Using Compound Microscopes in Biology

• Objectives:
  • Discuss the importance of the microscope in biology
  • Identify and describe the function of the parts of a compound microscope
  • Properly handle and care for a microscope
  • Exhibit the proper technique when using and focusing a microscope
  • Determine the total magnification of a compound microscope using different objectives
  • Properly prepare a wet mount
  • Calculate the field of view at low and high power
  • Define magnification, resolving power, working distance, parfocal, illumination, plane of focus, and depth of field; describe their interrelationships
• The microscope is an important tool in biology to study cells and microscopic objects
• Compound microscopes extend vision more than a thousand times
• Transmission electron microscopes magnify objects up to 1 million times
• Microscopes have improved the understanding of cells, tissues, disease, and ecology for over 300 years

Types of Microscopes

• Light microscopes are the most common type used in biology labs today
• Simple light microscopes utilize a single lens
• Compound microscopes utilize two sets of lenses to magnify objects
• Compound microscopes magnify objects in the range of 10–2,000X with a resolution of 300 nanometers
• Bright field microscopes transmit light directly through the specimen; the specimen appears dark against a light background
• Bright field microscopes are used to examine cells, microscopic organisms, and tissues
• Compound dark field microscopes use a special condenser that reflects light rays off the specimen at an angle
• In a dark field microscope the specimen appears bright against a dark background
• Dark field microscopes are useful when viewing specimens lacking contrast
• Fluorescence microscopes use ultraviolet light and fluorescent dyes to study specimens
• Fluorescence microscopes magnify objects in the range of 10–3,000X, with a resolution of 200 nanometers
• Fluorescence microscopes are often used in advanced biological and medical laboratories to study cells, antibodies, microscopic organisms, and tissues
• Phase contrast microscopes use regular light for illumination and a special condenser to highlight minute differences in the refractive index of structures within a specimen
• Phase contrast microscopes magnify objects in the range of 10–1,500X and yield a resolution of 200 nanometers
• Nomarski microscopes use differences in the refractive index of specimens to study structures
• Nomarski microscopes provide better resolution than phase contrast microscopes and produce nearly three-dimensional images
• Electron microscopes use beams of electrons to magnify a specimen

Transmission Electron Microscope (TEM)

• Transmission electron microscopes (TEM) use very thin sections of specimens treated with heavy metal salts
• Transmission electron microscopes (TEM) are capable of magnifications in the range of 200–1,000,000X
• TEM has a resolution of 0.1 µm because of the short wavelength of the electron beam
• TEM are used to study the ultrastructure of cells and certain biochemicals

Scanning Electron Microscope (SEM)

• Scanning electron microscopes (SEM) provide three-dimensional views of objects and have a greater depth of focus
• Scanning electron microscopes (SEM) achieve magnifications in the range of 10–500,000X with a resolution of 5–10 nanometers
• SEM are useful in studying the surface features of specimens

Scanning Transmission Electron Microscope (STEM)

• Scanning transmission electron microscope (STEM) is a combination of TEM and SEM
• Scanning transmission electron microscope (STEM) are used in the analysis of specimens and various chemicals

Parts of a Compound Microscope

• 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
• Draw tube: Connects the ocular to the body tube
• Body: Holds the nosepiece at one end and includes the draw tube
• Arm: Serves as a handle
• Nosepiece: Revolves and holds the objectives
• Objectives: Lower lenses attached to the nosepiece
• Scanning Objective: Used for viewing larger specimens or searching for a specimen; the shortest objective usually magnifies an object 4Χ
• Low-Power Objective: Used for coarse and preliminary focusing; magnifies an object approximately 10X
• High-Power Objective: Used for final and fine focusing, magnifies an object approximately 40X
• Oil-Immersion Objective: Uses the optical properties of immersion oil to help magnify a specimen; capable of magnifications of 100X
• Stage: Platform on which slides are placed; some microscopes have a mechanical stage to accurately control the movement of slides; stage clips secure the slide
• Light Source (Illuminator): Serves as the source of illumination for the microscope
• Iris Diaphragm: Regulates light entering the microscope
• Condenser: A lens system found beneath the stage; used to focus the light on the specimen
• Coarse-Focus Adjustment Knob: Used to adjust the microscope on scanning and low power only
• Fine-Focus Adjustment Knob: Used to adjust the specimen into final focus
• Base: The supportive portion of the microscope, which rests on the laboratory table
• Aperture: Hole in the stage that permits light to pass from the illuminator to the object being examined
• Slide Position Adjustment Knobs: Paired knobs below the stage that move the slide holder either forward and back or left and right

