Spotlight
¾ Basic Introduction of Light Microscopy
光學顯微鏡與螢光顯微鏡基本原理
¾ What is the difference between the light microscope and the magnifier?
¾ Two kinds of beam path: reflected light and transmission light
The Basic Concept of Light Microscope
¾ Transmitted Techniques in Light Microscope
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台灣儀器行 許紹君
Bright Field
Dark Field
Phase Contrast
Polarization
Difference Interference Contrast
¾ Reflected Techniques in Light Microscope
ƒ What is ‘crosstalk’?
ƒ How to resolve the ‘crosstalk’ problem?
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The basic concept of light microscope
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The incident angle determines the size that we see
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All the magnifier are designed for extending the incident angle
¾Microscopy means seeing a large image of something small.
•A, microscope beam path
•B, see the object directly from a distance of approx. 25 cm.
•1, object
•2, objective (image projected at infinity)
•3, tube lens (produce a magnified intermediate image)
•4, intermediate image
•5, eyepiece
•6, eye
¾ICS principle (Infinity Color-corrected System)
•The objective projects an image at an ‘infinite’ distance, the tube
lens with its fixed focal length then forms the intermediate image
from these parallel beams. Let us assume that nothing decisive for
the image formation happens in the space between objective and
tube lens. The light rays coming from the focused specimen plane
are parallel in this space anyway.
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The basic concept of light microscope
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The incident angle is magnified by lens.
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The incident angle is magnified by lens.
¾Microscopy means seeing a large image of something small.
¾Microscopy means seeing a large image of something small.
•A, microscope beam path
•A, microscope beam path
•B, see the object directly from a distance of approx. 25 cm.
•B, see the object directly from a distance of approx. 25 cm.
•1, object
•1, object
•2, objective (image projected at infinity)
•2, objective (image projected at infinity)
•3, tube lens (produce a magnified intermediate image)
•3, tube lens (produce a magnified intermediate image)
•4, intermediate image
•4, intermediate image
•5, eyepiece
•5, eyepiece
•6, eye
•6, eye
¾ICS principle (Infinity Color-corrected System)
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The basic concept of light microscope
¾ICS principle (Infinity Color-corrected System)
•The objective projects an image at an ‘infinite’ distance, the tube
•The objective projects an image at an ‘infinite’ distance, the tube
lens with its fixed focal length then forms the intermediate image
lens with its fixed focal length then forms the intermediate image
from these parallel beams. Let us assume that nothing decisive for
from these parallel beams. The light rays coming from the focused
the image formation happens in the space between objective and
specimen plane are parallel in this space anyway. It makes it
tube lens. The light rays coming from the focused specimen plane
possible to project intermediate images in the same Z position from
are parallel in this space anyway.
different objectives.
The basic concept of light microscope
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The basic concept of light microscope
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1
Besides the multi-lens, there are still other parts important
for the resolution.
What does ‘resolution’ actually mean?
¾A bright disk with shapely defined edges, but as a slightly
blurred spot surrounded by diffraction rings, called ’Airy
disks’ are formed from a spot light through the lens.
¾The light incident from the objects is deflected from the
original direction.
¾To obtain sharp images of small structures, the objective in
¾The resolving power, the limit up to which two small
objects are still seen separately.
the microscope must collect as much of this diffracted light as
possible. This works particularly well if the objective covers a
large solid angle. The term aperture (opening) describing this
property.
¾Numerical aperture: a measure of the solid angle covered
by an objective.
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Plan-Apochromat
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Plan-Neofluar
N.A.=1.518* sin 67.3°=1.4
N.A.=1.518* sin 59°=1.3
Plan-Neofluor
-an universal objective
-perfectly designed for general fluorescence
microscope
-chromatic correction from 435 nm to 670nm
-transmission starts at 365 nm
-brilliant view of field up to 25mm
-made by low auto- fluorescence glass type
Plan-Apochromat
-Top of the objectives
-Perfect image flatness for fields up to 25mm
-Oil immersion objectives of highest numerical
apertures
-With the chromatic correction from 420 to 670 nm
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The basic concept of light microscope
The basic concept of light microscope
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C-Apochromat
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The basic concept of light microscope
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The way of increasing the optical resolving power
¾Choose a large angle of the ray cone on the illumination side.
N.A.=1.33* sin 64.5°=1.2
¾To use immersion liquids between the front lens of the objective and the cover slip.
The aperture of the objective and the
resolving power would be reduced by the
reflection.
