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Microscope Techniques Page 1

Light, Optics and the Microscope

I. Why we should consider these subjects:II. General outline of the subject:

A. Description of lightB. The electromagnetic spectrum

C. Refraction, refractive indexD. LensesE. Simple and compound microscopesF. Aberrations and their correctionsG. Resolution and magnification

III. Light (slides 1-3, 1-4):A. Electric fieldB. Magnetic fieldC. Direction of propagationD. Speed of propagation. = 3 x 10^8 m/sec in vacuumE. The simplified representation: Usually a light ray is drawn in a simplified manner. In this

format only the electric component is shown in a planar waveform. The height of thewaves is an indication of the intensity of the light ray.

F. Wavelength: Varies over a wide range, only a few of which are normally seen as "visiblelight". I have listed below a number of parts of the electromagnetic spectrum and theirrespective wavelength. (You do not have to memorize this.)

type of radiation wavelength

gamma rays 10-4 to 10-2 nmx-rays 10-2 to 10 nmU.V. light 10-400 nm

Violet 400-450 nmBlue 450-500 nmGreen 500-560 nmyellow 560-600 nmred 600-700 nminfrared 700 nm to 1 mmradio waves up to km

G. Interference: When two light waves of the same polarity are traveling in the samedirection, their amplitudes can be added together at each point to obtain a resultant wave.If the two light waves in question have the same wavelength, this addition could result in

constructive or destructive interference.1. Constructive interference (slide 1-5): If the two or more waves are in phase, than

the resultant wave has an amplitude that is the sum of the original waves. Becauseamplitude of the wave is an indicator of energy, this wave would look bright.

2. Destructive interference (slide 1-6): If two or more waves are exactly out of phase,the resultant wave will be the difference between the amplitudes of the originalwaves. If the two original waves were nearly the same amplitude, the resultantwave will have a low amplitude and thus would not look bright.

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3. Partial destructive interference: If two waves are partially out of phase, the resultantwave will have an amplitude lower than that of the wave produced by constructiveinterference, but higher than that produced by destructive interference. Theamplitude of the resulting wave is determined by the relative phase shift; rays nearlyin phase will produce a relatively large amplitude resultant, rays nearly out of phasewill produce a relatively small amplitude resultant.

4. Interference phenomenon determine the ultimate theoretical resolving power of themicroscope, and are exploited in both phase contrast microscopy and differentialinterference microscopy. We will consider these in more detail in the next fewweeks.

IV. Refractive index:A. Although all electromagnetic waves travel at the same speed in vacuum, the waves are

slowed down by matter (slide 1-7). The ratio of the velocity of the wave in vacuumdivided by the velocity of the wave in a given substance is called the refractive index.Different substances can have markedly different refractive indices (slide 1-8).

B. A beam (ray) of light bends as it moves from a substance of one refractive substance intoanother of a different refractive at any angle other than the normal (slide 1-9)

C. Going from a substance of lower refractive index to one of higher refractive index, it willbend towards the normal. Going from a substance of higher refractive index to lower, itwill bend away from the normal. This is why a fish underwater looks as if it is in adifferent position from where it really is.

V. The lens: Now lets what happen when two parallel beams of light pass through a convex lens.A. As the rays enter the lens each ray bends to the normal of the air/glass interface. Because

the surface is curved the rays are no longer traveling in parallel paths, but bend towardsone another (slide 1-10).

B. Further, as the rays exit the glass, they are bent away from the normal (slide 1-11), againbending towards one another.

C. Thus a convex glass lens in air is a converging lens (slide 1-12).

VI. Real and virtual images:A. The real image:1. A lens or lens system can be used to form a real image that is larger or smaller than

the actual object. Such an image is called real because it is located in a particularspace. A translucent screen held at the right place would allow you to see the imagefrom many angles.

2. An example of a real image: Note that the slides we have used make a real image onthe screen. If the screen was translucent we would be able to see it from all sides.

3. It turns out that the objective of the compound microscope also projects a real imageof the specimen much like the slide projector.

