Basic Concepts of Micros

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
Outline

Upright microscopy
Inverted microscopy
Stereomicroscopy

Bright Field
Dark Field
Phase Contrast
Polarization
Fluorescence

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.)
Resolution
Resolving power, the limit up to which two

small objects are still seen separately.
Outline

Upright microscopy
Inverted microscopy
Stereomicroscopy

Bright Field
Dark Field
Phase Contrast
Polarization
Fluorescence
Upright Microscopy
Eclipse 80i-Epi
Transmitted Light path of Upright Microscopy

Eclipse 80i
Inverted Microscopy
TE2000U-Epi
Transmitted Light path of Inverted Microscopy

TE2000U
Stereomicroscopy
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
Outline

Upright microscopy
Inverted microscopy
Stereomicroscopy

Bright Field
Dark Field
Phase Contrast
Polarization
Fluorescence
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.
, 10X
Dark Field
Fine structures can often not be seen in front of a bright background.
Phase Contrast
Hoffman Modulation Contrast
D : dark, 1% transmittance
G : gray, 15% transmittance
B : bright, 100% transmittance
Increase visibility and

contrast in unstained and living
material by detecting optical
gradients (or slopes) and
converting them into variations
of light intensity.
Diatoms
Differential interference contrast (DIC)
Analyzer
Objective
Nomarski prism
Condenser
Nomarski prism
Polarizer
HeLa Cell Culture
Heliozoans (Actinophrys sol)

Polarization
Analyzer
Polarizer
Glutaric Acid Crystallites
Dinosaur Bone

The Principle of Fluorescence and
Configuration of Fluorescence
Accessories
The Principle of Fluorescence

40X
Stoke shift
1. Quartz Glass bulb

2. Cathode
3. Anode
C
3
C
B
3
2
Specimen
A
1. Light from HBO Lamp

2. Monochromatic Light
3. Fluorescence Light returning from
the Specimen
Bandpass emission filter
Longpass emission filter
Reflected Light path of Inverted Microscopy

TE2000U
Reflected Light path of Upright Microscopy

Eclipse 80i
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
Detector
(PMT)
Widefield versus Point scanning
WideField
Confocal
Basic Concept of Laser Scanning Microscope

Microscope
Laser light source
Laser
focal plane
Objective lens
specimen
Scan Head
Confocal
pinhole Detector
Dichroic mirror
Light emitted from the focal plane

Light emitted from the out-of-focus region
Detector
(PMT)
Detector module
Scan Head
Laser module
C1s
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,

DsRed, Alexa 546, Alexa 568,
BOBO-3, Calcium Orange, DiI,
Mitotracker Orange, DS Red
Laser
408nm
488nm
543nm
Detector
Blue
Green
Red
C1 on Eclipse 90i with 3 lasers
Scan Head
3 Laser Unit
Detector
PC System
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 loaded
Objective: CFI Apo TIRF 100_ oil, NA 1.49
Outline

Upright microscopy
Inverted microscopy
Stereomicroscopy

Bright Field
Dark Field
Phase Contrast
Polarization
Fluorescence
Image-Pro Family Software Series
The Ultimate Imaging Package,

Includes Support for Macros and
Plug-Ins
Perfect for Basic Imaging or

Capture Stations
EDF: Extended Depth of Focus
Grey scale threshold
Size Distribution
Particles:
Area,
Diameter
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 Micros

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Basic Concepts of

Basic concepts of microscopy

The Concept of Magnification

Basic Introduction of Microscopy

Transmitted and Reflected Techniques in Light Microscopy

Image Analysis Software & Digital Camera system

Basic concepts of microscopy

The Concept of Magnification

Basic Introduction of Microscopy

Transmitted and Reflected Techniques in Light Microscopy

Image Analysis Software & Digital Camera system

Basic concepts of microscopy

The Concept of Magnification

Microscope = M Objective X M Eyepiece X M Intermediate Factor

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

Resolving power, the limit up to which two

The characteristics of objectives

Basic concepts of microscopy

The Concept of Magnification

Basic Introduction of Microscopy

Transmitted and Reflected Techniques in Light Microscopy

Image Analysis Software & Digital Camera system

Basic concepts of microscopy

Basic concepts of microscopy

Basic concepts of microscopy

Transmitted Light path of Upright Microscopy

Basic concepts of microscopy

Basic concepts of microscopy

Basic concepts of microscopy

Transmitted Light path of Inverted Microscopy

Basic concepts of microscopy

Basic concepts of microscopy

Basic concepts of microscopy

Differences between light and stereomicroscopy

- Including a small angle ( Greenough Concept )

Light Microscopy : ( Upright / Inverted )

- Infinity Optics Design

The Concept of Magnification

Basic Introduction of Microscopy

Transmitted and Reflected Techniques in Light Microscopy

Image Analysis Software & Digital Camera system

Basic concepts of microscopy

Basic concepts of microscopy

Basic concepts of microscopy

Basic concepts of microscopy

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

Basic concepts of microscopy

Basic concepts of microscopy

Hoffman Modulation Contrast

Basic concepts of microscopy

Increase visibility and

Basic concepts of microscopy

Differential interference contrast (DIC)

Basic concepts of microscopy

HeLa Cell Culture