OVERVIEW

In this chapter we examine the role of diffraction in determining spatial resolution and

image contrast in the light microscope. In the previous chapter we emphasized that

Abbe’s theory of image formation in the light microscope is based on three fundamental

actions: diffraction of light by the specimen, collection of diffracted rays by the

objective, and interference of diffracted and nondiffracted rays in the image plane. The

key element in the microscope’s imaging system is the objective lens, which determines

the precision with which these actions are effected. As an example, examine the remarkable

resolution and contrast in the image of the diatom, Pleurosigma, made with an

apochromatic objective designed by Abbe and introduced by Carl Zeiss over 100 years

ago (Fig. 6-1). To understand how such high-resolution images are obtained, we examine

an important parameter of the objective, the numerical aperture, the angle over

which the objective can collect diffracted rays from the specimen and the key parameter

determining spatial resolution. We also investigate the effect of numerical aperture on

image contrast. In examining the requirements for optimizing resolution and contrast,

we make an unsettling discovery: It is impossible to obtain maximal spatial resolution

and optimal contrast using a single microscope setting. A compromise is required that

forces us to give up some spatial resolution in return for an acceptable level of contrast.

NUMERICAL APERTURE

Implicit in the Overview is an understanding that the objective aperture must capture

some of the diffracted rays from the specimen in order to form an image, and that lenses

that can capture light over a wide angle should give better resolution than an objective

that collects light over a narrower angle. In the light microscope, the angular aperture is

described in terms of the numerical aperture (NA) as

NA _ n sin_,

85

where _ is the half angle of the cone of specimen light accepted by the objective lens and

n is the refractive index of the medium between the lens and the specimen. For dry

lenses used in air, n _ 1; for oil immersion objectives, n _ 1.515.

The diffraction angles capable of being accepted by dry and oil immersion objective

lenses are compared in Figure 6-2. By increasing the refractive index of the medium

between the lens and coverslip, the angle of diffracted rays collected by the objective is

increased. Because immersion oil has the same refractive index as the glass coverslip

86 DIFFRACTION AND SPATIAL RESOLUTION

Figure 6-1

Resolution of the pores in a diatom shell with an apochromatic objective lens. Joseph Gall

described these historic photographs of the diatom Pleurosigma prepared over 100 years

ago using one of Abbe’s lenses (Molecular Biology of the Cell, vol. 4, no. 10, 1993). “The

photographs . . . are taken from a Zeiss catalog published in 1888 in which Abbe’s

apochromatic objectives were advertised. Both figures show the silica shell of the diatom

Pleurosigma angulatum. Because of the regular patterns of minute holes in their shells,

diatoms have long been favorite objects for testing the resolution of microscope objectives.

The top figure shows an entire shell at 500_, a magnification beyond which many 19thcentury

objectives would show little additional detail. The bottom figure, reproduced here and

in the original catalog at a remarkable 4900_, was made with an apochromatic oil

immersion objective of 2.0 mm focal length and a numerical aperture of 1.3. The center-tocenter

spacing of the holes in the shell is 0.65 _m, and the diameter of the holes themselves

is about 0.40 _m. Almost certainly this objective resolved down to its theoretical limit of 0.26

_m in green light.”

(1.515), refraction of specimen rays at the coverslip-air interface is eliminated, the

effective half angle is increased, and resolution is improved. The reader can refer to

Pluta (1988) for more details on this important phenomenon.

SPATIAL RESOLUTION

For point objects that are self-luminous (fluorescence microscopy, dark-field microscopy),

or for nonluminous points that are examined by bright-field microscopy in

transmitted light where the condenser NA is ? the objective NA, the resolving power of

the microscope is defined as

d _ 0.61ë/NA,

where d is the minimum resolved distance in _m, ë is the wavelength in _m, and NA is

the numerical aperture of the objective lens.

In the case of bright-field microscopy, where the condenser NA _ objective NA

(the condenser aperture is closed down and/or an oil immersion condenser is used in the

absence of oil), the resolution is given as

d =

+

1.22

.

