Student Learning Outcomes

At the completion of this exercise, students will be able to:

1. Discuss the importance of the microscope in biology.2. Label the parts of a compound light microscope and a stereomicroscope (dissecting

microscope) and describe their functions.3. Properly handle and care for a compound light microscope and a stereomicroscope.4. Exhibit the proper technique when using and focusing a compound light microscope and

a stereomicroscope.5. Determine the total magnification of a compound light microscope using different

objectives.6. Properly prepare a wet mount.7. Explain the direction of movement of objects while using a compound light microscope.8. Calculate the field of view of low and high power.9. Estimate the size of objects seen under the compound light microscope.10. Define and display an understanding of the following: magnification, resolving power,

working distance, parfocal, illumination, depth of field.11. Compare and contrast compound light microscopes, fluorescence microscopes, phase

contrast microscopes, Nomarski microscopes, transmission electron microscopes, scanning electron microscopes, scanning transmission electron microscopes, and stereomicroscopes.

OVERVIEW

The microscope is one of the most important and frequently used tools in the biological sciences. It allows the user to peer into the world of the cell, as well as discover the fascinating world of microscopic organisms. A typical compound microscope, similar to the one that we will use in today’s activity, is capable of extending the vision of the observer more than a thousand times. Other microscopes, such as the transmission electron microscope, can magnify objects up to one million times. Since its invention more than 300 years ago, the microscope has greatly improved our understanding of the cell, tissues, disease, and ecology.

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CHAPTER 4. MICROSCOPY: USING THE TOOLS OF BIOLOGY

MICROCOPE COMPARISONS

A microscope that uses any kind of light to view a specimen is a called a light microscope. A simple light microscope can have a single lens; similar to the early microscopes made be Anton Von Leeuwenhoek. A compound microscope uses two sets of lenses to magnify an object.

The compound bright field microscope is capable of a magnification range of 10-2000x and resolution of 300 nanometers. Light is transmitted directly through the specimen, and the specimen generally appears as a dark object against a light background. This microscope is used to examine various types of cells, microscopic organism, and tissues.

The compound dark field microscope is similar to the bright field microscope except that a special condenser causes light rays to reflect off the specimen at an angle, making the specimen appear bright against a dark background. This type of microscope is particularly useful when viewing specimens that lack contrast with a bright field microscope.

The fluorescence microscope uses ultraviolet light and fluorescent dyes to study specimens. This microscope is capable of magnifications from 10-3000x and has a resolution of 200 nanometers. It is used in advanced biological laboratories and medical laboratories to study cells, antibodies, microscopic organisms, and tissues.

The phase contrast microscope uses regular light for illumination but possesses a special condenser to accent minute differences in the refractive index of structures within a specimen. As a result, it is useful in studying cellular components and microscopic organisms. It is capable of magnifications from 10-1500x and has a resolution of 200 nanometers.

The Nomarski microscope uses differences in the refractive index of a specimen to study structures. This microscope has better resolution than a phase contrast microscope and produces nearly three-dimensional images. It is used to study the finer details of the internal structure of organisms.

Electron microscopes utilize beams of electrons to magnify a specimen. They are capable of greater magnification than compound microscopes.

o The transmission electron microscope (TEM) uses extremely thin sections of specimens treated with heavy metal salts and is capable of magnification of 200-1,000,000x. The TEM has a resolution of 0.1micrometers because of the shorter wavelength of the electron beam. This instrument is used to study the ultra-structure of cells and certain biochemical.

o The scanning electron microscope (SEM) provides three-dimensional views of objects and has a greater depth of focus. It is capable of magnifications from 10-500,000x and a resolution of 5.0 to 10.0 nanometers. The SEM is useful in studying the surface features of specimens.

o The scanning transmission electron microscope (STEM) is a combination of the TEM and SEM. It is used in the analysis of specimens and various chemicals.

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THE ANATOMY OF A COMPOUND LIGHT MICROSCOPE

Microscopes are expensive and delicate scientific instruments, so proper care and handling procedures must be followed when working with a microscope. Carefully remove the microscope from the case according to the instructor’s directions, and place it on the laboratory table. Identify the following parts of the compound microscope.

