Basic Concepts of Microscopy
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Transcript of Basic Concepts of Microscopy
Basic concepts of microscopy
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 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
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
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
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.)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 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
Basic concepts of microscopy
Upright MicroscopyUpright Microscopy
Basic concepts of microscopy
目鏡
鼻輪
載物台聚光鏡
視野光圈
物鏡
鹵素燈燈室
視野光圈調鈕
光路切換桿螢光濾片槽
Eclipse 80i-Epi Eclipse 80i-Epi 正立顯微鏡各部位名稱正立顯微鏡各部位名稱
汞燈燈室
數位影像擷取系統
Basic concepts of microscopy
Transmitted Light path of Upright MicroscopyTransmitted Light path of Upright Microscopy
Eclipse 80iEclipse 80i
Basic concepts of microscopy
Inverted MicroscopyInverted Microscopy
Basic concepts of microscopy
TE2000U-Epi TE2000U-Epi 倒立顯微鏡各部位名稱倒立顯微鏡各部位名稱
目鏡
載物台
聚光鏡
鼻輪
汞燈燈室
視野光圈
光路切換鈕
螢光濾片槽
鹵素燈燈室
數位影像擷取系統
Basic concepts of microscopy
Transmitted Light path of Inverted MicroscopyTransmitted Light path of Inverted Microscopy
TE2000UTE2000U
Basic concepts of microscopy
StereomicroscopyStereomicroscopy
Basic concepts of microscopy
Basic concepts of microscopy
Differences between light and stereomicroscopyDifferences 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
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
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
Basic concepts of microscopy
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
Basic concepts of microscopy
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
Basic concepts of microscopy
Dark Field
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% transmittanceG : gray, 15% transmittanceB : bright, 100% transmittance
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.
Basic concepts of microscopy
Differential interference contrast (DIC)
Analyzer
Polarizer
ObjectiveNomarski prism
CondenserNomarski prism
Basic concepts of microscopy
HeLa Cell Culture
Heliozoans (Actinophrys sol)
Basic concepts of microscopy
Polarization
Analyzer
Polarizer
Basic concepts of microscopy
Dinosaur Bone Glutaric Acid Crystallites
Basic concepts of microscopy
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
40X
Stoke shift
Basic concepts of microscopy
1. Quartz Glass bulb
2. Cathode
3. Anode
321
Basic concepts of microscopy
1. Light from HBO Lamp2. Monochromatic Light3. Fluorescence Light returning from
the Specimen
12
23
3
3
A
B
C
A
B
C
Specimen
Basic concepts of microscopy
Bandpass emission filter Longpass emission filter
Basic concepts of microscopy
Reflected Light path of Inverted Microscopy
TE2000UTE2000U
Basic concepts of microscopy
Reflected Light path of Upright Microscopy
Eclipse 80iEclipse 80i
Basic concepts of microscopy
Basic concepts of microscopy
Light emitted from the focal plane
Light emitted from the out-of-focus region
Objective lens
Dichroic mirror
Detector(PMT)
Confocalpinhole
specimen
focal planeLaser light source
Laser Scanning Microscope (Confocal System)
Basic concepts of microscopy
WideField Confocal
Widefield versus Point scanningWidefield versus Point scanning
Basic concepts of microscopy
Light emitted from the focal plane
Light emitted from the out-of-focus region
Objective lens
Dichroic mirror
Detector(PMT)
Confocalpinhole
specimen
focal planeLaser light source
Basic Concept of Laser Scanning MicroscopeBasic Concept of Laser Scanning Microscope
Microscope
Scan Head
Laser
Detector
Basic concepts of microscopy
C1’sOptical System
Detector moduleDetector module
Laser moduleLaser module
Scan HeadScan Head
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
Laser 408nm 488nm 543nm
Detector BlueBlue GreenGreen RedRed
Compatible Lasers, Wavelengths, and DyesCompatible Lasers, Wavelengths, and Dyes
Basic concepts of microscopy
Scan Head
Detector
3 Laser Unit
PC System
C1 on Eclipse 90i with 3 lasersC1 on Eclipse 90i with 3 lasers
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: HeLa cells with Fluo4 loadedObjective: CFI Apo TIRF 100_ oil, NA 1.49
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
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
Basic concepts of microscopy
EDF: Extended Depth of FocusEDF: Extended Depth of Focus
Basic concepts of microscopy
Grey scale threshold Grey scale threshold
Basic concepts of microscopy
Particles:
Area, Diameter
Size Distribution Size Distribution
Basic concepts of microscopy
Evolution VF - cooled & uncooled- mono & color
Features
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 FireWire™ digital interface allows ease of use
Basic concepts of microscopy
Evolution MP™ Integrated Color Camera Kit
High resolution Color Camera (Sony 282 Sensor)
Features Fully integrated with your
choice of Image-Pro software FireWire (IEEE 1394)
C-mount camera Max 25 fps at 640x480 (30-bit)
Real Time Video (RTV) Option for cooling No interpolation or Pixel shifting Laptop kit
Basic concepts of microscopy
Evolution QEi™ Integrated Camera Kit
High Quantum Efficiency Camera
Features
Fully integrated with your choice of Image-Pro software
FireWire (IEEE 1394) C-mount camera
Fast FireWire Live 10 fps at 1360x1036(12-bit)
Basic concepts of microscopy
Thanks for your attention!