ë

condenser NA objective NA

SPATIAL RESOLUTION 87

Air Oil

NA = 0.95

72°

NA = 1.4

67°

n = 1 n = 1.515

Figure 6-2

Effect of immersion oil on increasing the angular extent over which diffracted rays can be

accepted by an objective lens. Numerical aperture is directly dependent on the wavelength _

and the sine of the half angle of the cone of illumination _ accepted by the front lens of the

objective. For dry lenses, NA is limited, because rays subtending angles of 41° or greater are

lost by total internal reflection and never enter the lens (dotted line). The practical limit for a

dry lens is _39°, which corresponds to an acceptance angle of 72°, and an NA of 0.95. By

adding high-refractive index immersion oil matching that of the glass coverslip (n _ 1.515), an

oil immersion objective can collect light diffracted up to 67°, which corresponds to NA _ 1.4.

These equations describe the Rayleigh criterion for the resolution of two closely

spaced diffraction spots in the image plane. By this criterion, two adjacent object points

are defined as being resolved when the central diffraction spot (Airy disk) of one point

coincides with the first diffraction minimum of the other point in the image plane

(Fig. 6-3). The condition of being resolved assumes that the image is projected on the

retina or detector with adequate magnification. Recording the real intermediate image on

film or viewing the image in the microscope with a typical 10_ eyepiece is usually adequate,

but detectors for electronic imaging require special consideration (discussed in

Chapters 12, 13, and 14). The Rayleigh resolution limit pertains to two luminous points in

a dark field or to objects illuminated by incoherent light. For a condenser and objective

with NA _ 1.3 and using monochromatic light at 546 nm under conditions of oil immersion,

the limit of spatial resolution d _ 0.61ë/NA _ 0.26 _m. Numerical apertures are

engraved on the lens cap of the condenser and the barrel of the objective lens.

An image of an extended object consists of a pattern of overlapping diffraction

spots, the location of every point x,y in the object corresponding to the center of a diffraction

spot x,y in the image. Imagine for a moment a specimen consisting of a

crowded field of submicroscopic particles (point objects). For a given objective magni-

88 DIFFRACTION AND SPATIAL RESOLUTION

(a) (b) (c)

a

b

c

Figure 6-3

Rayleigh criterion for spatial resolution. (a) Profile of a single diffraction pattern: The bright

Airy disk and 1st- and 2nd-order diffraction rings are visible. (b) Profile of two disks

separated at the Rayleigh limit such that the maximum of a disk overlaps the first minimum

of the other disk: The points are now just barely resolved. (c) Profile of two disks at a

separation distance such that the maximum of each disk overlaps the second minimum of

the other disk: The points are clearly resolved.

fication, if the angular aperture of a microscope is increased, as occurs when opening

the condenser diaphragm or when changing the objective for one with the same magnification

but a higher NA, the diffraction spots in the image grow smaller and the image

is better resolved (Fig. 6-4). Thus, larger aperture angles allow diffracted rays to be

included in the objective, permitting resolution of specimen detail that otherwise might

not be resolved (Fig. 6-5).

The optical limit of spatial resolution is important for interpreting microscope

images. Irregularities in the shapes of particles greater than the limiting size (0.52 _m

diameter in the example cited previously) just begin to be resolved, but particles smaller

than this limit appear as circular diffraction disks, and, regardless of their true sizes and

shapes, always have the same apparent diameter of 0.52 _m. (The apparent variability

in the sizes of subresolution particles is due to variations in their intensities, not to variability

in the size of their diffraction spots.) Thus, whereas minute organelles and filaments

such as microtubules can be detected in the light microscope, their apparent

diameter (for the lens given previously) is always 0.52 _m, and their true diameters are

not resolved. It should therefore be apparent that two minute objects whose center-tocenter

distance is less than 0.26 _m cannot be resolved, but that two objects with physical

radii smaller than this size can easily be resolved from each other if they are farther

apart than 0.26 _m.

It must be remembered that adjusting the condenser aperture directly affects spatial

resolution in the microscope. Since a large aperture angle is required for maximum

resolution, the front aperture of the condenser must be fully illuminated. Stopping down

SPATIAL RESOLUTION 89

Back aperture stop

of objective lens

Objective

Specimen

Condenser

Condenser

diaphragm

a

b

a?

b?

