REPORT: BASIC OF BIOLOGY PRACTICAL “The Procedure of Microscope”

BASIC OF BIOLOGY PRACTICAL REPORT

“The Procedure of Microscope”

By:

ZAKYAH

120220153086

STUDY PROGRAM OF BIOLOGY EDUCATION

FACULTY OF TEACHER TRAINING AND EDUCATION

UNIVERSITY OF JEMBER

2012

I.                   TITLE : THE PROSEDURE OF MICROSCOPE

II.                PURPOSE

1.      Introducting the components of the microscope andhow to use them

2.      Determining wide the field of view from the microscope

3.      Learn how to prepare materials that would be observed under microscope.

III.             BASIC TEORY

A microscope (from the Ancient Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see") is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope.

There are many types of microscopes, the most common and first to be invented is the optical microscope which uses light to image the sample. Other major types of microscopes are the electron microscope (both the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscope. (http://en.wikipedia.org/wiki/Microscope)

The microscopes first used by Renaissance scientists, as well as the microscopes you are likely to use in the laboratory, are all light microscopes.In a light microscope (LM), visible light is passed through the specimen and then through glass lenses. The lenses refract (bend) the light in such away that the image of the specimen is magnified as it is projected into the eye, onto photographic film or a digital sensor,or onto a video screen. (See the diagram of microscope structurein Appendix D.)

Two important parameters in microscopy are magnification and resolving power,or resolution. Magnification is the ratio of an object's image size to its real size. Resolution is a measure of the clarity of the image ; it is the minimum distance two points can be separated and still be distinguished as two points. For example, what appears to the unaided eye as one star in the sky may be resolved as twin stars with a telescope.

Just as the resolving power of the human eye is limited, the light microscope can not resolve detail finer than about 0.2 micrometer (mm), or 200 nanometers (nm), the size of a small bacterium, regardless of the magnification factor. This resolution is limited by the shortest wavelength of light used to illuminate the specimen. light microscopes can magnify effectively to about 1,000 times the actual size of the specimen ; at greater magnifications, additional details can not be seen cearly. A third important parameter in microscopy is contrast, which accentuates differ-ences in parts of the sample. In fact, most improvements in light microscopy in the last hundred years have involved new methods for enhancing contrast, such as staining or labeling cell compo-nents to stand out visually. (Campbell, 2000: 95)

The parts of a compound microscope

www.microbehunter.com

Here is a quick overview of the most important parts of a compound microscope (biological microscope) and their function.

The following list of terms can also be found in the glossary:

·       Condenser: This is a system of different lens elements which is mounted beneath the stage of the microscope. It contains an iris diaphragm which controls the diameter of the light beam. The light beam should be adjusted to be larger or equal to the numerical aperture of the objective in use. Condensers can be moved up and down. The normal operating position is up.

·       Base: This is the bottom part of the microscope, it contains the lamp.

·       Coarse Focus: Also referred to as rough focus, this knob raises and lowers the microscope stage quickly. It should only be used in connection with the low magnification lenses.

·       Eyepiece Lens: Also known as ocular lenses, they magnify the image of the objective. The eyepiece is the lens into which a person looks into when observing. The total magnification of a microscope is calculated by multiplying the magnification of the objective by the magnification of the eyepiece. Many eyepiece lenses have a magnification of 10x ot 15x.

·       Fine Focus: This focus knob moves the stage up and down in small steps. It is used to focus at different layers of the specimens.

·       Head: This is the top part of the microscope. It carries the eyepiece(s) and other optical elements. There are several different types of heads: a monocular head is designed to carry only one eyepiece, a binocular head carries two (but does not give stereoscopic vision in compound microscopes) and a trinocular head is designed to carry a camera as well.

·       Mechanical Stage: This type of stage is equipped with a slide holder and two knobs to turn. One knob moves the stage backwards and forwards, the other one moves the slide sideways.

·       Nosepiece (or revolving nosepiece, turret): This part carries the objectives. It can be rotated.

·       Objective Lens: This is a highly magnifying lens system, it is located close to the specimen to be observed. The image of the objective is then magnified again by the ocular lens which is close to the eye.