C-Apochromat
-special water immersion objectives
-using water immersion is the closest match to the
refractive index of biological tissue and popular
embedding media
-with chromatic range from 360~700nm, therefore
they work especially well for extended Z-scans in
biological tissue and for spectral imaging with the
MEAT detector.
-compensation of different temperature (21°～37°)
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The basic concept of light microscope
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Condenser
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The basic concept of light microscope
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Besides the multi-lens, there are still other parts important
for the resolution.
The way of increasing the optical resolving power
¾Choose a large angle of the ray cone on the illumination side.
¾The light incident from the objects is deflected from the
¾To use immersion liquids between the front lens of the objective and the cover slip.
original direction.
¾To obtain sharp images of small structures, the objective in
The aperture of the objective and the
resolving power would be reduced by the
reflection.
the microscope must collect as much of this diffracted light as
possible. This works particularly well if the objective covers a
large solid angle. The term aperture (opening) describing this
property.
¾Numerical aperture: a measure of the solid angle covered
by an objective.
Condenser
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The basic concept of light microscope
Upright Light Microscope
Illumination
equipment for
fluorescence
Eyepiece
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The basic concept of light microscope
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Inverted Light Microscope
Lamp for
fluorescence
Illuminator
Luminous-field and
aperture diaphragm
for fluorescence
Eyepiece
Transmitted
Condenser with
aperture diaphragm
Binocular tube
Specimen stage
Binocular tube
Filter turret
Specimen stage
Nosepiece
Objective
Transmitted
Condenser with
aperture diaphragm
Objective
Nosepiece
Light Control
Luminous-Field
Diaphragm
Lamp for
fluorescence
Light control of
transmitted
illuminator
Illuminator
Stand
Stand
Filter turret
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The difference between the upright and inverted microscope
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The basic concept of light microscope
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The difference between the upright and inverted microscope
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The Right Climate for Live Cell Imaging
The difference between the upright and inverted microscope
The design of Carl Zeiss leaves all the control icons out of the incubation system. Brings users the
convenience about switch between electronic and PC control. The released space can be freely
used to arrange other instruments.
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The basic concept of light microscope
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The basic concept of light microscope
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Transmitted Light Path of Upright Microscope
Different Beam Path of Image Formation
Eyepiece
Binocular tube
Specimen stage
Nosepiece
Objective
Transmitted
Condenser with
aperture diaphragm
Light Control
Luminous-Field
Diaphragm
Illuminator
Stand
Reflected-light (Fluorescence)
Transmitted-light
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Transmitted Techniques in Light Microscopy
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Bright Field
¾ 明視野 Bright field – 有染色、較厚的樣本
¾ 暗視野 Dark field – 對比差、極小樣本
¾ 相位差 Phase contrast – 透明、無染色、對比差樣本
¾ 偏光 Polarization – 結晶體、澱粉、聚合物、有偏光反應之樣本
¾ 干涉位相差 Differential Interface Contrast – 對比差、表面形態
觀察
¾Bright Field is the most universal technique used in light microscope.
¾Usually used in samples with colorimetric staining or good contrast.
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4
Operation of light microscope
Dark Field
¾Fine structures can often not be seen in front of a bright background.
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Adjust parts of dark field on upright microscope
Dark Field
¾It is necessary for the objective aperture to be a smaller
than the inner aperture of the illuminating light cone.
Objective
¾If there is no sample, the image seen in the eyepieces
remains completely dark.
Sample
¾If objectives are in the objective plane, light is laterally
diffracted away from the straight path.
Condenser optics
¾The object becomes brightly visible in front of a dark
background.
Annular stop
¾Objectives with an integrated variable iris diaphragm are
available to shutter out the indirect light even if it falls into
the aperture cone of the objective. This permits the use of
very high apertures for darkfield.
Variable iris
diaphragm
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The basic concept of light microscope
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Phase contrast
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Phase Contrast
¾Phase contrast is ideal for thin unstained
objects, for example culture cells on glass,
which are approx. 5 bis 10 um “thick”
above the cell nucleus, but less than 1um
“thick” at the periphery, and which barely
exhibit any light absorption in the visible
part of the spectrum.
¾The eye can scarcely see them in bright
field and dark field. However, very small
differences exist between the refractive
indices of the cells and the surrounding
aqueous solutions and within the cells
between the cytoplasm and the cell
nucleus.
¾The higher the refractive index of a medium, the
smaller the speed or velocity of light in the medium.
¾It translates the tiny differences into differences in
intensity.
New path
Phase ring
Phase stop
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Adjust parts of phase contrast on upright microscope
The characteristics of objectives
PH1
PH1
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Adjust parts of Polarization on upright microscope
Polarization
¾The polarizer privileges light source with specific direction of
vibration.