è

Figure 6-4

Role of the condenser diaphragm in determining the effective numerical aperture. Closing

the front aperture diaphragm of the condenser from position b to a limits the angle _ of the

illumination cone reaching the objective, and thus the effective numerical aperture. Notice

that the back aperture of the objective is no longer filled at the reduced setting.

the condenser diaphragm limits the number of higher-order diffracted rays that can be

included in the objective and reduces resolution. In an extreme case, the condenser aperture

may be nearly closed in a mistaken effort to reduce light intensity. Then the half

angle of the light cone entering the lens is greatly restricted, and resolution in the image

is reduced significantly. The proper way to reduce light intensity is to turn down the

voltage supply of the lamp or insert neutral density filters to attenuate the illumination.

DEPTH OF FIELD AND DEPTH OF FOCUS

Just as diffraction and the wave nature of light determine that the image of a point object

is a diffraction disk of finite diameter, so do the same laws determine that the disk has a

measurable thickness along the z-axis. Depth of field Z in the object plane refers to the

thickness of the optical section along the z-axis within which objects in the specimen are

in focus; depth of focus is the thickness of the image plane itself. Our present comments

are directed to the depth of field. For diffraction-limited optics, the wave-optical value

of Z is given as

Z _ në/NA2,

where n is the refractive index of the medium between the lens and the object, ë is the

wavelength of light in air, and NA is the numerical aperture of the objective lens. Thus,

90 DIFFRACTION AND SPATIAL RESOLUTION

Figure 6-5

Effect of numerical aperture on spatial resolution. The diatom Pleurosigma photographed

with a 25_, 0.8 NA oil immersion lens using DIC optics. (a) Condenser aperture open,

showing the near hexagonal pattern of pores. (b) The same object with the condenser

diaphragm closed. The 1st-order diffracted rays from the pores are not captured by the

objective with a narrow cone of illumination. Spatial resolution is reduced, and the pores are

not resolved. Bar _ 10 _m.

the larger the aperture angle (the higher the NA), the shallower will be the depth of field.

The concept of depth of field is vivid in the minds of all of those who use cameras.

Short-focal-length (fast) lenses with small focal ratios (_f/4) have a shallow depth of

field, whereas the depth of field of long-focal-length (slow) lenses (_f/16) is relatively

deep. At one extreme is the pinhole camera, which has an infinitely small NA and an

infinite depth of field—all objects, both near and far, are simultaneously in focus in such

a camera.

The depth of field along the z-axis is determined by several contributing factors,

including principles of geometrical and physical optics, lens aberrations, the degree of

physiological accommodation by the eye, and overall magnification. These variables

and quantitative solutions for each are described in detail by Berek (1927), and are

reviewed by Inoué (in Pawley, 1995) and Pluta (1988). Calculated values of the wave

optical depth of field for a variety of objective lenses are given in Table 4-1.

The depth of field for a particular objective can be measured quickly and unambiguously

using the microscope. A planar periodic specimen such as a diffraction grating

is mounted obliquely on a specimen slide by propping up one end of the grating

using the edge of a coverslip of known thickness. When the grating is examined in the

microscope, it will be seen that only a narrow zone of grating will be in focus at any particular

setting of the specimen focus dial. The depth of field z is then calculated from the

width w of the focused zone (obtained photographically) and the angle of tilt _ of the

grating through the relationship

Z _ nw tan_,

where n is the refractive index of the medium surrounding the grating.

OPTIMIZING THE MICROSCOPE IMAGE: A COMPROMISE

BETWEEN SPATIAL RESOLUTION AND CONTRAST

Putting these principles into practice, let us examine two specimens using transmitted

light illumination and bright-field microscope optics: a totally opaque object such as a

copper mesh electron microscope grid and a stained histological specimen. These

objects are called amplitude objects, because light obscuring regions in the object

appear as low-intensity, high-contrast regions when compared to the bright background

in the object image. In comparison, transparent colorless objects such as living cells are

nearly invisible, because the amplitude differences in the image are generally too small

to reach the critical level of contrast required for visual detection. We discuss methods

for visualizing this important class of transparent objects in Chapter 7.

With the microscope adjusted for Koehler illumination using white light, begin by

opening the condenser front aperture to match the diameter of the back aperture of the

with inherently low contrast. For color objects such as histological specimens that are to

be examined visually or recorded with a gray-scale camera or black-and-white film,

contrast can be improved dramatically by selecting filters with complementary colors: a

green filter for pink eosin dye or a yellow filter for blue hematoxylin stain. A green filter,

for example, removes all of the pink eosin signal, reducing the amplitude of eosinstained

structures and making them look dark against a bright background in a

gray-scale image.

