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

Hemlock Leaf

, 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
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
Objective
Nomarski prism
Condenser
Nomarski prism
HeLa Cell Culture
Heliozoans (Actinophrys sol)
Polarization
Analyzer
Polarizer
Dinosaur Bone Glutaric Acid Crystallites

The Principle of Fluorescence and Configuration of
Fluorescence Accessories

The Principle of Fluorescence
40X
Stoke shift
1. Quartz Glass bulb
2. Cathode
3. Anode

3 2 1
1. Light from HBO Lamp
2. Monochromatic Light
3. Fluorescence Light returning from
the Specimen

1
2
2
3
3
3
A
B
C
A
B
C
Specimen
Bandpass emission filter Longpass emission filter
Reflected Light path of Inverted Microscopy

TE2000U
Reflected Light path of Upright Microscopy

Eclipse 80i
Light emitted from the focal plane
Light emitted from the out-of-focus region
Objective lens
Dichroic mirror
Detector
(PMT)
Confocal
pinhole
specimen
focal plane
Laser light source
WideField Confocal
Widefield versus Point scanning
Light emitted from the focal plane
Light emitted from the out-of-focus region
Objective lens
Dichroic mirror
Detector
(PMT)
Confocal
pinhole
specimen
focal plane
Laser light source
Basic Concept of Laser Scanning Microscope
Microscope
Scan Head
Laser
Detector
C1s
Optical System
Detector module
Laser module
Scan 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 Blue Green Red
Compatible Lasers, Wavelengths, and Dyes
Scan Head
Detector
3 Laser Unit
PC System
C1 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 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
Perfect for Basic Imaging or
Capture Stations
The Ultimate Imaging Package,
Includes Support for Macros and
Plug-Ins

EDF: Extended Depth of Focus
Grey scale threshold
Particles:
Area,
Diameter
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!

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Basic concepts of microscopy

Basic Concepts of Microscopy

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