BME 210 Lecture 2 Optical Microscopy

BME 210 Lecture 2 - 2 Imaging of cells and bio-molecules Main method characteristics • Physical principles • Detected biological properties •

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2. Imaging of cells and bio-molecules
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Main method characteristics • Physical principles • Detected biological properties • General instrument design; critical elements • Sensitivity, resolution • Biological applications • Limitations
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Microscopy Microscopy – a group of methods for observation of objects smaller than ~100 μm (eye resolution limit) Main microscopic methods used in biology: 1. Optical Microscopy 2. Fluorescence Microscopy 3. Confocal Microscopy 4. Electron Microscopy 5. Atomic Force Microscopy Less important: 1. X-ray Microscopy 2. Scanning Tunneling Microscopy The majority of microscopic methods employ Electromagnetic Radiation
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Electromagnetic Radiation (EMR) EMR (light) – a physical entity with properties of both waves and particles Light waves - oscillating electrical (E) and magnetic (B) fields, orthogonal to each other and to the direction of their propagation Wave parameters: amplitude (A), frequency ( f ), wavelength ( λ ), propagation speed (c), phase λ = c/f
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EMR Spectrum Parts of EMR spectrum used in biological imaging: 1. λ = 1-10 μm (infrared light) - Infrared imaging 2. λ = 450-700 nm (visible) - Optical, fluorescence, confocal 3. λ = 50-300 nm - Ultraviolet microscopy 4. λ = 1-10 nm (soft x-rays) - X-ray microscopy 1 2 3 4
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Optical Microscopy “Optical” - observation in visible light: λ 450-700 nm • Instrument – optical microscope • Main component - lens • Principle of image formation – bending of light due to light refraction
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Light refraction Refraction – change of direction of light propagation when passing from one medium to another with a different light speed Snell’s law: n – refractive index, the ratio of the speed of light in vacuum to the speed of light in a medium
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Lens A lens has a curved surface; the law of refraction can be applied to every point on the surface A concave lens bends light rays towards the lens center (“positive” lens) With proper lens shape (near-spherical), all light rays emitted from a point will be focused by the lens at another point perpendicular
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Lens focal point Moving the light source away brings the image closer to the lens When the source is at infinity, it sends parallel beam of light which is focused by at a point - “ focal” point (F) Parallel beams with different angles of incidence are focused at focal points laying in a “focal” plane Lens definition: element which focuses parallel beam of light into a point L F L F
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Using lens definition: • Object at a > F • Every point of the object sends rays in every direction • To build an image, it is sufficient to find image location of the object tip by drawing 2 rays: 1. Central ray 1 – it is a straight line 2. A ray 2 parallel to the main axis – it is refracted to the focal point F • Intersection of two rays defines the tip
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This note was uploaded on 04/28/2010 for the course BME 210 taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

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BME 210 Lecture 2 - 2 Imaging of cells and bio-molecules Main method characteristics • Physical principles • Detected biological properties •

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