To our surprise, closing down the condenser diaphragm also has a pronounced effect:

It increases the contrast and greatly improves the visibility of the scene (Fig. 6-6). The

grid bars now look black and certain features are more readily apparent. There are several

reasons why this happens: (1) Part of the improvement comes from reducing the

amount of stray light that becomes reflected and scattered at the periphery of the lens.

(2) Reducing the aperture increases the coherence of light; by selecting a smaller portion

of the light source used for illumination, the phase relationships among diffracted

rays are more defined, and interference in the image plane results in higher-amplitude

differences, thus increasing contrast. (3) With reduced angular aperture, the unit diffraction

spots comprising the image become larger, causing lines and edges to become

thicker and cover a greater number of photoreceptor cells on the retina, thus making the

demarcations appear darker and easier to see. Thus, the benefits of improved contrast

and visibility might lead us to select a slightly stopped-down condenser aperture, even

though spatial resolution has been compromised slightly in the process. If the condenser

aperture is closed down too far, however, the image loses significant spatial resolution

and the dark diffraction edges around objects become objectionable.

Thus, the principles of image formation must be understood if the microscope is to

be used properly. A wide aperture allows maximal spatial resolution, but decreases contrast,

while a smaller, constricted aperture improves visibility and contrast, but

decreases spatial resolution. For all specimens, the ideal aperture location defines a balance

between resolution and contrast. A useful guideline for beginners is to stop down

the condenser aperture to about 70% of the maximum aperture diameter, but this is not

a rigid rule. If we view specimens such as a diffraction grating, a diatom, or a section of

striated muscle, stopping down the diaphragm to improve contrast might suddenly obliterate

periodic specimen details, because the angular aperture is too small to allow diffracted

light to be collected and focused in the image plane. In such a situation, the

aperture should be reopened to whatever position gives adequate resolution of specimen

detail and acceptable overall image visibility and contrast.

Catalase is a non-digestive enzyme produced by the liver. It breaks down the toxin hydrogen peroxide in the body according to the equation H2O2 H2O + O2. The presence of the flammable gas oxygen can be used to detect this reaction. The volume produced can be used to calculate the rate of the enzyme’s activity. In this investigation, you will design experiments to test for factors that may affect the rate of this reaction. Factors to consider include pH, temperature, the quantity of the substrate hydrogen peroxide, and the quantity of the enzyme. Test for each factor separately.

Problem

How do different factors affect the rate of enzyme activity?

Hypothesis

Make hypotheses about three factors you would like to test.

CAUTION: Hydrogen peroxide is a bleaching agent and an irritant. Take care not to get it, the vinegar, or the sodium bicarbonate solutions in your eyes or on your skin or clothes. Wash spills away immediately with lots of water and inform your teacher. Exercise caution when testing for flammable gas. Make sure there are no cracks in the glassware you use.

Materials

30 mL square bottle stock solution of puréed

one-holed rubber stopper liver (contains catalase) to fit above medicine dropper

100 mL graduated cylinder vinegar (acid)

10 mL graduated cylinder sodium hydrogen

plaster tray or pneumatic carbonate solution (base)

trough graph paper

forceps dilute hydrogen peroxide

600 mL beaker solution (substrate) (3%)

watch or clock matches

absorbent paper disks wooden splints

dissecting-zoom microscope

Experimental Plan

1. Using the materials and considering the set-up shown in the illustration, prepare a list of possible ways in which you can test your hypotheses.

2. Decide on an approach that you can carry out in your classroom.

3. Make sure your approach will test for only one possible factor (independent variable) at a time. Prepare to collect and record quantitative data for at least three variables on the graph paper, and to summarize this data in a data table (like the one shown here) that can be interpreted by others.

4. Outline a procedure for your experiment listing each step. Include all necessary safety procedures. Provide a list of materials and the quantities you will require. Obtain our teacher’s approval before starting any reaction. One step you must do is soak as many of the paper disks in the puréed liver extract as you think you will need. Make sure you soak them all for the same length of time. To begin the reaction, the bottle containing the hydrogen peroxide and disks is turned over. Wash your hands thoroughly at the end of the investigation.

Checking the Plan

1. What will be the dependent variable for each of the independent variables you want to test? What will the controlled variables be?

