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Microscopy Lecture I. Three branches of Microscopy Optical Electron Scanning Probe Optical and Electron microscopy measure refraction, diffraction, and.

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Presentation on theme: "Microscopy Lecture I. Three branches of Microscopy Optical Electron Scanning Probe Optical and Electron microscopy measure refraction, diffraction, and."— Presentation transcript:

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

4 Units of Measure μm - Micrometer – 1,000,000 micrometers = 1 meter Strand of hair has a diameter of ~ μm 10 6 nm - Nanometer – 1,000,000,000 nanometers = 1 meter 10 9 Wavelength of visible light ( nm) Ǻ - Angstrom – 10,000,000,000 Angstroms = 1 meter 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 (θ 1 ) is related to the refraction angle (θ 2 ) by Snell’s Law n 1 sin(θ 1 )=n 2 sin(θ 2 ) 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 1.Ocular lens 2.Objective turret 3.Objective 4.Coarse Adjustment 5.Fine Adjustment 6.Stage 7.Light source 8.Condenser 9.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 utorials.html utorials.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

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