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


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


The characteristics of objectives


Numerical Aperture (N.A.)Numerical Aperture (N.A.)


Resolution

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


Upright MicroscopyUpright 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


Transmitted Light path of Upright MicroscopyTransmitted Light path of Upright Microscopy

Eclipse 80iEclipse 80i


Inverted MicroscopyInverted 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


Transmitted Light path of Inverted MicroscopyTransmitted Light path of Inverted Microscopy

TE2000UTE2000U


StereomicroscopyStereomicroscopy


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


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


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


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


Dark Field

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


Phase Contrast


Hoffman Modulation Contrast

D : dark, 1% transmittanceG : gray, 15% transmittanceB : bright, 100% transmittance


Diatoms

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


Differential interference contrast (DIC)

Analyzer

Polarizer

ObjectiveNomarski prism

CondenserNomarski prism


HeLa Cell Culture

Heliozoans (Actinophrys sol)


Polarization

Analyzer

Polarizer


Dinosaur Bone Glutaric Acid Crystallites


螢光原理與配件解說

The Principle of Fluorescence and Configuration of Fluorescence Accessories


The Principle of Fluorescence

40X

Stoke shift


1. Quartz Glass bulb

2. Cathode

3. Anode

321


1. Light from HBO Lamp2. Monochromatic Light3. Fluorescence Light returning from

the Specimen

12

23

3

3

A

B

C

A

B

C

Specimen


Bandpass emission filter Longpass emission filter


Reflected Light path of Inverted Microscopy

TE2000UTE2000U


Reflected Light path of Upright Microscopy

Eclipse 80iEclipse 80i


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)


WideField Confocal

Widefield versus Point scanningWidefield versus Point scanning


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


C1’sOptical System

Detector moduleDetector module

Laser moduleLaser module

Scan HeadScan Head


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


Scan Head

Detector

3 Laser Unit

PC System

C1 on Eclipse 90i with 3 lasersC1 on Eclipse 90i with 3 lasers


Total Internal Reflection Fluorescence (TIRF)


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


Image-Pro Family Software Series

• Perfect for Basic Imaging or Capture Stations

• The Ultimate Imaging Package, Includes Support for Macros and Plug-Ins


EDF: Extended Depth of FocusEDF: Extended Depth of Focus


Grey scale threshold Grey scale threshold


Particles:

Area, Diameter

Size Distribution Size Distribution


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


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


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)


Thanks for your attention!

basic concepts of microscopy basic concepts of microscopy

Documents

tools light microscopy

x slide

objective x

magnification example

eyepiece x

x eyepiece

inverted single objective

characteristics of objectives