B. The virtual image

1. In contrast a virtual image is an image made in the mind. Virtual images can have aperceived position, but will not project an image onto a card.2. An example of a virtual image: when you look in a mirror, you appear to see another

person on the other side of the mirror. However, the apparent position of the imagemay be several feet in back of a solid wall.

3. When you look into a microscope (or telescope) the enlarged image will appear to belocated about a foot from the eye. However, there is no real image in that position.No one else can see that image even if you hold a translucent screen where yousee the image.

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VII. Simple microscopes (the magnifiers; slide 1-13):A. The eye cannot focus at very close distances, so there is a limit to the ability to magnify an

object by bringing an object closer to the eye.B. However consider a converging lens placed between the object and your eye. If the

distances between the object and the lens and the lens and the eye are suitable, a sharpimage of the object will be projected to the eye. The mind will form a virtual image at

about a foot that will be much larger than the object.C. The usable magnification of this system is rather limited. Perhaps the most impressive useof such a system was the microscopes of Leeuwenhoek. Incidentally, to use hismicroscopes the lens must be held right up against the eye, certainly an uncomfortableposition.

VIII. The compound microscope (slide 1-14):A. This is what we think of when we talk about a microscope.B. Notice that it is a two-step process:

1. First, the microscope objective makes a magnified real image of the object.2. Then, the ocular is used to further magnify the image.3. As we saw with the simple microscope, the mind creates a virtual magnified image

about a foot from the eye. In fact, it is possible to place a piece of paper at thatdistance, and, while looking in the microscope with one eye, trace the outline of theimage by looking at the pencil and paper with the other.

IX. Aberrations and their corrections: Up to now, we have considered only "ideal" lenses. However,lenses are never really ideal. Aberrations are not the result of poor design, but are the result ofbasic physical principles inherent in simple lenses and lens systems. Most aberrations cannot becompletely eliminated even in very high quality optics.

A. Spherical aberration:1. Cause: most lenses are composed of segments of a sphere as the result of the

manufacturing process (slide 1-16). In such a lens the rays that go through theperiphery of the lens will focus in front of rays that go through the center (Slide 1-

17)2. Possible remedies:a) Use only central portion of lens.b) Use aspherical surfaces. Generally difficult to grind lenses this way and

therefore such lenses are expensive to make. Also, it is difficult to solve foraberrations so it generally works better for simple lens systems than complexones. Aspheric lenses are sometimes used for the condenser and ocular, but Iknow of no microscope manufacturer who does this with regular objectives.

c) Use combinations of lenses. This is the way good microscope lenses arecorrected.

B. Chromic (or chromatic) aberration (slide 1-17): 1. Cause: Different wavelengths are

slowed to a different degree as they enter the glass, and thus light of each wavelength isbent to a different degree. This is exactly what happens in a prism.) This in turn resultsin different focal lengths for the different wavelengths. The blue focuses in front othercolors while red focuses in back.1. Remedies:

a) Use a filter to give monochromatic light. This is often done with inexpensivemicroscopes.

b) Combine several lenses to make a lens system with reduced chromaticaberration (see 1-18). For example, in b. a relatively weak diverging lens

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with high dispersion is combined with converging lenses of greater totalpower, but approximately equal dispersion.

C. Field curvature (slide 1-19):1. Cause: the image plane produced by a curved lens is normally also curved. This

results in only part of the field being in focus at any one time. With thin flatspecimens (such as a blood smear) only a small portion of the field may be in focus

at any one time. Field curvature is especially a problem in photography, where theultimate viewer cannot use the fine focus knob to bring the edges into focus.2. Remedy: Use of lens systems to correct for flat field.

D. Other faults: The following are not inherent in the design of simple lenses, but are the resultof imperfect design or manufacturing of lenses or lens systems. Therefore, strictlyspeaking they are not aberrations, although in common language they are frequentlyreferred to in that way.1. Coma: Off axis points do not look like a point but rather like a comet.2. Astigmatism: Horizontal and vertical lines have different levels of focus. Common in

the human eye and some of you may have glasses to correct for this condition. Inmicroscope lenses, this results in a fuzzy image.

