Microscopy Lecture I

1. Microscopy Lecture I

2. Three branches of Microscopy • Optical • Electron • Scanning Probe • Optical and Electron microscopy measure refraction, diffraction, and reflection of the source radiation • Optical uses white light, fluorescent light, or lasers • Electron uses electromagnetic radiation/electron beams • Scanning uses a physical probe to interact with the surface of the specimen

3. Imaging Techniques

4. Units of Measure • μm - Micrometer • 1,000,000 micrometers = 1 meter • Strand of hair has a diameter of ~ 20-180 μm • 106 • nm - Nanometer • 1,000,000,000 nanometers = 1 meter • 109 • Wavelength of visible light (400-700 nm) • Ǻ - Angstrom • 10,000,000,000 Angstroms = 1 meter • 1010 • Used to measure the size of atoms/bond lengths • Length of a C-H bond in methane is ~1 Angstrom

5. 0.75% Collagen Crosslinked

6. 2% Collagen

7. Handspun Collagen

8. Optical Microscopy

9. Properties of light • Reflection • Refraction • Numerical Aperture

10. Refraction • Change in the direction of a wave (light) due to a change in speed • The straw in the picture looks bent because the light is bending as it moves from the water to the air

11. Refractive Index (RI) • RI of a material a measure of the speed of light in material • RI is the ratio of the velocity of light in a vacuum to the speed of light in the specified material • Incident angle (θ1) is related to the refraction angle (θ2) by Snell’s Law • n1sin(θ1)=n2sin(θ2) • Used in calculating focusing power of lenses and dispersion properties of prisms

12. Reflection • Reflection is defined as a change in direction of a wave at an interface between 2 different media so that the waveform returns to the media from which it came • Used in focusing light waves to increase transmitted light

13. Numerical Aperture • NA of a microscope objective is a measure of its ability to gather light • The more light (higher NA) the better the resolving power of the lens • Better resolution • NA = (n)sin(θ) • n = Refractive Index • θ = ½ the maximum cone of light than can enter the lens • Usually the NA of an objective increases with its magnifying power. • The smallest detail that can be resolved is proportional to: • λ/NA

14. Optical Microscope • Ocular lens • Objective turret • Objective • Coarse Adjustment • Fine Adjustment • Stage • Light source • Condenser • X-Y Control

15. Phase Contrast • Uses phase shifted waves of through transparent specimens cause changes in amplitude (contrast) in structures of the specimen • One of the most widely used in biology • No staining required

16. Compound Light

17. Phase Contrast

18. Fluorescence • Fluorescence utilizes fluorescent dyes/stains that fluoresce when radiated with specific wavelengths of light • Typically use mercury or xenon lamps • Fluorescent dyes are extremely useful in identifying/highlighting specific parts of cells that can otherwise go undetected using simple phase contrast • http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

19. Filter Cube

20. Live Dead Assay

21. Confocal Image of Schwann Cells

22. Green Fluorescent Protein • Class of proteins that naturally fluoresce • First isolated from the jellyfish • 238 amino acid long protein that naturally fluoresces green (509 nm) in the presence of blue (488 nm) light • Through genetic engineering, scientists have artificially engineered many variations of GFP

23. Guess who

24. Guess who

25. Guess who

26. Guess who

27. Guess who