principle of optical microscopy
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Transcript of principle of optical microscopy
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8/7/2019 principle of optical microscopy
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HKUSTHKUST
Introduction to Optical MicroscopeIntroduction to Optical Microscope
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8/7/2019 principle of optical microscopy
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HKUSTHKUST
Introduction to Optical MicroscopeIntroduction to Optical Microscope
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8/7/2019 principle of optical microscopy
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HKUSTHKUST
Introduction to Optical MicroscopeIntroduction to Optical Microscope
Casting microstructure of Ni-Cr alloy Microstructure of powder metallurgy Al alloy
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8/7/2019 principle of optical microscopy
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Types of Optical Microscopes:Types of Optical Microscopes:
(1) Simple OM: One lens; 25x; 10 m resolution
(2) Stereo OM: Two lens trains; 6-8x;
(3) Compound OM: Objective + eyepiece + condenser lenses
1300x; 1 m resolution
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Stereo Optical Microscope:Stereo Optical Microscope:
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Compound Optical MicroscopeCompound Optical Microscope
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HKUSTHKUST
Introduction to Optical MicroscopeIntroduction to Optical Microscope
Optical Principles of MicroscopeOptical Principles of Microscope
Image formation in the transmission illuminationImage formation in the transmission illumination
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8/7/2019 principle of optical microscopy
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Optical Principles of MicroscopeOptical Principles of Microscope
Image formation in the reflected illuminationImage formation in the reflected illumination
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Light source systemLight source system
Light source: light or electron
Condenser lens: the lens collects the light to direct it atthe small area of the object which is to be examined.
It makes the object brighter (better contrast) and enable
to control the angle at which the illumination reaches theobject.
The condenser lens can converge the light beam on object or
can illuminate it with parallel rays.
Condenser aperture: controls the area of specimen to be
illuminated
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
MagnificationMagnificationObjective lens: the lens provides an inverted image of object at B with magnification,
M1 = (v1f1)/f1
Projective lens: the lens gives a final upright image at a further magnification,M2 = (v2f2)/f2
Recall basic optics: image distance (v) and focal distance (f)
The total magnification:M = M1 x M2
The size of a microscope is more fixed (little variation of v), thus we usually changelens with different f to change magnification of microscope.
A higher magnification lens has a shorter focal distance!
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
ResolutionResolution
Is there any limitation of magnification in microscopes?
The effective magnification is limited by a microscopes resolution.
The resolution of microscope is controlled by diffraction effect of light.
Light must pass through a series of restricted openings in microscope.
Diffraction occurs while light passing limited openings.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
ResolutionResolution: the ability to discern fine details. It is represented by the
minimum distance between two points such that the two points are
perceived as separated image.
R.P.= 0.61 / N.A.
N.A.= sin
R.P.- Resolving PowerN.A. - Numerical Aperture
- wavelength of the light
- the half acceptance angleof the lens.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
N.A.N.A. - the numerical aperture (N.A.) is basically a value which describes thequality of a lens.
N.A. = sindepends on size of the lens; working distance; refractive index of mediumbetween object and objective lens ().
Effective Magnification of an objective = 1000 x N.A.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Resolution limit of light microscopeResolution limit of light microscope
can decrease to 400 nm (green light) sin is limited to ~1.6,thus R.P. = 0.61 / N.A. = 0.61 / sin = 0.61x400/1.6 = 152 nm
Thus, the maximum resolution is about 150 nm (0.15 m).
Resolution of electron microscopeResolution of electron microscope
can decrease to 0.001 nm sin is very small, because is unity and is about a few degrees. Thus,
0.61 R.P. =
For wavelength of 0.0037 nm and = 0.1 radians, the resolution isabout 0.02 nm.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Resolution limit of microscopeResolution limit of microscope:
: wavelength of light
: refractive index of medium between object and objective lens
: semi-angle of aperture
The product, sin, is called numerical aperture (NA).
sin
61.0=R.P.
To obtain the better resolution:
Decrease , (electron wavelength
is much less than light)
Increase and sin, (oil refractiveindex is higher than air)
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Steps for optimum resolutionSteps for optimum resolution
Use objective lens with highest NA.
Use high magnifications. Use eyepiece compatible with the chosen objective lens.
Use the shortest possible wavelength light.
Keep the light system properly centered.
Use oil immersion lenses if available.
Adjust field diaphragm for maximum contrast and aperture diaphragmfor maximum resolution and contrast.
Use dark-field and interference-contrast if additionalcontrast is required.
Adjust brightness for best resolution.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Effective magnificationEffective magnification
Resolution of microscopyxeffective magnification= Resolution of naked eye
Resolution of naked eyes is only about 0.2 mm.
The effective magnification of light microscope is about
Meff= 0.2/0.00015 = 1333
A higher magnification than the effective one only makesthe image bigger, but not providing more details of image.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Depth of fieldDepth of fieldis the area in front of and behind the specimenthat will be in acceptable focus.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Depth of fieldDepth of field
The range of position for the object in which our eye cannot detect change in
sharpness of the image.
The image is only accurately in focus when the object lies in the appropriate plane.
The image is out of focus when the object lies above or below this plane.
Since the diffraction effect is limited the resolution to r1 , it does not
make any difference to the sharpness of image if the object is anywhere
within the range, h..
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
The expression of depth of field is given as
Note: The large depth of field and high resolution cannot beobtained simultaneously.
Because Large h large r1 (worse resolution)
For a light microscope, is about 45 degree. Thus, the depth
of field is not much different from resolution.
For electron microscope, is very small, also the wavelength
The depth of field reaches about 10 times of resolution.
tansin
22.1
tan
1 ==d
h
2
61.0
=h
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Depth of Field
Improved Depth of Field by reducing Objective N.A.
Effect of Focal Length on the Depth of Field
*Wavelength is also a factor (Change N.A.)
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Steps to improve depth of fieldSteps to improve depth of field
Reduce NA by closing down aperture diaphragm, or use a lower
NA objective lens.
Lower the magnification for a given NA.
Use a high-power eyepiece with a low-power, high-NA objective lens.
Reduce zoom factor.
Use the longest possible wavelength light.
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
Depth of focus
The range of image plane position at which the image
can be viewed without appearing out of focus for a fixed
position of object.
This is often confused with depth of field. This is not so important as depth of field.Depth of focus is M2 times of depth of field!
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Introduction to Optical MicroscopeIntroduction to Optical Microscope
ContrastContrastis number of shades found in an image. A high contrastpicture will have only two shades, black and white. The more
shades you have, the less contrast, but more information.
ColorColoris also considered a form of contrast.
Absorption contrast
Diffraction contrast
Interference contrast