3. Distortion: Objects in the field do not display their true shape. Most common kindsare "pincushion" and "barrel" (slide 1-20).X. Magnification and resolution:

A. Magnification is simply the relative size of the image (virtual or real) and the object. Byusing more powerful lenses or a greater distance between the objective and the ocular, animage can be made as large as you want. We could even project and image of abacterium on the moon (assuming that we had a bright enough light). However,magnification, by itself is not very useful to a biologist.

B. As a first approximation, resolution is the ability to see detail. Resolution is the reason weuse a microscope.

C. An example of the difference between magnification and resolution: (slide 1-21).

D. We will return to resolution and factors lead that influence it after we have talked about thecomponents of the compound microscope.

The Brightfield Microscope

I. The concept of the optical train (slides 1-23 1-27)A. Light source including bulb, collector lens, field diaphragm.B. Conditioner condenser iris, DIC prisms. phase annulus, filters, etc.C. CondenserD. SpecimenE. ObjectiveF. Image filters (DIC prism, phase ring, barrier filters, analyzer etc.)G. OcularH. Receiver (eye, film camera, video camera, etc.)

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II. Strategy for study: We will start with the objective and ocular, than work through the specimen,condenser, and light source. If you really understand the brightfield microscope, it will berelatively easily to understand the other types of light microscopy such as phase, DIC, polarizingetc.

III. ObjectiveA. Purpose:

The objective magnifies the object and supplies a magnified real image to the ocular. It is herethat the constraints placed on the optics are the most severe: thus it is this component thatis most responsible for the resolution of the system. It is also one of the most expensiveportions of the microscope, good lenses commonly cost $1,000-3,000 each!

B. Objectives come in a bewildering array of types. One manufacturer may make thousands ofdifferent objectives. Objectives can differ in:1. Magnification2. Numerical aperture:

The numerical aperture is defined as the half angle of the cone of light entering orleaving a lens system (times the refractive index of the immersion medium -- one inthe case of air). Thus, the numerical aperture will generally be larger with higher

magnification objectives (slide 1-29). As we shall see, the larger the numericalaperture, the higher the resolving power (assuming perfect lenses). Thus the higherthe N.A. the better (and the more expensive) the lens.

3. Working distance:In general the working distance decreases as the magnification and resolutionincreases. However, this is not an optical law, and most manufactures also makespecial longer working distance condensers and objectives for use inmicromanipulation, tissue culture etc.

4. Immersion medium:a) air: usually not marked or marked dryb) water

c) glycerold) oil5. Types of chromatic and spherical aberration corrections (slide 1-30)

(The following specifications are minimum. Many manufacturers correct theirlenses for more wavelengths then suggested below)a) Achromats (achromatic lens: chromatic aberration corrected for 2 wavelengths,

usually red and blue. The paths of the other wavelengths are presumed to beclose to one or the other (but see slide 1-18!) Spherical aberration is correctedfor one wavelength.

b) Fluorites, Semi-apochromats: chromatic aberration corrected for 3 wavelengths.Spherical aberration is corrected for a single wavelength. Semi-apochromats

often have fluorite glass in one or more optical elements.c) Apochromats: chromatic aberration corrected for at least three wavelengths andfor spherical aberration corrected for 2 wavelengths. (See slide 1-31 for anexample of the construction difference between an achromat and aapochromat.)

6. Presence of other correction or special attributes.a) Flat field (= plan)b) Strain free (used in DIC, polarizing)c) Modified for special contrasting methods such as phase contrast.

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IV. Oculars:A. Purpose: the ocular further magnifies the real image produced by the objective to produce a

magnified virtual image (when viewing through the microscope) or real image (whenphotographing). The optical constraints are not nearly so great as in the case of theobjective, so the ocular are usually much less expensive.

B. Differences in oculars:

1. Tube length: mentioned previously. Obviously, the real image projected by theobjective must be in the proper place or it cannot be imaged by the ocular.2. Final correction for chromatic aberration. Many manufactures use the ocular to

correct residual lateral chromatic aberration. Oculars so designed are referred to ascompensating oculars. So a microscopist can use a single set of oculars with allobjective lenses, many manufacturers design the ocular to correct for the worstchromatic aberration produced by any of their objectives. They then purposelydesign the other objectives to add chromatic aberration to match the correctionpresent in the oculars.