·       Stage: This is the flat surface on which the slides are placed on. It can be moved up and down for focusing.

·       Stage Clips: These are clips that hold the slide.

·       Trinocular Head: This microscope head has three exits, two for viewing (for binocular vision) and a third exit to connect a camera. Some microscopes also allow for taking photographs through a special adapter at the eyepiece, but a trinocular head offers more stability and is to be preferred for photographic work. (http://www.microbehunter.com/2008/12/31/parts-of-a-compound-microscope/)

The following procedures are to be carefully followed in any laboratory where a light microscope is used:

1.      Two hands should always be used when carying a microscope to or from a storage cabinet.

2.      Use lens paper to clean the ocular or objective lens of a microscope. If immersion oil is to be removed from the objectif lens, a small amount of alcohol may be utilized to expedite this cleaning.

3.      For maximum resolution, adjust the consender lens to the posisition closest to the stage, and set the iris diaphgram lever in the wideopen posisition. To incrase contrast, adjust the iris diaphgram toward the closed position. The use of an oil immersion lens also increase resolution by preventing light loss due to difraction between the objective lens and the speciment.

4.      The magnification of an object viewed through the microscope can be calculated by multiplying the power of the ocular by the power of the objective lens. For example, a 10x ocular used with an objective lens rated at 45x yields a total magnification of 450x.

5.      Always begin the microscopic observation of a slide using low power. Obtain a clearly focaused image and make any observations at this magnification that are possible. Than rotate the nosepiece and click into place the mediumpower objective lens. Your microscope id of parfocal design; your speciment should be in focus (or nearly so) if it was in focus under lower power. Minor adjustments may be made with the fine adjustment control knob.

6.      When the high-power or oil immersion objective lenses are in place, never adjust the focus using the coarse adjustment. This can cause severe damage to the objectives.

7.      If the image is lost while under high power, do not keep turning the fine adjustment, but return to low power and work your way back to a focused high-magnification image. Remember that adjustment of the iris diaphgram can help to procedure the proper contrast for the speciment being viewed.

8.      The oil immersion lens is quite useful for viewing material of small size and low contrast. Some fine adjustment may be necessary and be sure to open the iris diaphgram to its maximum aperture.

9.      Always take a few moments to check on your microscope before returning it to the storage cabinet. Clean off the ocular and objective lenses. Rotate the low-power objective into the clicked posisition. Adjust the mechanical stage so that it does not project too far on either side of the microscope. Wrap around and secure the power cord to the base of the microscope. Make sure the microscope is returned to the same berth in the cabinet where it was originally stored. (John S. Choimski, Jr., 1992 : 3)

Resolution and Magnification

The most important feature that limits the effecttiveness of a light microscope is the resolving power of the lens system. A basic definition of resolution is the ability of an instrument (or the eye) to discern distinict images of points close together. As resolution increases, this has the practical effect of allowing a microscopist to study cell structure with greater effectiveness. The limit of resolution (LR) may be quantified by Abbe’s equation:

The resolution is limited by   (lambda or the wavelenght of incoming light) and the NA or numeric aperture. Numeric aperture is a quality of the lens system (NA = n sin α ; n = refractive index of the medium, and sin α = sine of the semiangle of aperture). The NA of a very good lens never exceeds. Therefore, the LR can be expressed simply as a function of the wavelength of the incoming light:

 

The shortest wavelenght of visible light is monochromatic violet light where = 400 nm. So:

The resolution of the unaided eye is approximately 0.1 mm or 100 mm. Thus, a quality light microscope has the theoretical capability to increase resolution over 580x that of the naked eye. Unfortunately, many cell consistuents are closer together than 0.17 mm and meaningful viewing requires an instrument of greater resolution. The electron microscope (EM) serves this purpose with a theoretical LR of approximately 0.2 nm. This purpose is achieved because the “illumination” of the EM does not employ any wavelength of visible light, but instead utilizes an electron beam (  = 0.005 nm).

