Basic Concepts of Microscopy Basic concepts of microscopy

Outline n The Concept of Magnification n Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy n 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) n Image Analysis Software & Digital Camera system Basic concepts of microscopy

n. M Microscope = M Objective X M Eyepiece X M Intermediate Factor The Concept of Magnification M = Magnification n. Example: Objective = 60 x Eyepiece = 10 x Intermediate Factor = 1 x Overall M = 600 x Basic concepts of microscopy

The characteristics of objectives Basic concepts of microscopy

Basic concepts of microscopy

The characteristics of objectives Basic concepts of microscopy

Numerical Aperture (N. A. ) Basic concepts of microscopy

Resolution Resolving power, the limit up to which two small objects are still seen separately. Basic concepts of microscopy

The characteristics of objectives Basic concepts of microscopy

Outline n The Concept of Magnification n Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy n 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) n Image Analysis Software & Digital Camera system Basic concepts of microscopy

Upright Microscopy Basic concepts of microscopy

Transmitted Light path of Upright Microscopy Eclipse 80 i Basic concepts of microscopy

Inverted Microscopy Basic concepts of microscopy

Transmitted Light path of Inverted Microscopy TE 2000 U Basic concepts of microscopy

Stereomicroscopy Basic concepts of microscopy

Basic concepts of microscopy

Differences between light and stereomicroscopy Stereomicroscopy : Two Identical Objectives with axes n - 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 n - Infinity Optics Design - Wide range of Observation - Short working Distance - High Magnification - Thin and small Specimen Basic concepts of microscopy

Outline n The Concept of Magnification n Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy n 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) n Image Analysis Software & Digital Camera system Basic concepts of microscopy

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. Basic concepts of microscopy

Dark Field n Fine structures can often not be seen in front of a bright background. Basic concepts of microscopy

Phase Contrast Basic concepts of microscopy

Hoffman Modulation Contrast D : dark, 1% transmittance G : gray, 15% transmittance B : bright, 100% transmittance Basic concepts of microscopy

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

Differential interference contrast (DIC) Analyzer Objective Nomarski prism Condenser Nomarski prism Polarizer Basic concepts of microscopy

He. La Cell Culture Heliozoans (Actinophrys sol) Basic concepts of microscopy

Polarization Analyzer Polarizer Basic concepts of microscopy

Glutaric Acid Crystallites Basic concepts of microscopy Dinosaur Bone

螢光原理與配件解說 The Principle of Fluorescence and Configuration of Fluorescence Accessories Basic concepts of microscopy

The Principle of Fluorescence 40 X Stoke shift Basic concepts of microscopy

1 2 3 1. Quartz Glass bulb 2. Cathode 3. Anode Basic concepts of microscopy

3 C 3 1 2 C B 3 2 B A Specimen A 1. Light from HBO Lamp 2. Monochromatic Light 3. Fluorescence Light returning from the Specimen Basic concepts of microscopy

Bandpass emission filter Basic concepts of microscopy Longpass emission filter

Reflected Light path of Inverted Microscopy TE 2000 U Basic concepts of microscopy

Reflected Light path of Upright Microscopy Eclipse 80 i Basic concepts of microscopy

Basic concepts of microscopy

Laser Scanning Microscope (Confocal System) Laser light source focal plane Objective lens specimen Confocal pinhole Dichroic mirror Light emitted from the focal plane Light emitted from the out-of-focus region Basic concepts of microscopy Detector (PMT)

Widefield versus Point scanning Wide. Field Basic concepts of microscopy Confocal

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

Detector module Scan Head Laser module Basic concepts of microscopy C 1’s Optical System

Compatible Lasers, Wavelengths, and Dyes Fluorescence DAPI, Dyes Hoechst 33542 FITC, Fluo-3, GFP, Cy-2, Alexa Fluor 488, BODIPY, Calcium Green, Acridine Orange, BCECF, Oregon Green TRITC (Rhodamine), Cy-3, PI, Ds. Red, Alexa 546, Alexa 568, BOBO-3, Calcium Orange, Di. I, Mitotracker Orange, DS Red Laser 408 nm 488 nm 543 nm Detector Blue Green Red Basic concepts of microscopy

C 1 on Eclipse 90 i with 3 lasers Scan Head 3 Laser Unit Detector PC System Basic concepts of microscopy

Total Internal Reflection Fluorescence (TIRF) 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: He. La cells with Fluo 4 loaded Objective: CFI Apo TIRF 100_ oil, NA 1. 49 Basic concepts of microscopy

Outline n The Concept of Magnification n Basic Introduction of Microscopy Upright microscopy Inverted microscopy Stereomicroscopy n 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) n Image Analysis Software & Digital Camera system Basic concepts of microscopy

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

EDF: Extended Depth of Focus Basic concepts of microscopy

Grey scale threshold Basic concepts of microscopy

Size Distribution Particles: Area, Diameter Basic concepts of microscopy

Evolution VF - cooled & uncooled - mono & color n Features q q q Designed for high resolution brightfield scientific and industrial applications. A progressive scan interline CCD sensor gives a resolution of 1. 3 million pixels in a 12 -bit digital output. (Sony 205 Sensor) High-speed low noise electronics provide linear digital data Live Frame rates of up to 110 fps with reduced region of interest and binning. The IEEE 1394 Fire. Wire™ digital interface allows ease of use Basic concepts of microscopy

Evolution MP™ Integrated Color Camera Kit n High resolution Color Camera (Sony 282 Sensor) n Features q Fully integrated with your choice of Image-Pro software q Fire. Wire (IEEE 1394) C-mount camera q Max 25 fps at 640 x 480 (30 -bit) Real Time Video (RTV) q Option for cooling q No interpolation or Pixel shifting q Laptop kit Basic concepts of microscopy

Evolution QEi™ Integrated Camera Kit n High Quantum Efficiency Camera n Features q q q Fully integrated with your choice of Image-Pro software Fire. Wire (IEEE 1394) C-mount camera Fast Fire. Wire Live 10 fps at 1360 x 1036(12 -bit) Basic concepts of microscopy

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