3. Obviously, it is good practice to use an ocular from the same company that youpurchase the objective.

C. Choice of magnification for oculars:The ocular merely magnifies the real image produced by the objective, so the ocular does notdirectly contribute to resolution of the microscope. However, if the oculars are ofinsufficient power, the final image is not large enough for the human eye to pick out allthe information that is present in the image. Because people differ in their visual acuity,the minimum magnifying power needed is different for each person. If the ocular hasmore magnification power than needed, the extra magnification reduces the width of fieldwithout enabling the investigator to see any additional detail. For good microscopist andmost objectives, a 10x magnification is sufficient. Most oculars have a magnification ofbetween 5 and 20x.

V. Specimen:

One of the most overlooked parts of the optical path. For the following discussion, thespecimen will be taken to be anything between the condenser and the objective and willinclude the object, slide, coverslip, mounting medium, and immersion medium (slide 1-35).

A. The coverslip:1. The effect of a coverslip on image quality (slide 1-36).2. The reason for this effect (slide 1-37). As you can see, the coverslip affects the light

path, especially of rays at the periphery. The objective lens has been designed totake this in account, therefore trying to use an objective designed for a coverslipwithout one will result in a poor image. Most microscope objectives are designedfor a coverslip of 0.17 +/- 0.01 mm thick with refractive index of 1.515.

2. How to match coverslips with objectives:a) For critical work can measure each coverslip individually with a micrometer,otherwise use #1 1/2 coverslips which come close to the ideal.

b) Some objectives have a correction collar so the microscopist can correct for eachcoverslip.

c) Objectives can be designed for use without coverslips, but are generallyexpensive and special purpose. For example, blood smears are frequentlyexamined without a coverslip, so for critical work an NC (no coverslip)objective should be used.

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d) Matching the coverslip to the objective is most critical for high dry objectives. Ifoil is used there is much less refraction at the surface of the coverslip.Therefore, oil lenses can be used with mismatched or absent coverslips with aminimal loss of resolution.

B. Oil immersion:1. The critical angle is the angle at which light, traveling from a substance of one

refractive index (usually higher) to another (usually lower), cannot pass, but isinstead reflected. (slide 1-38).2. It is important to microscopist because it limits the amount of light (and hence

information) that can be gathered (or delivered) by an objective (or condenser) lens(slide 1-39). Because the ultimate resolution of the system is dependent on thenumerical aperture of the objective and condenser and because the numericalaperture is dependent in large part on the refractive index of the immersion medium,oil immersion optics are used for the highest resolution possible.

3. How to use oil:a) Only use oil on objectives and condensers designed for it. Oil will not improve

the optics of dry objectives because they have not been corrected for the light

rays that would be introduced in the periphery of the lens. Oil can quicklyruin non-immersion lenses by getting between optical elements.b) Use only the immersion medium for which the lens is designed. The lens will

not necessary be corrected for immersion media of differing refractiveindexes.

c) Avoid bubbles in oild) If your condenser is designed to use oil, you should put oil between the

condenser and specimen any time you use oil between the specimen andobjective.

e) Be careful using oils manufactured some time ago. Some of these oils werefilled with PCBs, now known to be toxic.

VI. The condenser:A. The importance of the condenserThe condenser does three important things:

1. It focuses light so the specimen is brightly lit. This was the first use of the condenserin early microscopes. Remember that if the image is magnified 1000x, the lightgoing through the specimen has been spread over an area 1,000,000 as great as theoriginal specimen before viewing.

2. It conditions the light so that the light will interact optimally with the specimen.3. It can contain portions of imaging systems for control of contrast etc.

B. Considerations on the use of the condenser1. If you are responsible for purchase of a microscope system, do not skimp on the

condenser. It is nearly as important for a good image as the objectives.2. Condensers, like objectives, come in different degrees of correction. The most highlycorrected condensers are referred to as achromatic aplantic (corrected forchromatic and spherical aberrations). The best condensers also have a highnumerical aperture.

