Microscopy Lecture I

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

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)

Units of Measure μm - Micrometer nm - Nanometer Ǻ - Angstrom 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

0.75% Collagen Crosslinked

2% Collagen

Handspun Collagen

Optical Microscopy

Properties of light Reflection Refraction Numerical Aperture

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

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

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

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

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

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

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

Phase Contrast

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

Filter Cube

Live Dead Assay

Confocal Image of Schwann Cells

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

Guess who DRG of embryonic rat pups

Guess who Mouse Intestine

Guess who Cardiomyocyte

Guess who Mouse Purkinje cells

Guess who Hippocampal slice.