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Basic Concepts of Microscopy

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

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Basic Concepts of Microscopy

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  1. Basic Concepts of Microscopy

  2. 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

  3. 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

  4. The Concept of Magnification Magnification of the Microscope • MMicroscope = MObjective X M EyepieceX MIntermediate Factor M = Magnification • Example: Objective = 60 x Eyepiece = 10 x Intermediate Factor = 1 x Overall M = 600 x

  5. The characteristics of objectives

  6. The characteristics of objectives

  7. Numerical Aperture (N.A.)

  8. Resolution • Resolving power, the limit up to which two small objects are still seen separately.

  9. The characteristics of objectives

  10. 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

  11. Upright Microscopy

  12. Eclipse 80i-Epi 正立顯微鏡各部位名稱 數位影像擷取系統 汞燈燈室 目鏡 光路切換桿 螢光濾片槽 鼻輪 物鏡 載物台 聚光鏡 鹵素燈燈室 視野光圈 視野光圈調鈕

  13. Transmitted Light path of Upright MicroscopyEclipse 80i

  14. Inverted Microscopy

  15. TE2000U-Epi 倒立顯微鏡各部位名稱 鹵素燈燈室 視野光圈 聚光鏡 目鏡 載物台 鼻輪 螢光濾片槽 汞燈燈室 數位影像 擷取系統 光路切換鈕

  16. Transmitted Light path of Inverted MicroscopyTE2000U

  17. Stereomicroscopy

  18. 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

  19. 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

  20. Bright Field Hemlock Leaf • Bright Field is the most universal technique used in light microscope. • Usually used in samples with colorimetric staining or good contrast.

  21. 光軸中心點校正 • 選擇明視野位置, 使用10X 物鏡將樣品對焦 • 將視野光圈關到最小 • 上下移動聚光鏡使八角型邊緣清晰

  22. 判斷中心位置偏離方向，利用聚光鏡中心校正桿將調整至中心位置判斷中心位置偏離方向，利用聚光鏡中心校正桿將調整至中心位置 • 將光圈慢慢放大，最後全開以覆蓋全視野大小

  23. Fine structures can often not be seen in front of a bright background. Dark Field

  24. Phase Contrast

  25. Hoffman Modulation Contrast D : dark, 1% transmittance G : gray, 15% transmittance B : bright, 100% transmittance

  26. Increase visibility and contrast in unstained and living material by detecting optical gradients (or slopes) and converting them into variations of light intensity. Diatoms

  27. Differential interference contrast (DIC) Analyzer Objective Nomarski prism Condenser Nomarski prism Polarizer

  28. HeLa Cell Culture Heliozoans (Actinophrys sol)

  29. Polarization Analyzer Polarizer

  30. Glutaric Acid Crystallites Dinosaur Bone

  31. 螢光原理與配件解說The Principle of Fluorescence and Configuration of Fluorescence Accessories

  32. 40X Stoke shift The Principle of Fluorescence

  33. 1 2 3 1. Quartz Glass bulb 2. Cathode 3. Anode

  34. 3 C 3 1 2 3 B 2 A C B Specimen A 1. Light from HBO Lamp 2. Monochromatic Light 3. Fluorescence Light returning from the Specimen

  35. Bandpass emission filter Longpass emission filter

  36. Reflected Light path of Inverted MicroscopyTE2000U

  37. Reflected Light path of Upright MicroscopyEclipse 80i

  38. Light emitted from the focal plane Light emitted from the out-of-focus region Laserlight source focal plane Objective lens Confocal pinhole Detector (PMT) Dichroicmirror specimen Laser Scanning Microscope (Confocal System)

  39. Widefield versus Point scanning WideField Confocal

  40. Light emitted from the focal plane Microscope Light emitted from the out-of-focus region Laser Laser light source Scan Head focal plane Detector Objective lens Confocal pinhole Detector (PMT) Dichroicmirror specimen Basic Concept of Laser Scanning Microscope

  41. C1’s Optical System Detector module Scan Head Laser module

  42. Compatible Lasers, Wavelengths, and Dyes

  43. C1 on Eclipse 90i with 3 lasers Scan Head 3 Laser Unit Detector PC System

  44. Total Internal Reflection Fluorescence (TIRF)

  45. 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

  46. 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

  47. Image-Pro Family Software Series • The Ultimate Imaging Package, Includes Support for Macros and Plug-Ins • Perfect for Basic Imaging or Capture Stations

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