3. If you are going to use immersion objectives, get and use an immersion condenser.(The only exception is when epiillumination is used where no condenser isnecessary.) In biology, this type of illumination is used mainly in indirectimmunofluorescence (see The fluorescent microscope)). For discerning

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microscopy, make sure the condenser is matched your illuminating system and tothe objectives. The brightfield condenser has a built-in condenser diaphragm, Thisis used to regulate the working NA of the condenser-NEVER use to control lightintensity.

VII. The illuminating system:This is probably the most abused part of the microscope. The microscope image can never be

better than the system that supplies the light, but careful attention to the illuminating system canresult in a pretty good image from a marginal scope.A. Parts of the illuminating system (slide 1-40):

1. Light source: In general the brighter the better, especially if the microscope is capableof contrast control such as phase contrast etc.

2. Lamp condenser (field condenser, field lens): designed to focus the light into thecondenser.

3. Lamp iris (field diaphragm, field iris). This regulates the area of the specimen to beilluminated. It should not (and in a properly set up microscope cannot) be used tocontrol light intensity.

4. Diffusing screen: In inexpensive microscope can be used in place of 2 and/or 3 above.

B. Types of illumination systems:1. Diffuse:a) Least expensive, does not need lamp iris or lamp condenser.b) Easy to set-up, almost no adjustments possible or needed.c) Not a very satisfactory illumination system for exacting requirements. Image is

normally less bright and it is not possible to adjust illumination for utmostresolution.

d) Most applicable for use in microscopes that will be used by untrained people,beginning students.

2. Koehler (Slide 1-40):a) Basic premise: The image of the lamp filament is focused onto the plane of the

condenser diaphragm. In addition, the image of the field diaphragm is focusedonto the specimen plane.b) Advantages:

(1) The field is homogeneous and bright.(2) The working NA of the condenser and the size of the illuminated field can

be manipulated separately.(3) Gives the maximum lateral resolution.(4) Gives the finest optical sectioning (longitudinal resolution)(5) Flair resulting from optics and barrels is reduced(6) Gives optimal contrast

c) Disadvantages:

(1) Is more difficult to set up than diffuse illumination(2) Set up incorrectly it will not give optimal results(3) Is more expensive than diffuse illumination

d) How to set up Koehler:Note: This will only work if the microscope illumination system has beendesigned for Koehler. This must be done every time you change objectives.(1) Focus on specimen (slide 1-42).(2) Close field diaphragm.

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(3) Raise or lower condenser so you see a crisp image of the field diaphragm(slide 1-43).

(4) Center the image of the field diaphragm (slide 1-44).(5) Open the field diaphragm so that the edges are just visible at the edges of

the field (slide 1-45).(6) Adjust condenser diaphragm:

(a) Replace the ocular with a focusing telescope and adjust telescope tovisualize the condenser diaphragm.or

Move Bertrand lens into positionor

Remove ocular and look down eyepiece tube(b) Adjust the condenser diaphragm so that it results in a lit area of

about 2/3 of the total area. Now the working condenser numericalaperture is slightly less than that of the objective.

(c) Replace the focusing telescope with the objectiveor

Remove Bertrand lensorReplace ocular

(7) Check image and contrast. The maximum resolution is with the workingNA of the condenser equal to that of the objective, so if resolution iscritical the condenser diaphragm can be opened a little. On the otherhand, closing the condenser further will increase contrast. In general, thecondenser diaphragm should be open as far as possible consistent withthe required contrast (but never further than is required to make the NAof the condenser equal to that of the objective).

(8) A warning:

Note that neither the field diaphragm not the condenser diaphragmshould be used to regulate light intensity, because both are adjusted forthe proper optical requirements. Instead use light intensity controls orneutral density filters.

VIII. How to recognize brightfield:A. As the name suggests, the surround is normally bright (white).

1. But occasionally a microscopist will place a colored filter over the light source so thebackground (and the specimen) would be colored.