A. Ocular (eyepiece): The uppermost lens or series of lenses through which a specimen is viewed. Most oculars have a magnification of 10X. A microscope with one ocular is called a monocular microscope, and a microscope with two oculars is called a binocular microscope. The distance between the oculars can be adjusted to match different distances between pupils. Many times, a trinocular head is added to a microscope for teaching and photography purposes. Some oculars contain a pointer or a micrometer disc that is used to determine the size of an object. NEVER TOUCH THE OCULAR LENS WITH YOUR FINGERS.

B. Draw Tube: Connects the ocular to the body tube.C. Body: Holds the nosepiece at one end and includes the draw tube.D. Arm: Serves as a handle.E. Nosepiece: Revolves and holds the objectives. When changing objectives, always turn the

nosepiece instead of using the objectives themselves. The objectives will click into place when properly aligned.

F. Objectives: Lower lenses attached to the nosepiece. The magnification of each objective is stamped on the housing of the objective. The magnification may vary with different brands. NEVER TOUCH THE OBJECTIVE LENSE (use the NOSEPIECE to rotate them to the desired position).

1. Scanning objective: Used for viewing larger specimens of searching for a specimen; the shortest the objective and usually magnifies an object 4x or 5x.

2. Lower – power objective: Used for coarse and preliminary focusing; magnifies an object approximately 10x

3. High – power objective: Used for final and fine focusing, magnifies and object approximately 43x or 45x

4. Oil immersion objective: Uses the optical properties of immersion old to help magnify a specimen. Oil immersion objectives are capable of magnifications of 93x, 95x or 100x (LACC). Care should be taken with oil immersion objectives in that the oil should not be allowed to build up on the objective or contaminate other objectives. After use, the oil immersion objective and slide should be thoroughly wiped off and cleaned.

G. Stage: Platform on which slides are placed. Some microscopes have a mechanical stage to accurately control the movement of slides. Stage clips secure the slides in place.

H. Light source (illuminator): Serves as the source of illumination for the microscope.I. Iris diaphragm: Regulates light entering the microscope; usually is controlled by a mechanical

level or rotating disc.J. Condenser: A lens system found beneath the stage; used to focus the light on the specimen.K. Coarse adjustment knob: Used to adjust the microscope on scanning and low power only.L. Fine adjustment knob: Used to adjust the specimen into final focus.M. Base: The supportive portion of the microscope that rests on the laboratory table.

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PROCEDURE 4.1

USING THE MICROSCOPE

1) Ensure that your work area is clean and uncluttered. 2) After procuring your microscope from the cabinet, carry the microscope close to your body in an

upright position with one hand placed under the base and the other hand holding the arm. Place the microscope directly in front of you on the table. Once in place, do no drag the microscope across the table for other to view. Doing so can damage the intricate optics and mechanisms of the microscope.

3) Examine the anatomy of the microscope.4) Carefully clean the ocular and objectives with lens paper only. If a smudge or scratch persists,

consult the laboratory instructor.

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Illuminator

Collector lens with field diaphragm

Mechanical Ocular Rotator Knob

Mechanical Stage Rotator KnobMechanical Stage Clip

Iris Diaphragm

5) Make sure that the microscope was stored with the scanning or low – power objective in place. Never begin a session with the high power objective in place. If necessary, use the revolving nosepiece to click the low – power objective in place.

6) Inspect the electric cord, making sure that it is not frayed or damaged. Plug in the electric cord as instructed so it will not get in your way, trip other students, or damage the microscope. Turn the switch to the “on” position.

7) A good quality clean slide will be provided for observation. Carefully place your slide on the stage, and use the stage clips or the mechanical stage to hold it in place. Center the specimen under the objective.

8) If using a binocular microscope, adjust the distance between the oculars to match the distance between your pupils. One of the oculars on some binocular microscopes can be focused individually for precision. If using a monocular microscope, keep both eyes open to view the object, as closing one eye will result in eyestrain and a headache. If you are having difficulty keeping just one eye open, consult your laboratory instructor.

9) Use the coarse adjustment knob to focus the specimen. This knob is to be used to view specimens under scanning and low power only. Depending upon the brand of the microscope, either the nosepiece will move toward the stage or the stage will move toward the nosepiece. Practice your microscopy skills by viewing various parts of the slide. While viewing the slide, do not rest your hand on the stage.

10) Reposition the slide to attain the desired view. Use the iris diaphragm and condenser to focus and regulate the light entering the microscope. On scanning and low power, the fine adjustment knob may be used to fine – tune the specimen.

