 9-20 Molecular Biology of the Cell (© Garland Science 2008)

Results Figure 9-22 Molecular Biology of the Cell (© Garland Science 2008)

Mitochondria

Mitochondrial Structure Budding Yeast Mouse Sperm Mammalian COS-7 Mouse B lymphocyte Human RBC Fission Yeast

Mitochondria are Dynamic

Wild-typeΔMdv1