2. Also sometimes people put ink around the nominal specimen to block out light thatwould otherwise make up the image. This does not break the rule, b/c the inkparticles are themselves specimens, even if they are not the main point of the

micrograph.B. Many biological specimens are normally near colorless, so the specimen will often beartificially stained.

C. The lack of characteristic features that go with the other contrasting techniques.D. A gallery (slides 1-62 to 1-65)

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Important Optical Characteristics of the Light Microscope

I. Resolution: Perhaps the most important optical characteristic:A. Definitions:

1. Informal:Up to now we have simply referred to resolution as the ability to see detail. This is

still a good working definition but is sometimes not sufficient.2. Formal (slide 1-47 -- 1-48):

The resolution of a system is the minimum distance at which two very small butbright (or dark) objects can be distinguished from one another. Note that an objectsmaller than the resolving power of the system can be observed but it is not possibleto visually separate two or more of these objects if they are closer to one anotherthan the limit of resolution.

B. What determines resolution?1. Wavelength of light used:

a) The shorter the wavelength, the better the theoretical resolution. However thewavelength of visible light varies over a small range, so one can only make

minor improvements over white light by using blue or green rather than redlight. It is possible to use U.V. light for a slight increase in resolving powerbut one must use quartz lenses (U.V. does not penetrate most optical glass)and a special viewing system (U.V. cannot be seen by the human eye and isvery bad for eyes. However, the relatively small increase in resolving powerdoes not make up for the considerable expense and complexity necessary forsuch as microscope. Consequently U.V. microscopes are very rare.

2. The numerical aperture of the system: The numerical aperture of a objective lens orcondenser is defined as the sine of the half cone angle of light entering or leavingthe lens system times the refractive index of the immersion medium. The higher thenumerical aperture of a lens system, the higher the amount of information that can

be captured by the lens system and the greater the potential resolution.C. The formula to determine maximum theoretical resolution:

We have talked about resolution in general terms until now. However, we finally have achance to quantify it for a given system. The theoretical resolution (that is the smallestdistance between two points that still results in those points being resolved) is:

r = 1.2 (wavelength of light used) / (NA obj + NA cond).

Where the NAs are working NAs; not necessarily the ones marked on the lenses.Sometimes this equation takes different forms, especially if the author is talking aboutself-luminous objects, or other simplified or specialized cases.

D. Example: About the best one can do with a light microscope is:Wavelength = 450 nm (blue/violet)

NA obj. = 1.4NA cond. = 1.3

(1.2) (450 nm)/(1.4 + 1..3) = 200 nm = 0.2 umE. Note that this is the best resolution possible and requires perfect optics and careful

microscope adjustment. Fortunately, the optical microscope comes closer to perfectionthan any other machine I know of. Resolution close to the maximum theoreticallypossible is obtainable, although expensive, with microscopes from several manufactures.

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F. Also note again that the objective and condenser are the two most important components ofthe system. The oculars should be good enough that they do not degrade the imagesubstantially while supplying further magnification, but normally do not have a majoreffect on the resolution of the microscope.

II. Depth of field:A. Definition: The distance between the closest and furthest objects in focus at the same time.

A lens system with a large depth of focus will be able to form images of overlappingstructure simultaneously.B. Desirability (or undesirability) of a large depth of field: Where the specimen is arranged in

a nearly planar manner, a large depth of field allows all objects to be in focussimultaneously. Fore example, this could be an advantage when differentiating cells ispreferred. This allows fine optical sectioning and 3- dimensional reconstruction ofcomplex objects such as cells (slide 1-52). In addition, the image at any given focus isclearer because objects immediately above or below the level of true focus do notimpinge on the image.

C. Factors that influence depth of field:1. The numerical aperture of the objective: the higher the numerical aperture, the smaller

the depth of field. Thus, good quality objectives tend to have a shallow depth offield.2. The setting of the condenser aperture: the further the condenser diaphragm is closed

the smaller the working condenser numerical aperture, and the greater the depth offield. (Note: this is another reason why the condenser diaphragm should be set upfor Koehler illumination.)