11) Using the revolving nosepiece ring, rotate the high power objective (oil immersion) into position until you feel it click into place. Sketch and record the name and magnification of your specimen below (Note: use Diatoms Prepared Slide).

12) Many microscopes are parfocal (once the image is focused with one objective, it should be in focus with others) and require only minor adjustments in focusing. Using the fine adjustment knob only, focus your specimen. You may have to reposition your specimen carefully and adjust the iris diaphragm and condenser. Sketch and record the name and magnification of your specimen below (Note: use Mammalian Blood Prepared Slide).

________________________________ ___________________________________

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10) Diatoms at 1000X 12) Mammalian Blood at 1000X

DEVELOPING THE SKILLS OF MICROSCOPY

To become proficient with the microscope you must understand fundamental principles of microscopy and acquire basic skills. The key to becoming skillful with a microscope is practice. Using your laboratory microscope, perform the following tasks.

Understanding Magnification

Microscopes are designed to magnify objects. The magnification of a specimen is the product of the power of the ocular and the power of the objective. Microscopes generally are equipped with a 10x ocular, a 4x or 5x scanning objective, a 10x low – power objective, a 43x or 45x high power objective, and a 93x, 95x or 100x oil immersion objective. Thus, the total magnification of a microscope on low power is the 10x ocular times the 10x low – power objective, or 100x.

Check your Understanding:

1) What is the total magnification of a typical microscope using the high – power objective?

________________________________________________________________________

2) If a scanning objective is used, what is the total magnification of a microscope?

________________________________________________________________________

3) What is the total magnification of a microscope using on oil immersion objective (100x)?

________________________________________________________________________

UNDERSTANDING RESOLVING POWER

The ability to resolve objects is an important characteristic of microscopes and a measure of lens quality. The resolving power of a microscope depends upon the design and quality of the objectives lenses. Quality lenses have a high resolving power, which is the ability to deliver a clean image in detail. In comparing microscopes, lower values represent better resolving power.

When using the microscope, magnifying a specimen is not always accompanied by an increase in the clarity of the detail with the specimen. At a certain point, greater magnification of the specimen yields progressively fuzzy images. Oil immersion objectives can increase the resolving power or a microscope.

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PROCEDURE 4.2

UNDERSTANDING IMAGE ORIENTATION

1) Place a prepared slide of the letter “e” on the stage of your microscope. Using low power 40X and then switch to Medium power 400X , observe the slide. Sketch and describe your slide.

2) What is the difference between the orientation of the letter between the unaided eye and the microscope?________________________________________________________________________

3) Move the slide to the left and to the right. Describe the direction of the movement of the image. ________________________________________________________________________

UNDERSTANDING ILLUMINATION

When using a microscope, proper illumination is essential as improper illumination can result in poor images and eyestrain. Some microscopes are equipped with a mirror to gather and concentrate light. Generally, the mirror has a smooth side and a concave side. The smooth side should be used with it is necessary to concentrate light on the specimen. If your microscope used a mirror, adjust the mirror to deliver maximum light on the specimen, and use the iris diaphragm to regulate the intensity of the light. Thus, the iris diaphragm is used to cut off light scattering into the objective lens. This "flair" lowers contrast and degrades the image.

Most microscopes are equipped with an illuminator, attached to the base. The iris diaphragm is used to adjust the amount of light entering the objective lens by rotating a disc or moving an aperture adjustment control. The iris diaphragm can be used to regulate the light passing through an object, as well as the contrast of the object. Partially closing iris increases contrast and depth of f i eld . Although closing the diaphragm results in poorer resolution it can be useful in viewing certain specimens. The iris has another function when viewing transparent subjects. The iris aperture can be reduced in order to increase contrast, making otherwise clear subjects stand out. Reducing the aperture in this way reduces resolution, but the improvement in contrast often provides much more information about the subject.

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Some microscopes have a substage condenser. For an introductory laboratory, the condenser should be left in its uppermost position. Many microscopes have a light intensity control; use this control as directed by instructor.

PROCEDURE 4.3

ILLUMINATION

1) Carefully place a slide (Instructor’s choice: Diatoms prepared sli de) on the stage, and focus using low power (total magnification of 40X).

2) Use the iris diaphragm to attain the best illumination for the specimen. Open and close the iris diaphragm and notice changes in the contrast of the specimen.