2. What will be your control?

3. What will you measure, and how will you record this information on the graph paper?

4. How will you safely test whether any resulting gas is flammable?

Data and Observations

Conduct your investigation and make your measurements. Graph your results first, and then enter your summary data in the table.

Analyze

1. Changes in which factors (independent variables) influenced the rate at which gas was produced? Which changes, if any, meant that little or no gas was produced?

2. Changes in which factor produced the greatest amount of gas in the shortest amount of time?

3. Why was it necessary to test only one variable at a time?

Conclude and Apply

4. Based on your results, which factors affect the rate at which enzymes such as catalase act?

5. Explain how variations in these factors could affect digestive enzymes, and how they could affect the general well-being of a person.

Exploring Further

6. Ask a pharmacist at a local drugstore to show you some of the products intended for use by customers with digestive problems.

How many of these products contain digestive enzymes, mild acids, or mild bases? Explain why these ingredients might be used in these products, in light of what you have learned about factors that affect the rate at which enzymes work.

7. Why do you suppose the human body is kept at a near constant temperature of 37°C? Could it have anything to do with enzymes?

Do some research to find out more.

Pre-lab Questions

What is the pH value of an acid solution, a neutral solution, and a dilute acid solution?

What function(s) do peroxisome enzymes serve?

Adding hydrogen peroxide to cut potatoes causes foam to be produced, Why?

Problem

How can you demonstrate the effect of pH on the catalase enzyme found in potato peroxisomes?

Prediction

Predict what pH condition will best support enzyme function in peroxisomes.

CAUTION: Acids are corrosive; avoid contact with skin and use water to flood spills. If you are using a computer pH probe, ensure that the probe remains stable so that it does not fall and cause a spill.

Materials

3 graduated cylinders 60 mL 3% hydrogen

2 droppers peroxide

knife 0.1 mol/L hydrochloric

dissecting probe acid solution

pH probe or indicator distilled water

paper potato

dissecting zoom microscope

Procedure

1. Prepare 3 hydrogen peroxide solutions of different pH as follows:

(a) Measure 50 mL of 3% hydrogen peroxide into each of the 3 graduated cylinders.

(b) Label the cylinders 1, 2, and 3.

(c) Use one dropper to add 10 drops of 0.5 mol/L hydrochloric acid solution to cylinder 1 and 5 drops of 0.5 mol/L hydrochloric acid solution to cylinder 2.

(d) Use the other dropper to add 5 drops of distilled water to cylinder 2 and 10 drops of distilled water to cylinder 3.

(e) Record the contents of each cylinder.

2. Check and record the pH of each cylinder. If you are using a pH probe, keep a running record of the pH.

3. Peel the potato, and cut it into 3 equal cubes. Mince each cube (sample) to an equal fineness.

4. Add one potato sample to each graduated cylinder. Use the dissecting probe to push all of the potato into each hydrogen peroxide solution, rinsing the probe between each use.

5. Observe the contents of each cylinder through a dissecting zoom microscope, and record your observations once a minute for 5 minutes.

Then test and record the final pH for each cylinder. Use a chart to organize your observations. Make a graph to compare the reaction of each sample.

Post-lab Questions

1. In terms of their initial reactions, order the hydrogen peroxide solutions from strongest to weakest.

2. Which solution had the strongest final reaction?

Conclude and Apply

3. How does pH affect the rate of the catalase reaction?

4. In terms of pH, what kind of internal environment would allow peroxisomes to function most efficiently?

5. Identify the dependent and independent variables in this experiment.

Exploring Further

6. What do you predict the natural pH of a potato to be? How could you test this?

7. Conduct research to find out what other factors can affect the rate of an enzyme reaction.

pancreas slides, H&E stain
immersion oil
lens tissue and cleaner
eyepiece reticules
stage micrometers
dissecting stereo-zoom microscope

Examine a histological specimen to practice proper focusing of the condenser and setting of the field stop and condenser diaphragms. A 1 m thick section of pancreas or other tissue stained with hematoxylin and eosin is ideal. A typical histological specimen is a section of a tissue or organ that has been chemically fixed, embedded in epoxy resin or paraffin, sectioned, and stained with dyes specific for nucleic acids, proteins, carbohydrates, and so forth. In hematoxylin and eosin (H&E) staining, hematoxylin stains the nucleus and cell RNA a dark blue or purple color, while eosin stains proteins (and the pancreatic secretory granules) a bright orange-pink. When the specimen is illuminated with monochromatic light, the contrast perceived by the eye is largely due to these stains. For this reason, a stained histological specimen is called an amplitude specimen and is suitable for examination under the dissecting-zoom microscope using bright field optics. A suitable magnification is 10–40.

