Download Optical microscopy laboratory practice 2012

Optical microscopy laboratory practice
2012
Construction of the microscope
The main parts of the microscopes are similar, but there are application dependent parts too.
The biological microscope (Fig.1.) illuminates the transparent subject through the condenser
under the stage. Since the metal samples are not transparent, the metallurgical microscope
illuminates the subject from above, parallel to the viewing direction.
1 eyepiece lens
2 binocular head
3 revolving
objective lenses
4 filter support
5 truss
6 subject-table
7 condenser
height
adjustment
8 condenser
9 aperture
adjustment
10 condenser
centralizer
11 subject
movement
(x-y direction)
13 illumination
aperture
14 and 15 rough
and fine contrast
adjustment
16 lamp housing
Fig.1. The main parts of the biological microscopes [4]
In Fig. 1. the binocular head (2) shows the same picture for both eyes: it is for comfort viewing,
and it gives no stereo picture. The interpupillary distance of the eyes can be adjusted via rail
mechanism.
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Construction of the metallurgical microscope
Metallurgical samples, and other not-transparent subject can only be observed with the aid of
reflected light. Since the metallurgical microscope illuminates the subject from above, parallel
to the viewing direction, the examination light is reflected to the sample via mirrors. Most cases
the illuminating mirrors are integrated into the objective lens. The main construction of the
metallurgical microscope can be observes on Fig.2.: it can based on bright-field or dark-field
illumination principle. Our Zeiss metallurgical microscope (Fig 4.) uses dark-field illumination.
Fig 2. bright-field (left) or dark-field (right) illumination principle. [3]
Microscope Parts and Functions [7]
1. Eyepiece: The eyepiece (sometimes called the 'ocular') is the lens of the microscope closest to
the eye that you look through. It is half of the magnification equation (eyepiece power
multiplied by objective power equals magnification), and magnifies the image made by the
objective lens... sometimes called the virtual image. Eyepieces come in many different powers.
One can identify which power any given eyepiece is by the inscription on the eyecup of the
lens, such as "5x", "10x", or "15X". Oculars are also designed with different angles of view; the
most common is the wide field (W.F.).
2. Eyepiece Holder: This simply connects the eyepiece to the microscope body, usually with a setscrew to allow the user to easily change the eyepiece to vary magnifying power.
3. Body: The main structural support of the microscope which connects the lens apparatus to the
base.
4. Nose Piece: This connects the objective lens to the microscope body. With a turret, or rotating
nose piece as many as five objectives can be attached to create different powers of
magnification when rotated into position and used with the existing eyepiece.
5. Objective: The lens closest to the object being viewed which creates a magnified image in an
area called the "primary image plane". This is the other half of the microscope magnification
equation (eyepiece power times objective power equals magnification). Objective lenses have
many designs and qualities which differ with each manufacturer. Usually inscribed on the
barrel of the objective lens is the magnification power and the numerical aperture (a measure of
the limit of resolution of the lens).
6. Focusing Mechanism: Adjustment knobs to allow coarse or fine (hundredths of a millimeter)
variations in the focusing of the stage or objective lens of the microscope.
7. Stage: The platform on which the prepared slide or object to be viewed is placed. A slide is
usually held in place by spring-loaded metal stage clips. More sophisticated high-powered
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microscopes have mechanical stages which allow the viewer to smoothly move the stage along
the X (horizontal path) and Y (vertical path) axis. A mechanical stage is a must for high-power
observing.
8. Illumination Source: The means employed to light the object to be viewed. The simplest is the
illuminating mirror which reflects an ambient light source to light the object. Many
microscopes have an electrical light source for easier and more consistent lighting. Generally
electrical light sources are either tungsten or fluorescent, the fluorescent being preferred
because it operates at a cooler temperature. Most microscopes illuminate from underneath,
through the object, to the objective lens. On the other hand, stereo microscopes use both top
and bottom illumination.
9. Base: The bottom or stand upon which the entire microscope rests or is connected.
10. Photography unit with CMOS or CCD sensor able to make pictures via microscope.
Stage
Camera
Eyepiece
Illumination
Source
Objective
Illumination
Source
Focusing Mechanism
Objective
Stage
Focusing Mechanism
Fig. 3. Zeiss metallurgical microscope [3]
Fig 4.. Inverse-system metallurgical
microscope [3]
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Microscope Objective Specifications [8]
Identification of the properties of individual objectives is usually very easy because important
parameters are often inscribed on the outer housing (or barrel) of the objective itself as
illustrated in Figure 5.
Manufacturer - The name of the objective manufacturer is almost always included on the
objective.
Linear Magnification - In the case of the apochromatic objective in Figure 5, the linear
magnification is 60x, although the manufacturers produce objectives ranging in linear
magnification from 0.5x to 250x with many sizes
in between.
Optical Corrections - These are usually listed as
Ac Achromat (achromatic), Field curvature
corrections are abbreviated Plan, and as Apo
(apochromatic) for the highest degree of
correction for spherical and chromatic aberrations.
