principle of optical microscopy

8/7/2019 principle of optical microscopy

1/24

HKUSTHKUST

Introduction to Optical MicroscopeIntroduction to Optical Microscope


2/24

HKUSTHKUST



3/24

HKUSTHKUST


Casting microstructure of Ni-Cr alloy Microstructure of powder metallurgy Al alloy


4/24HKUSTHKUST


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


5/24HKUSTHKUST


Stereo Optical Microscope:Stereo Optical Microscope:


6/24HKUSTHKUST


Compound Optical MicroscopeCompound Optical Microscope


7/24

HKUSTHKUST


Optical Principles of MicroscopeOptical Principles of Microscope

Image formation in the transmission illuminationImage formation in the transmission illumination


8/24

HKUSTHKUST


Optical Principles of MicroscopeOptical Principles of Microscope

Image formation in the reflected illuminationImage formation in the reflected illumination


9/24

HKUSTHKUST


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


10/24

HKUSTHKUST


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!


11/24

HKUSTHKUST


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.


12/24

HKUSTHKUST


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.


13/24

HKUSTHKUST


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.


14/24

HKUSTHKUST


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.


15/24

HKUSTHKUST


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)


16/24

HKUSTHKUST


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.


17/24

HKUSTHKUST


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.


18/24

HKUSTHKUST


Depth of fieldDepth of fieldis the area in front of and behind the specimenthat will be in acceptable focus.


19/24

HKUSTHKUST


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..


20/24

HKUSTHKUST


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


21/24

HKUSTHKUST


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.)


22/24

HKUSTHKUST


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.


23/24

HKUSTHKUST


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!


24/24

HKUSTHKUST


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

principle of optical microscopy

Documents