Basic concepts of microscopy Basic Concepts of Microscopy.

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Transcript of Basic concepts of microscopy Basic Concepts of Microscopy.

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  • Basic concepts of microscopy Basic Concepts of Microscopy
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  • Basic concepts of microscopy Outline The Concept of Magnification Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy Transmitted and Reflected Techniques in Light Microscopy Bright Field Dark Field Phase Contrast Hoffman Modulation Contrast (HMC) Differential Interference Contrast (DIC) Polarization Fluorescence Laser Scanning Microscope (Confocal System) TIRF (Total Internal Reflection Fluorescence Microscope) Image Analysis Software & Digital Camera system
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  • Basic concepts of microscopy Outline The Concept of Magnification Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy Transmitted and Reflected Techniques in Light Microscopy Bright Field Dark Field Phase Contrast Hoffman Modulation Contrast (HMC) Differential Interference Contrast (DIC) Polarization Fluorescence Laser Scanning Microscope (Confocal System) TIRF (Total Internal Reflection Fluorescence Microscope) Image Analysis Software & Digital Camera system
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  • Basic concepts of microscopy The Concept of Magnification Magnification of the Microscope M Microscope = M Objective X M Eyepiece X M Intermediate Factor M = Magnification Example: Objective = 60 x Eyepiece = 10 x Intermediate Factor = 1 x Overall M = 600 x
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  • Basic concepts of microscopy The characteristics of objectives
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  • Basic concepts of microscopy
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  • The characteristics of objectives
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  • Basic concepts of microscopy Numerical Aperture (N.A.)
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  • Basic concepts of microscopy Resolution Resolving power, the limit up to which two small objects are still seen separately.
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  • Basic concepts of microscopy The characteristics of objectives
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  • Basic concepts of microscopy Outline The Concept of Magnification Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy Transmitted and Reflected Techniques in Light Microscopy Bright Field Dark Field Phase Contrast Hoffman Modulation Contrast (HMC) Differential Interference Contrast (DIC) Polarization Fluorescence Laser Scanning Microscope (Confocal System) TIRF (Total Internal Reflection Fluorescence Microscope) Image Analysis Software & Digital Camera system
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  • Basic concepts of microscopy Upright Microscopy
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  • Basic concepts of microscopy Eclipse 80i-Epi
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  • Basic concepts of microscopy Transmitted Light path of Upright Microscopy Eclipse 80i
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  • Basic concepts of microscopy Inverted Microscopy
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  • Basic concepts of microscopy TE2000U-Epi
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  • Basic concepts of microscopy Transmitted Light path of Inverted Microscopy TE2000U
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  • Basic concepts of microscopy Stereomicroscopy
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  • Differences between light and stereomicroscopy Stereomicroscopy : Two Identical Objectives with axes - Including a small angle ( Greenough Concept ) - Parallel share a common Objective ( Telescope Concept ) - Stereo Imaging Obtain - Longer Working Distance - Low Magnification - Huge Samples - Specimen Prepare Tools Light Microscopy : ( Upright / Inverted ) Single Objective with axis - Infinity Optics Design - Wide range of Observation - Short working Distance - High Magnification - Thin and small Specimen
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  • Basic concepts of microscopy Outline The Concept of Magnification Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy Transmitted and Reflected Techniques in Light Microscopy Bright Field Dark Field Phase Contrast Hoffman Modulation Contrast (HMC) Differential Interference Contrast (DIC) Polarization Fluorescence Laser Scanning Microscope (Confocal System) TIRF (Total Internal Reflection Fluorescence Microscope) Image Analysis Software & Digital Camera system
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  • Basic concepts of microscopy Bright Field Bright Field is the most universal technique used in light microscope. Usually used in samples with colorimetric staining or good contrast. Hemlock Leaf
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  • Basic concepts of microscopy , 10X
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  • Basic concepts of microscopy
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  • Basic concepts of microscopy Dark Field Fine structures can often not be seen in front of a bright background.