Numerical Aperture - This is a critical value that
indicates the light acceptance angle, which in turn
determines the light gathering power, the
resolving power, and depth of field of the
objective.
Fig 5. Objective Specifications [8]
Characterization of microscope picture [9]
Definition of Magnification in Microscopy
Microscope magnification is how large the object will appear compared to its actual size.
Finding the Overall Magnification
To find the overall magnification of the microscope, multiply the magnification of the eyepiece
by the magnification of the objective lens you are using. For example, if the magnification of
the eyepiece is 10x and the magnification of the objective lens is 40x, the overall magnification
is 400x.
What is Microscope Resolution?
Resolution refers to the ability of a microscope to distinguish two separate points. Magnifying
an object without good resolution will simply produce a large image of the object where details
cannot be identified.
What is Depth of Focus?
The depth of focus of an optical microscope is the range of image plane position at which the
image may be viewed without appearing out of focus for an object or specimen
The bigger the overall magnification, the lower the depth of focus.
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The numerical aperture of the objective lens
In most areas of optics, and especially in microscopy, the numerical aperture of an optical
system such as an objective lens is defined by
where n is the index of refraction of the medium in which the lens is, and θ is the half-angle of
the maximum cone of light that can enter or exit the lens.
There are several equations that have been derived to express the relationship between
numerical aperture, wavelength, and resolution:
Resolution (r) =
Where
/(2NA)
is the average wavelength of the illuminating light
Fig 6. Objectives with Low and High numerical
aperture
The table 1 describes the relationship between the NA and the resolution, and between the NA
end the limits of useful magnification.
NA
Resolution
Resolution
pairs / mm
0,04
0,12
0,25
0,5
0,65
0,75
0,95
1,3
1,4
6,9
2,3
1,1
0,55
0,42
0,37
0,29
0,21
0,19
145
436
910
1820
2380
2730
3450
4750
5090
Table 1
5
line Lower and Upper limits of
useful magnification
500*NA
1000*NA
20
40
60
120
125
250
250
500
325
650
375
750
475
950
650
1300
700
1400
Stereoscopic microscope A stereo microscope is an optical microscope fitted with two
sets of lenses, each positioned to view an object from a slightly different angle. The result is a
three-dimensional image. It is widely used in industry for quality control of small pieces, or for
manual SMD soldering.
Estimating the average grain size diameter as determine from a random
cross section.
The average grain size diameter (d) can be calculated by the following equation, where L0 is a
straight line crossed N grains :
d=
Lo
N
Monitoring questions
1.
2.
3.
4.
5.
6.
What are the main parts, and its functions of a metallurgical microscope?
Bright-field and dark-field illumination principle
Microscope Objective Specifications
Definition of the numerical aperture
Definition of the Overall Magnification, Resolution and Depth of focus
Estimating the average grain size diameter as determine from a random cross section.
Literature:
[1]. Bernolák-Szabó-Szilas: A mikroszkóp (zsebkönyv). 1979, Mőszaki Könyvkiadó,
Budapest
[2]. Lovas Béla: Mikroszkóp-mikrokozmosz. 1995. Gondolat Kiadó, Budapest
[3]. A korábbi laboratóriumi útmutató: Dr. Csiszár Sándor: Mikroszkóp Villamosipari
anyagismeret (Laboratóriumi gyakorlat)
[4]. Determann – Lepusch: Das Mikroskop und seine Anwendung Ernst Leitz Wetzlar
GmbH
[5]. www.zeiss.de/micro
[6]. http://micro.magnet.fsu.edu/
[7]. http://wiki.answers.com/Q/What_are_the_microscope%27s_parts_and_functions
[8]. http://www.microscopyu.com
[9]. www.ehow.com
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Report of the optical microscopy Made by:
…………………………………… ………
laboratory practice
Name
neptun code, group identification
Leader of laboratory practice
Date:
Note:
1. What are the main parts, and its functions of a metallurgical microscope?
[ ] Zeiss metallurgical microscope [ ] Inverse-system metallurgical microscope
2. Calculation of the Overall Magnification of the microscope using the data of a given
objective lens:
3. Measuring of the field of view using the given objective lens:
:
measuring instrument:
field of view diameter in millimeters:
4. Microscope Objective Specifications (don’t touch the surface of the lens!):
Data
Explanation
Manufacturer
Optical corrections
Magnification
Numerical aperture
Cover glass thickness
Infinity correction
Antireflex layer color
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5. Based on NA look out the resolution and the Lower and Upper limits of useful magnification
values from Table 1:
•
Resolution:
•
Lower and Upper limits of useful magnification:
6. What is the relation between the of the Overall Magnification (Point 2) and the Lower and
Upper limits of useful magnification.
7. Identify 3 different metallurgical cross sections:
8. Estimating the average grain size diameter as determine from a random cross section.
The horizontal measure of the field of view (millimeters):
How many grains are on the given horizontal line of the field of view :
Calculation of the average grain size diameter:
9. Measuring 2 holes on printed circuit board via image analysis software
10. Specimens inspected with Stereomicroscope:
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