Equipment and Procedure –for upload.

Three items are required: a focusable eyepiece, an eyepiece reticule, and a stage micrometer. The eyepiece reticule is a round glass disk usually containing a 10 mm scale divided into 0.1 mm (100 _m) units. The reticule is mounted in an eyepiece and is then calibrated using a precision stage micrometer to obtain a conversion factor (m/reticule unit), which is used to determine the magnification obtained for each objective lens. The reason for using this calibration procedure is that the nominal magnification of an objective lens (found engraved on the lens barrel) is only correct to within 5%. If precision is not of great concern, an approximate magnification can be obtained using the eyepiece reticule alone. In this case, simply measure the number of micrometers from the eyepiece reticule and divide by the nominal magnification of the objective. For a specimen covering 2 reticule units (200 m), for example: 200 _m/10__20 _m.

The full procedure, using the stage micrometer, is performed as follows:

To mount the eyepiece reticule, unscrew the lower barrel of the focusing eyepiece and place the reticule on the stop ring with the scale facing upward. The stop ring marks the position of the real intermediate image plane. Make sure the reticule size matches the internal diameter of the eyepiece and rests on the field stop. Carefully reassemble the eyepiece and return it to the binocular head. Next focus the reticule scale using the focus dial on the eyepiece and then focus on a specimen with the dissecting zoom microscope focus dial. The images of the specimen and reticule are conjugate and should be simultaneously in sharp focus.

All materials and media used for culturing microorganisms (especially bacteria) must be sterilized before using. This is most efficiently done in a device called an autoclave that heats the material to high temperature (250 F) under pressure (~15 PSI) for a time long enough (15-20 min) to kill all microorganisms that may be present. Autoclaves are also used to sterilize old, contaminated materials prior to safe disposal via normal waste streams.
If you are not paying attention carefully, it is easy to cross-contaminate cultures by forgetting to flame a tool or changing swabs. Never use the same tool in two different cultures without first flaming it to sterilize - when in doubt, flame it. With disposable swabs or loops, when in doubt, use a new one.

• Examine the stage micrometer slide, rotating the eyepiece so that the micrometer and reticule scales are lined up and partly overlapping. The stage micrometer consists of a 1 or 2 mm scale divided into 10 _m units, giving 100 units/mm. The micrometer slide is usually marked 1/100 mm. The conversion factor we need to determine is simply the number of _m/reticule unit. This conversion factor can be calculated more accurately by counting the number of micrometers contained in several reticule units in the eyepiece. The procedure must be repeated for each objective lens, but only needs to be performed one time for each lens.• Returning to the specimen slide, the number of eyepiece reticule units spanning the diameter of a structure is determined and multiplied by the conversion factor to obtain the distance in micrometers.

Exercise

1. Calibrate the magnification of the objective lens/eyepiece system using the stage micrometer and an eyepiece reticule. First determine how many micrometers are in each reticule unit.

2. Determine the mean diameter and standard deviation of a typical cell, a nucleus, and a cell organelle (secretory granule), where the sample size, n, is 10. Examination of cell organelles requires a magnification of 40–100X.

3. Why is it wrong to adjust the brightness of the image using either of the two diaphragms? How else (in fact, how should you) adjust the light intensity and produce an image of suitable brightness for viewing or photography?

Even with frame averaging and high gain, the detection limit (as judged from a noisy, barely acceptable image) corresponds roughly to an object of moderate fluorescence intensity as seen through the eyepieces of a fluorescence microscope. Faint fluorescent objects cannot be detected at all. The solution is to use a low-light-level zoom microscope or a zoom microscope coupled to an image intensifier. (more…)

A digital image processors (digital signal processor) are a valuable tool for increasing the contrast, smoothness, and signal-to-noise ratio of the raw microscopy image and for performing complex operations such as background subtraction and enhancement of image contrast. This unit (a dedicated computer) is positioned between the zoom microscope and the TV monitor and other recording devices. The processor converts the raw image into digital form by an A/D converter, performs image processing operations digitally, and reconverts the processed signal back to analogue form through a D/A converter. (more…)