cell biology tutorial

1. HCB 1019 Cell Biology Trimester 1, 2012/13
Osama Barayan - 1091105869
HCB 1019 – CELL BIOLOGY
LECTURE : TAN CHAI HONG
Practical 5
Basic light Microscopy Technique
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2. HCB 1019 Cell Biology Trimester 1, 2012/13
Practical 5:
Basic Light Microscopy Techniques
Introduction:
Because biological objects can be very small, a microscope is often
used to view them. We have two kinds of light microscopes available
in the lab. Light microscopes use light rays that are magnified and
focused by means of lenses. The binocular dissecting microscope is
designed to study entire objects in three dimensions at low
magnification. The compound light microscope is used for examining
small or thinly sliced sections of objects under magnification that is
higher than that of the dissecting light microscope. Illumination is
from below, and the light passes through clear sections but does not
pass through opaque sections. To improve contrast, the microscopic
uses stains or dyes that bind to cellular structures and absorb light.
Objective:
• To learn the general techniques on using microscope
Methods:
Setting up Kohler Illumination
1. Place slide on the stage and bring to focus. If the microscope is way out of
alignment, this may require roughly aligning the illumination by centering the
condenser, opening the condenser aperture diaphragm, opening the field
diaphragm and focusing the condenser to give initial reasonable illumination so
you can see well enough to focus on the specimen.
2. Stop down the field diaphragm. This is the diaphragm in the fixed part of the
microscope, the part that bears a constant distance from the lamp source.
3. Focus the condenser. Turn the focus knob on the condenser until the (edge) image
of the field diaphragm is sharp – there may be red fringe on one side of focus and
blue on the other: go for the center between these.
4. Open the field diaphragm to the edge of the field and center using the condenser
centering controls
5. Adjust the condenser aperture diaphragm. This is the diaphragm that is located in
the condenser unit that can be focused up and down. Two possibilities: either
from fully open stops down until the image flares, if any just disappears, or
remove an ocular and look at the back of the objective. Close the CAD control
until diaphragm reduces the aperture to about 2/3 of full open.
Note: for critical work, it is necessary to touch up the alignment if objectives are
switched or even for large variations in focus plane in the sample.
Disscsion :
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3. HCB 1019 Cell Biology Trimester 1, 2012/13
A beginner tends to think that the challenge of viewing small objects lies in
getting enough magnification. In fact, when it comes to looking at living things
the biggest challenges are, in order,
• obtaining sufficient contrast
• finding the focal plane
• obtaining good resolution
• recognizing the subject when one sees it
HOW TO SET UP A MICROSCOPE FOR TRANSMITTED LIGHT ILLUMINATION
1. Turn on the light source and swing in a 10x objective.
2. Place a well-stained slide on the stage, checking that the coverslip is uppermost.
3. Decrease the size of the illuminating field diaphragm (IFD), sometimes called the
field iris, located nearest the light source so that you can see its edges.
4. When the edge of the field diaphragm silhouette is sharply defined, centre it with
the two knobs (usually knurled knobs) coming out diagonally from the condenser.
5. The correct height of the condenser has now been set.
The primary advantage of Kohler illumination is the extremely even illumination
of the sample. This reduces image artifacts and provides high sample contrast.
Even illumination of the sample is also critical for advanced illumination
techniques such as phase contrast and differential interference
contrast microscopy. What should be done whenever you are finished using a
microscope is Remove the slide ,return the scope to low powered and ,turn off the
light. How to prevent fungal growth m Store the microscope every night in a
cabinet fitted with an electric bulb (5 W or 40 W). The bulb should be preferably
fitted on the top of the cabinet so that it is near the tube (head of the microscope).
Keep the bulb switched on overnight. If this technique is used, the cabinet should
have holes for ventilation so that air flows freely. If an objectives magnifies 40X
and each binocular lens magnifies 15X, the total magnification of the object being
viewed is 40x15 = 600x. In this experiment we are going to use oil when we
magnify a very small structures , the reason we use the oil is that the structures
always when they being magnified they don't appear clear caused The refractive
index of the index-matching material is higher than air allowing the objective lens
to have a larger numerical aperture (greater than 1) so that the light is transmitted
from the specimen to the outer face of the objective lens with minimal refraction .
Usually the dry objective lenses loss of resolution prevent using magnification of
above 400x. When the condenser is in perfect focus it has the numerical aperture
of the objective. The denominator of the Resolving Distance equation is really the
numerical aperture of the objective plus the numerical aperture of the condenser.
If one assumes that "average blue light" is being used (wavelength = 400 nm),
using an oil lens of numerical aperture of 1.25 will allow you to resolve distinctly
objects as small as the resolving distance: R.D. = 400 nm/(2 x 1.25) = 160 nm or
0.16 μm This means that under the most ideal conditions, this lens is capable of
distinguishing two objects as separate if they are 0.16 um or greater apart. If the
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4. HCB 1019 Cell Biology Trimester 1, 2012/13
two objects are less than 0.16 μm apart, say 0.10 μm, then they will be blurred
together at the point where they are 0.10 μm apart.
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5. HCB 1019 Cell Biology Trimester 1, 2012/13
1. Ocular Lens : is what you look through at the top of the microscope.
2. Objectives: are the primary optical lenses on a microscope.
3. Stage : is where the specimen to be viewed is placed.
4. Stage Clips : are used when there is no mechanical stage.
5. Base : is the base of the microscope
6. Diaphragm : controls the amount of light reaching the specimen.
Reference:
http://wiki.answers.com
http://answers.yahoo.com
http://intranet.landmark.edu
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6. HCB 1019 Cell Biology Trimester 1, 2012/13
1. Ocular Lens : is what you look through at the top of the microscope.
2. Objectives: are the primary optical lenses on a microscope.
3. Stage : is where the specimen to be viewed is placed.
4. Stage Clips : are used when there is no mechanical stage.
5. Base : is the base of the microscope
6. Diaphragm : controls the amount of light reaching the specimen.
Reference:
http://wiki.answers.com
http://answers.yahoo.com
http://intranet.landmark.edu
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