Resolution limits usable magnification. No matter how large an image is magnified, the information to be learned from examining these enlargements depends entirely on the resolution of the image. Light microscope are generally only effective to about 1500x, whereas the EM can (potentially) yeild useful information to about 1,000,000x. (John S. Choimski, Jr., 1992 : 3)

IV.             TOOLS AND MATERIAL

v  TOOLS

a.       Microscope

b.      Object glass and cover glass

c.       Pipette

v  MATERIALS

a.       The cut of paper writing “d” and “b”

V.                PROCEDURES

1.      Observation cuts the latter "b" and "d"

Put the piece of the letter "b" or "d" in object glass

Describe the image of letter "b" or "d"

Look into the eyepiece

Compare the location of the image with the location of the object that observed

Move the preparation from left to right with scale player

Close slowly with a cover glass

 

2.      Measure the broad field of view

Put the piece of latter "b" or "d" in objct glass

Observe preparation by using a weak magnification objective glass

Move preparation to left side until the last limit of the letter look like in the previous step

Move the preparation with scale to the right until the last limit of letter visible

Mark on what number the location of the point by looking at number on scale

Calculate the area of field of view by calculating the difference between the two dots (Diametre resurrected of view) with formula

Observe the first location of the object by the scale on the left side and back of the table object

Close slowly with cover glass

 

VI.             RESULT OF OBSERVATION

Result of letter “b”

b

q

 

The latter “b” is begining of the letter “q”. Characteristic of shadow is illusion, erect, and enlargement.

v  After move to right side   (S₁) = 139 mm

After move to left side      (S­₂) = 137 mm

So,       d    = S₁ – S₂

= 139 mm – 137 mm

                  = 2 mm

             r    = 1 mm

L    =  r²

      = (3,14) (1)²

      = 3,14 mm²

v  After move to upper side  (S₁) = 18 mm

After move to tower side  (S₂) = 13 mm

So,       d    = S₁ – S₂

= 18 mm – 13 mm

                  = 5 mm

             r    = 2,5 mm

L    =  r²

      = (3,14) (2,5)²

      = 19,6 mm²

Result of letter “p”

p

d

 

The latter “b” is begining of the letter “q”. Characteristic of shadow is illusion, erect, and enlargement.

v  After move to right side   (S₁) = 140 mm

After move to left side      (S­₂) = 145 mm

So,       d    = S₂ – S₁

= 145 mm – 140 mm

                  = 5 mm

             r    = 2,5 mm

L    =  r²

      = (3,14) (2,5)²

      = 19,6 mm²

v  After move to upper side  (S₁) = 13 mm

After move to tower side  (S₂) = 17 mm

So,       d    = S₂ – S₁

= 17 mm – 13 mm

                  = 4 mm

             r    = 2 mm

L    =  r²

      = (3,14) (2)²

      = 12,6 mm²

VII.          DISCUSSION

This practical work explained about how to use microscope. When we observed the object letter “b” for instance, the shadow which was formed in microscope was letter “q”. The shadow which was in microscope had its charasteristic such as illusion, erect, and enlargement. If we moved the object to the left, the shadow would be moved to the right. In spite of it. If we moved the object into upon, the shadow would be moved into beneath. In spite of it.

This picture explain when we moved the object to the left, the shadow would be moved to the right.

This picture explain when we moved the object into upon, the shadow would be moved into beneath.

When we observed the object letter “p” for instance, the shadow which was formed in microscope was letter “d”. The shadow which was in microscope had its charasteristic such as illusion, erect, and enlargement. If we moved the object to the left, the shadow would be moved to the right. In spite of it. If we moved the object into upon, the shadow would be moved into beneath. In spite of it.

This picture explain when we moved the object to the left, the shadow would be moved to the right.

This picture explain when we moved the object into upon, the shadow would be moved into beneath.