Microscopy

• Magnification: Ability to make objects bigger
• The magnification of a specimen is a product of the power of the ocular and the power of the objective lenses
• Total magnification on low power = 10X ocular times the 10X low-power objective, thus 100X magnification
• Resolving Power: The ability to resolve objects, or distinguish two closely spaced objects as separate entities
• Resolving power is a measure of lens quality; quality lenses have a high resolving power, which gives a clear image in detail
• Lower values mean better resolving power
• Human eye resolution: 0.1 mm
• Quality magnifying glass resolution: 0.01 mm
• Resolving power can be increased with oil-immersion objectives
• Working distance: Distance between the objective lens and the specimen
• As working distance decreases, magnification increases
• More light is needed when the working distance decreases
• Field of view: Circular field seen when looking through the ocular
• Plane of focus: Specific distance from the lens where the specimen can be sharply focused
• Depth of field: Thickness of the specimen in focus at any one time
• The greater the magnification, the less is the depth of field
• Parfocal: After the image is focused with one objective, it should be in focus with others, requiring only minor adjustments

Proper Microscope Usage

• Area used must be clean and uncluttered
• Carry the microscope close to the body with one hand under the base and the other hand holding the arm
• Place the microscope directly in front
• Do not drag the microscope across the table
• Clean the ocular and objectives with lens paper
• Scanning or low-power objective should be in place when getting a microscope
• Inspect the electrical cord
• Place the slide on the stage and secure it with the stage clips
• Center the specimen under the objective and over the aperture using the slide position knobs
• Adjust the distance between oculars on binocular microscopes
• Keep both eyes open when using a monocular microscope to avoid eyestrain and headaches
• Use the coarse-focus adjustment knob to focus the specimen on scanning and low power only
• Practice microscopy skills by viewing various parts of the slide
• Do not rest your hand on the stage
• Reposition the slide to attain the desired view
• Movement of the slide is in reverse direction when viewing an image under the microscope
• Use the iris diaphragm and condenser to focus and regulate the light entering the microscope
• Proper illumination is essential
• The iris diaphragm regulates light passing through an object and the contrast of the object
• The substage condenser also regulates light
• Fine-focus adjustment knob may be used to fine-tune the specimen on scanning and low power
• Rotate the high-power objective into position using the revolving nosepiece ring
• Adjust the iris diaphragm and condenser after repositioning the specimen carefully
• Return the microscope to the lowest objective before removing the slide

Slides

• Prepared slides are commonly used in biology labs
• Slides must be carried by the edge or the end; never place fingers over the viewing area
• Note the type of mount you are viewing (whole mount, cross section, or longitudinal section)
• Do not become dependent on dye colors when learning specimens
• Treat prepared slides carefully and report any broken or damaged slides
• Wet mounts are often made in the laboratory to view fresh specimens
• Place a drop of solution on the slide and the specimen in the center of the slide, then lower the coverslip at a 45-degree angle to minimize air bubbles
• Always place a coverslip over wet mounts
• Onion and cheek cells are often used for wet mounts (have a plasma membrane, cytoplasm and a nucleus)
• Plasma membrane: Phospholipid bilayer that provides support and regulates the movement of substances into and out of the cell
• Cytoplasm: Semi-fluid medium between the plasma membrane and nucleus that contains the organelles of a cell
• The nucleus stains very dark and is the control center of the cell
• Plant cells have a cell wall around the plasma membrane, providing protection and shape