3) Follow the same procedure using the high power objective (total magnification of 1000X)4) After you finish, return the slide to the slide tray.5) Briefly describe the changes in illumination and contrast that you observed.

________________________________________________________________________

________________________________________________________________________

UNDERSTANDING WORKING DISTANCE AND DIAMETER OF FIELD

Working distance is the distance between the objective lens and the specimen. As the working distance decreases, magnification increases. More light is needed when the working distance decreases. Thus, more light must be made available when using high power and oil immersion objectives.

The circular field that you see when you look through the ocular is known as the field of view. The field of view changes at different magnifications. The size of a specimen can be estimated if one knows the diameter of the field of view at each magnification. To do this, one must first determine the diameter of the field of view for both low and high power (see procedure 4.4.)

PROCEDURE 4.4

DISTANCE AND DIAMETER

1) Place a translucent plastic millimeter ruler (it is a prepared slide found on your box slides) on the microscope stage as if it were a slide. If no plastic ruler is available, mount a paper millimeter ruler. If your microscope has ocular micrometers use it instead of the ruler.

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2) Using the low – power objective, record the number of millimeter (to tenth) that you see along the field. Your value = ______________________ mm.

3) Convert the figure that you attained to micrometers (µm). (1 millimeter = 1000 micrometers). This is the diameter of the field of view for the low – power objective (LPD).

Your value (LPD) = ___________________ µm.4) The diameter of the field of view for the high – power objective will be difficult to attain using

this method. However, a simple inverse proportion can be developed to determine the diameter of the field of view for the high power objectives.

HPD = high – power objective diameter of field of viewLPD = low – power objective diameter of field of viewLPM = low – power magnification (or total magnification under low power)HPM = high – power magnification (or total magnification under high power)

5) Based on the formula, find the high – power objective field of view for your microscope.

LPD= _____________________________ µm.

LPM=_____________________________

HPD= LPD (x) LPM/HPM = _____________________________ µm.

6) Using the above numbers, approximate the size of the following specimens (Instructor’s choice - Use the prepared slide of the letter e.)

________________________________________________________________________

________________________________________________________________________

7) A more precise method for measuring the size of a microscopic object is to use a standardized ocular micrometer. These devices are found on more advanced microscopes. In some microscopes, the pointer that is attached to the inside of the ocular and appears in the field of view can be used for measuring objects.

UNDERSTANDING PLANES OF FOCUS AND DEPTH OF FIELD

Microscope lenses have a restrictive plane of focus , a specific distance from the lens where the specimen can be sharply focused. It is possible to focus through different planes of the specimen by changing the plane of focus. The plane of focus has some depth called the depth of field , the thickness of the specimen that is in focus at any one time. When using lens systems, including cameras, the greater the magnification, the less is the depth of field.

PROCEDURE 4.5

FOCUS AND DEPTH

1) Obtain a prepared a slide containing three different colored threads, and place it on the stage of your microscope.

2) Locate the point on the slide where the threads intersect, and center it under the objective.

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3) Using the low – power objective, slowly focus until the first thread comes into focus. The fine adjustment can be used in addition to the coarse adjustment.

What is the color of the first thread? _____________________________________________

4) Continue focusing through the threads, noting the order of the colored threads.

What is the color of the second thread? ________________________________________________

What is the color of the third thread? __________________________________________________

5) Switch to the high – power objective and notice the depth of field is shallower. If you constantly use the fine adjustment, the three – dimensional form of the threads can be determined.

6) The laboratory instructor will provide a slide of a tissue such as columnar epithelium or an organism such as a rotifer. Focus through your specimen and record your observations at 1000X.

When will an understating of planes of focus and depth of field be valuable in the laboratory?

_________________________________________________________________________________

_________________________________________________________________________________

Discuss planes of focus and depth of field based upon your observations of both the low – power and high – power objectives._________________________________________________________________________________

_________________________________________________________________________________

Using Oil Immersion

Many microscopes have an oil immersion objective for observing small organisms and the fine detail of specimens. It appears longer than the other objectives and is clearly marked. Oil immersion objectives are capable of magnifications of 93x, 95x, or 100x [Note: LACC Microscopes (Biology 03).]

So; what is the total magnification under oil immersion at LACC’s Biology 03 microscopes?

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(4) Draw the levels of the threads

(6) Draw or Sketch a Rotifer

___________________________________________________________________________

Property Definition

ParfocalOnce an objective has been focused, you can rotate to another objective and the image will remain in coarse focus, requiring only slight movement of the fine focus knob.