 The image formation in the microscopes is used to see objects that are very small, which is not visible to the naked eye. The microscope uses two positive lenses (convex lens). The lens is near the eye (the lens at the top) is called the ocular lens. While the lens is located close to the objects observed (bottom lens) is called the objective lens. The thing to remember is to focus on the objective lens is shorter than the focus on the ocular lens (f_ob < f_ok). The workings of a simple microscope objective lens is going to form the image of the object is real, inverted, and enlarged. Shadow objects will be captured by the objective lens as the object by the ocular lens. The shadow is visible to the eye. If described, traveling light on a microscope shown in this picture:

The function is similar to a microscope magnifying glass, which is to see small objects. However, the microscope can be used to view objects that are much smaller because it generates more magnification doubled compared with the loop. In microscopy, the object to be observed should be placed in front of the objective lens on the distance between the fob and 2f_ob that shadow will be formed at a distance greater than 2f_ob behind the objective lens to the nature of real and inverted. The shadow on the objective lens is viewed as an object by the ocular lens and forming a shadow on the ocular lens. In order to shadow the eyepiece can be seen or observed by the eye shadow should be in front of the ocular lens and illusory. This can occur if the shadow falls on the objective lens at a distance of less than f_ok of the ocular lens. The process of formation of a shadow on the microscope, as shown in the image above. In Figure shows that the final image formed by the microscope is virtual, inverted, and enlarged. The distance between the objective lens and eyepiece lens of a microscope to determine the short-term.

The length of the microscope or the distance between the objective lens and eyepiece same objective imagination distance plus the distance to the objective lens objective was to shadow the ocular lens or mathematically written

 d = S'_ob S_ok

by:        d   = length of the microscope,

S'_ob    = distance into the shadow of the objective lens objective lens,

    S_ok   = shadow distance objective to the ocular lens.

The resulting total magnification of the microscope is the product of the magnification produced by the objective lens angle and magnification produced by the eyepiece. Mathematically, the resulting total magnification of the microscope is written as follows.

M = M_ob × M_ok

by:     M    = total magnification microscope produced,
         M_ob  = The resulting magnification objective lens, and
         M_ok  = magnification eyepiece resulting corner.

Magnification produced by the objective lens meets

M_ok = Sn / f_ok

whereas the resulting magnification eyepiece angle similar to the angle magnification loop,  for observation without accommodation

M_ob = S'_ob / S_ob

and for observations with maximum accomodation

M_ok = [Sn / f_ok] + 1

with f_ok = focal length eyepiece.

For observations without accomodation eye, the image of the objective lens must fall in point focus eyepiece focus. So long to the eye is not accomodation microscope are:

d = s'_ob f_ok

Description:
fok = eyepiece focal point

VIII.       CLOSING

v  CONCLUTION

§  A microscope (from the Ancient Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see") is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope.

§  The parts of a compound microscope is Condenser, Base, Coarse Focus, Eyepiece Lens, Head, Fine Focus, Mechanical Stage, Nosepiece (or revolving nosepiece, turret), Objective Lens, Stage, Stage Clips, and Trinocular Head

§  A Microscope consists of two convex lenses. Convex lens close to the object is called the objective lens. Convex lens close to the eye is called the eyepiece. Objective lens and eyepiece on a microscope to determine the nature of shadows. The objective lens has the properties of the virtual image, reversed or minimized. While the nature of the ocular lens has a real image, upright, and enlarged. Ocular lens that determines the nature of the shadows at last.

v  SUGGEST

We should be careful in using the microscope. Cover glass is very thin, so be careful in using it. Use a lens with a smallest magnification of the first. Clean the lens only with soft tissue. After using the microscope, turn regulator srough so there is a distance between the objectivelens with table microscope, arrange the position of the mirror in an upright position. Clean the objective lens when struct emersi oil and clean up the table microscope from dirt or spills medium using a tissue.

IX.             REFERENCE

http://en.wikipedia.org/wiki/Microscope accessed at 13 october 2012

http://www.microbehunter.com/2008/12/31/ parts-of-a-compound-microscope/ accessed at 13 october 2012

http://budisma.web.id/ accessed at 13 october 2012

Choinski, John. S. 1992. Experimental Cell and Molecular Biology. Wm.C.Brown Publisher,

Campbell, Neil. A and Recce, Jane B. 2008. Biology. Benjamin Cummings, San Fransisco.