¾The analyzer arranged at an angle of 90° to the polarizer is
located behind the objective.
¾If no specimen is on the microscope stage, the image will
remain completely dark.
¾When illuminated, some specimens, such as starch , minerals
and polymers ,turn the vibration direction of the polarized light
out of the plane produced by the polarizer.
Analyzer
Polarizer
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Adjust parts of DIC on upright microscope
Differential Interference Contrast (DIC)
7. Analyzer
6. DIC prism (slider
behind the objective)
2. Condenser prism
1. Polarizer
7. Analyzer
DIC
6. DIC prism (slider
behind the objective)
Decomposition and
laterally shift the partial
light beams
2. Condenser prism
1. Polarizer
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Transmitted beam path on inverted light microscope
Plan-Neofluar
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The basic concept of light microscope
The basic concept of light microscope
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Adjust parts of phase contrast on inverted microscope
Adjust parts of dark field on inverted microscope
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Adjust parts of Polarization on inverted microscope
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Adjust parts of DIC on inverted microscope
7. Analyzer
6. DIC prism (slider
in the objective)
2. Condenser prism
1. Polarizer
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The reflected beam path on light microscope
The Principle of Fluorescence
Excited state
Ground state
40X
Absorption and fluorescence emission spectra of a
protein conjugate labeled with fluorescein-5isothiocyanate in pH 8.0 buffer.
(www.molecularprobes.com)
Stoke shift
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The excitation of conventional light microscope
The principle of fluorescence
1. Quartz Glass bulb
2. Cathode
3. Anode
4. Burning Chamber contains some
Mercury
5. Light Arc
zEmission intensity depends on excitation efficiency
zThe more efficient excitation induces the stronger signal of emitted light.
zHow to get the best fluorescence image?
zExcite samples with the appropriate excitation wavelength.
zDetect the strong and pure signals.
zEliminate the signals from the out-of-focus plane.
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How to get the best fluorescence image?
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The basic concept of light microscope
How to get the best fluorescence image?
¾Excite samples with the appropriate excitation wavelength.
¾ Excite samples with the appropriate excitation wavelength.
¾Detect the strong and pure signals.
¾ Detect the strong and pure signals.
¾Eliminate the signals from the out-of-focus plane.
¾ Eliminate the signals from the out-of-focus plane.
The spectra of FITC
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The emission spectra of FITC
The basic concept of light microscope
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The emission spectra of FITC and
other fluorophore
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Conventional microscope use wavelength filters to segment
the excitation light and emission light
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The basic concept of light microscope
Conventional microscope use wavelength filters to segment
the excitation light and emission light
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The filters used in conventional light microscope
Types of the filter
Ex BP 450-490
BS FT 510
EM LP 515
1. Light from HBO Lamp
2. Monochromatic Light
3. Fluorescence Light returning from the Specimen
A. Excitation Filter
B. Dichroic Beam Splitter
C. Emission Filter
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Understanding the spectra of filters
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Band pass Filter of Excitation and Emission
A. Excitation filter / B. Dichroic beam splitter / C. Emission filter
The conventional fluorescence microscope identifies the true signal by these filters.
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Adjust parts of fluorescence on upright microscope
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The basic concept of light microscope
Then you will get fluorescence images
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The crosstalk problem
The basic concept of light microscope
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The crosstalk problem
FITC / Rhod
FITC / Rhod
FITC
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FITC
The basic concept of light microscope
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The solution of crosstalk in conventional fluorescence
microscope -Multichannel unmixing
The crosstalk problem
FITC / Rhod
Raw CFP image
Raw GFP image
But In fact
Universal localized CFP
that accumulated at
nusleoli
Merged image composed by raw
images
GFP coupled with a
histone protein that
localized in the entire
nucleus and a lesser
degree of nucleoli.
FITC
Several signal in the GFP channel is bleeded from CFP dye.
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The steps of multichannel unmixing
Result of Multichannel unmixing
1. Prepare the CFP-stained sample and GFP-stained sample as reference
and double stained sample as the processed image.
2. Get the image through the filters, respectively.
GFP only sample
Double stained sample
CFP channel
CFP only sample
GFP channel
Raw CFP image
Universal licalized CFP that
accumulated at nusleoli
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Merged image composed by raw
images
Raw GFP image
GFP coupled with a histone
protein that localized in the entire
nucleus and a lesser degree of
nucleoli
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Merged image composed by unmixing
images
The basic concept of light microscope
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