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  • Basic concepts of microscopy Phase Contrast
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  • Basic concepts of microscopy Hoffman Modulation Contrast D : dark, 1% transmittance G : gray, 15% transmittance B : bright, 100% transmittance
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  • Basic concepts of microscopy Diatoms Increase visibility and contrast in unstained and living material by detecting optical gradients (or slopes) and converting them into variations of light intensity.
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  • Basic concepts of microscopy Differential interference contrast (DIC) Analyzer Polarizer Objective Nomarski prism Condenser Nomarski prism
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  • Basic concepts of microscopy HeLa Cell Culture Heliozoans (Actinophrys sol)
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  • Basic concepts of microscopy Polarization Analyzer Polarizer
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  • Basic concepts of microscopy Dinosaur BoneGlutaric Acid Crystallites
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  • Basic concepts of microscopy The Principle of Fluorescence and Configuration of Fluorescence Accessories
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  • Basic concepts of microscopy The Principle of Fluorescence 40X Stoke shift
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  • Basic concepts of microscopy 1. Quartz Glass bulb 2. Cathode 3. Anode 321
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  • Basic concepts of microscopy 1. Light from HBO Lamp 2. Monochromatic Light 3. Fluorescence Light returning from the Specimen 1 2 2 3 3 3 A B C A B C Specimen
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  • Basic concepts of microscopy Bandpass emission filterLongpass emission filter
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  • Basic concepts of microscopy TE2000U Reflected Light path of Inverted Microscopy TE2000U
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  • Basic concepts of microscopy Eclipse 80i Reflected Light path of Upright Microscopy Eclipse 80i
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  • Basic concepts of microscopy
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  • Light emitted from the focal plane Light emitted from the out-of-focus region Objective lens Dichroic mirror Detector (PMT) Confocal pinhole specimen focal plane Laser light source Laser Scanning Microscope (Confocal System)
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  • Basic concepts of microscopy WideFieldConfocal Widefield versus Point scanning
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  • Basic concepts of microscopy Light emitted from the focal plane Light emitted from the out-of-focus region Objective lens Dichroic mirror Detector (PMT) Confocal pinhole specimen focal plane Laser light source Basic Concept of Laser Scanning Microscope Microscope Scan Head Laser Detector
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  • Basic concepts of microscopy C1s Optical System Detector module Laser module Scan Head
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  • Basic concepts of microscopy Fluorescence Dyes DAPI, Hoechst 33542 FITC, Fluo-3, GFP, Cy-2, Alexa Fluor 488, BODIPY, Calcium Green, Acridine Orange, BCECF, Oregon Green TRITC (Rhodamine), Cy-3, PI, DsRed, Alexa 546, Alexa 568, BOBO-3, Calcium Orange, DiI, Mitotracker Orange, DS Red Laser408nm488nm543nm DetectorBlueGreenRed Compatible Lasers, Wavelengths, and Dyes
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  • Basic concepts of microscopy Scan Head Detector 3 Laser Unit PC System C1 on Eclipse 90i with 3 lasers
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  • Basic concepts of microscopy Total Internal Reflection Fluorescence (TIRF)
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  • Basic concepts of microscopy Figure.1 Rapid Ca++ imaging with white-light TIRF microscope (orange frames show PFS on) Enlargements of A and B above (particle adhered to the glass) show focus drift present with PFS off. Numbers indicate the time in second before or after the addition of the reagent. Specimen: HeLa cells with Fluo4 loaded Objective: CFI Apo TIRF 100_ oil, NA 1.49
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  • Basic concepts of microscopy Outline The Concept of Magnification Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy Transmitted and Reflected Techniques in Light Microscopy Bright Field Dark Field Phase Contrast Hoffman Modulation Contrast (HMC) Differential Interference Contrast (DIC) Polarization Fluorescence Laser Scanning Microscope (Confocal System) TIRF (Total Internal Reflection Fluorescence Microscope) Image Analysis Software & Digital Camera system
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  • Basic concepts of microscopy Image-Pro Family Software Series Perfect for Basic Imaging or Capture Stations The Ultimate Imaging Package, Includes Support for Macros and Plug-Ins
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  • Basic concepts of microscopy EDF