Parcentral The center of the field of view remains about the same for each objectiveField of View The circle of light you see when looking through the ocular lens

Working Distance The space between the objective and the slideTable 4.0 Microscopy Terms.

Objective ObjectiveColor

Objective Magnifying

Power (ObMP)

Ocular Magnifying

Power (OcMP)Total Magnifying Power

(ObMP x OcMP =___x)

Numerical Aperture (NA)

Low – Power(Scanning Power)

Red

Medium – Power YellowHigh – Dry Blue

Oil – Immersion(High Power)

White

Table 4.1 Objective present on a compound Light Microscope.

Property DefinitionMagnification The amount of the image of an object is enlarged.

Resolving Power The quality to which objective detail in an image is preserved during the magnifying process.

Contrast The degree to which images details stand out against their background.Table 4.2 Show some of the most important Lens and Image Properties.

What is the magnification range of your Compound Light Microscope?

__________________x to __________________ x

Objective Magnifying Power Diameter of Field of ViewLow – Power

(Scanning Power)Medium – Power

High – Dry

Oil – Immersion(High Power)

Table 4.3 Diameter of Field of View.

NOTE: To find the diameter of the field of view of higher magnification

Total Magnification of Diameter of Field of View of

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Low – Power Objective X Low – Power ObjectiveTotal Magnification of Higher Objective

STEREOMICROSCOPE (DISSECTING MICROSCOPE)

Main objective is to become familiar with the stereomicroscope (dissecting microscope.)

PROCEDURE 4.8

Materials

Ring Coin Dollar bill

Fossil Feather Leaf

Pond water Other specimens,

instructor’s choice

1) View the following objects with the stereo microscope: you fingernail, the surface of a stone on a ring, a coin, a dollar bill, a fossil, a feather, a leaf, several biological specimens provided by your instructors, and some pond water in a Petri dish.

2) Try using various light and magnification settings with your stereomicroscope.3) Sketch, draw or take a picture of your observations below.

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__________________________________________________________________

_________________________________________________________________

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__________________________________________________________________

PROCEDURE 4.9. Storage

To properly storage of a microscope:

1) At the completion of each laboratory experience, rotate the lowest power objective into place and remove the slide.

2) Clean the ocular and objectives with lens paper only. If you have been using oil immersion, clean the slide and objective as instructed in the procedure for using oil immersion.

3) If you have been using wet mounts, clean the stages with a cleaning tissue or a clean cloth.4) Turn off the light and unplug the microscope. Wrap the cord around the base as directed by the

instructor. If a plastic dust cover is available, cover the microscope and return it to the proper cabinet space number.

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REVIEW QUESTIONS

Review Questions Chapter 4: Microscopy: Using the Tools of Biology

1. What is the difference between a simple microscope and a compound microscope?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

2. What are some useful applications of a stereomicroscope?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

3. What are some of the advantages and disadvantages of using electron microscopy?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

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___________________________________________ ____________________________________________Last Name, First Name [lab partner N0. 1] Last Name, First Name [lab partner N0. 2]

_______________________________ _______________________________Last Name, First Name [lab partner N0. 3] Last Name, First Name [lab partner N0. 4]

___________________________ _______________ ____________________Section group # Date

______________________________________________________________________________

______________________________________________________________________________4. When should an oil immersion objective be used?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

5. Why do many biologists prefer a phase contrast microscope?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

6. What is the advantage of parfocal objectives?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

7. Discuss revolving power?

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

8. Discuss the direction of movement of the protozoans in pond water in relation to movement of the slide.

______________________________________________________________________________

______________________________________________________________________________

9. Discuss the relationships between plane of focus, depth of field, illumination, and magnification.

______________________________________________________________________________

______________________________________________________________________________

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______________________________________________________________________________

______________________________________________________________________________10. What is the magnification of a microscope with a 10 X ocular and a 95 X oil immersion objective?

______________________________________________________________________________

11. List three rules to remember when focusing a microscope.

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

12. Label the parts of the microscope:

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1. ______________________________

2. ______________________________

3. ______________________________

4. ______________________________

5. ______________________________

6. ______________________________

7. ______________________________

8. ______________________________

9. ______________________________

10. _____________________________

11. _____________________________

12. ____________________________

13. _____________________________

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