3. The contrasting method used: DIC in particular, will tend to have a relatively shallowdepth of field.

III. Field of view: this one term has two separate meanings, normally you can pick out the propermeaning by the context.

A. The trivial definition (see slides 1-55, 1-56): The absolute size of the specimen that can be

imaged by a particular optical system. In this definition, a 20x objective will normallyhave a field of view twice as great as an 40x objective and half of that of a 10x objectiveAS LONG AS THE REST OF THE OPTICAL SYSTEM REMAINS THE SAME.

B. The important definition (see slides 1-57, 1-58): The size of the image at the rear apertureof the ocular. This is related to the size of the virtual image that you see when you lookthrough a microscope. A narrow field of view will result in the microscopist feeling thathe is looking down a tunnel, while an exceptionally wide field of view will give theimpression of being surrounded by the specimen. It turns out that is much easier to makeand correct optics that will result in a smaller image at that plane, then one that will give alarger aberration-free image. This is one of the few recent major improvements in thelight microscope. All parts of the microscope must be optimized to give a wide field

including the condenser, the objective, the prisms, and most importantly, the ocular.Oculars that are designed to produce a wide field are frequently labeled wf. Olderoculars so marked may have a 15 mm field of view; more modern systems can boast afield of view up to 30 mm. Putting a wide field ocular in an older microscope may notgive a wider field if the rest of the optical system is not designed to transmit a wide fieldimage to the ocular.

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Practical Hints

I. Changing magnificationsA. Usually change by changing the objectives. However in rare cases you may choose to

change magnifications by changing the oculars.B. The objectives are almost always placed in a rotating nosepiece. Always rotate the

nosepiece by grasping the knurled surface -- NEVER BY GRASPING THEOBJECTIVES THEMSELVES. Rotating by means of the lenses could damage them.

C. After changing objective lenses, the microscope must be readjusted for Kohler illumination.If the microscope is well aligned, you can simply adjust the field diaphragm until you seeits image at the periphery of the field, and adjust the condenser diaphragm so that the lightintensity is just short of maximum brightness. (Of course, by now you know that thisworks because the light will be at maximum brightness with the condenser NA greaterthan or equal to the objective NA. As the condenser NA falls below the objective NA thebrightness will decrease. Optimal settings require that the condenser NA is slightly lessthan the objective NA.) For very critical work completely align as described previously.

II. Determining magnifications:

A. Definition: The magnification is the size of the image divided by the size of the object.Multiplying the magnifications of the ocular and objective lenses gives an approximatemagnification. Here the image is a virtual image, not a real one, so the magnification isonly approximate. The image is assumed to be located about 10 inches in front of theviewers eye. Obviously, this method is not satisfactory to determine the magnification ofa photograph, and the magnification on a camera negative is likely to be much less thanthis.

B. Use of a stage micrometer: To determine exact magnifications, you typically photograph aspecial slide (the stage micrometer) that has lines spaced at known intervals. Determiningthe negative (or video) magnification is a simple matter of dividing the line spacing of theimage (measured) by the line spacing of the object (known).

III. Cleaning the optics:A. The importance of clean optics (slide 1-60)B. By far the best way to clean optics is not to get them dirty. Every time you attempt to

clean the optics you run the risk of scratching them. Additionally, some commoncleaning agents can dissolve the cement that holds the glass lenses in the objective barrel.Probably as many lenses are ruined by cleaning attempts as any other way.

C. If they must be cleaned, follow manufactures directions. Note that different manufacturersused different cements and coatings, so what works well in one case could ruin optics inothers. If oculars etc. are dusty remove dust by (in order) blowing with an ear syringe,blowing with canned air, lightly brushing with a camels hair brush, or cleaning with amild solvent (see below). The last should be done only as a last resort.

D. Be especially sure never to get oil on one of the non-immersion lenses. (If you do so byaccident clean immediately with solvent.

E. Cleaning with solvent and cotton or solvent and lens paper:1. Be sure you need to undertake this chore.2. Be sure to use good quality cleaning materials. Lens paper is specially manufactured

to contain minimal amounts of abrasive. Never use Kleenex which looks soft but infact has abrasive particles inside. Likewise use good cotton and be sure it in un-oiled (some drugstore cotton has oils added).

