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. Lowest Resolvable Unit
Imaging Techniques
Technique
Image Formed By
Lowest Resolvable Unit
Approx Lower Limit
Optical Microscopy
Light Rays
Microns (μm)
1 μm
(monochromatic light)
Confocal Microscopy
Coherent Light Source (Laser)
.1 μm
(X-Y Direction)
Transmission
Electron Microscopy (TEM)
Electrons
Angstroms (Ǻ)
2 Ǻ
(high resolution TEM)
Scanning Electron Microscopy (SEM)
Nanometers (nm) to Angstroms (Ǻ)
10 nm
(100 Ǻ)
Atomic Force & Scanning Tunneling Microscopies (AFM/STM)
Molecular Mechanical Probes
40 Ǻ
(theoretical)

4. Units of Measure μm - Micrometer nm - Nanometer Ǻ - Angstrom
1,000,000 micrometers = 1 meter
Strand of hair has a diameter of ~ μm
106
nm - Nanometer
1,000,000,000 nanometers = 1 meter
109
Wavelength of visible light ( 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
Speed of light in a vacuum is a constant
Light travels slower through mediums that are not vacuums
Refractive Index is dependent on the frequency of light
Each color has a slightly different RI for a given material

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 Bright – absorbance of light by the sample
Dark – light scattered by the sample

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

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
Transgenic rats/mice
In the picture is bacteria transfected with FPs

23. Guess who
DRG of embryonic rat pups

24. Guess who
Mouse Intestine

25. Guess who
Cardiomyocyte

26. Guess who
Mouse Purkinje cells

27. Guess who
Hippocampal slice.