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

- 1 -

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.

- 2 -

HCB 1019 Cell Biology Trimester 1, 2012/13

Disscsion :

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

- 3 -

HCB 1019 Cell Biology Trimester 1, 2012/13

distinguishing two objects as separate if they are 0.16 um or greater apart. If the 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.

- 4 -

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.comhttp://answers.yahoo.comhttp://intranet.landmark.edu

- 5 -