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3. Before cleaning oil contaminated lenses, blot (NOT RUB) excess oil off the lens withclean lens paper.

4. Check with the manufacturer of the lenses and follow exactly what they recommend.Many of the solvents will dissolve lens cements and this will ruin lenses. Differentcements have different solubilities in the various solvents so what is recommendedmay differ from one manufacturer to another.

5. Start with the most mild solvent possible and see if that will work before moving on tomore aggressive solvents. In general, water is the least aggressive, ethanol ormethanol next, with ether, xylene, benzene etc most aggressive. Some people usecommercial lens cleaners and one even recommends Windex. Use only at amanufacturers suggestion.

6. The solvent should be applied to clean cotton or lens paper. Briefly brush over theelement working from the inside out. Never rub as this will grind dust particles intothe lens. Use each cleaning tool only one for a few seconds. This will minimize thedamage that the dirt can cause the lens. Remember that a Q-tip or piece of lenspaper is virtually free compared to the cost of a new lens.

D. Cleaning interior surfaces (inner surfaces of lenses, prisms, etc.):

1. Dont.IV. Carrying the microscope:A. Use two hands.B. Do not invert the microscope, pieces may fall out.

V. Storing the microscope:A. Keep out of dust.B. Do not store in humid area. Fungi can grow on the cement between the lens surfaces

making the lenses cloudy. Such lenses are not normally repairable.

Contrast Methods I

I. Overview:

A. The nature of the problem. Brightfield microscopy has as good a resolution as any othercontrasting method, and fundamentally better than some others. However, resolution isonly useful if objects can be seen, that is if they have enough contrast. An object that stopsonly 5% of the light rays that pass through it will seem invisible to the eye. Most biologicalobjects have inherently low contrast.

B. The solutions:1. Stain biological objects (slide 2-1): We will say more about this later, but it has the

obvious disadvantage that the most frequently used staining procedures requirekilling the specimens.

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2. Using optical methods to increase contrast: These methods result in an amplificationof one or more features of the light interaction with the specimen. We shall talkabout the following:a) Darkfield (slide 2.2)b) Polarizationc) Phase contrast (slide 2.3)

d) Differential interference contrast3. Using light emitted from the specimen itself. We will discuss this underFluorescence Microscopy.

II. The darkfield microscopeA. Principle

In brightfield microscopy, the image is made up predominately by light that has not beenrefracted or has been refracted very little. However, it is possible to exclude much of thislight, and conversely collect a portion of the light that has been refracted. This is done bypreventing those rays that normally pass through the specimen and directly into theobjective from reaching the specimen at all. Instead light is focused on the specimenfrom such a direction that it will not enter the objective unless it is refracted. Thus, the

only light that enters the objective has been refracted by the specimen (slides 2-4, 2-5).This results in a light image on a dark background - just the reverse of the brightfieldimage (slides 2-6, 2-7).

B. Darkfield microscopy in practice:1. To work, darkfield microscopy requires that the working numerical aperture of the

condenser is larger than that of the objective. Objectives designed for darkfield mayhave a diaphragm built into the objective so that the working numerical aperture canbe adjusted for best results (slide 2-5).

2. The direct (undiffracted) light is excluded from entering the objective by using adarkfield condenser (or a modified brightfield condenser) that illuminates thespecimen only from the sides. The simplest way to do this is to put a darkfield stop

immediately under the condenser (slide 2-4). This stop intercepts the light rays thatotherwise would have directly entered the objective but its transparent edges allowlight to illuminate the specimen from all sides. Special darkfield condensers reflectthe central rays to the periphery for greater brightness (2-5).

3. Only a small percentage of light that enters the condenser will enter the objective, thusthis method is very wasteful of light. Light intensity is often the limiting factor,especially at high magnifications (remember that the stop must block (or redirect)all the light that would have otherwise entered an objective of relatively highnumerical aperture).

4. The theoretical resolution of darkfield microscopy is smaller than bright fieldmicroscopy because of the necessity to restrict the numerical aperture of the

objective lens.C. How to identify darkfield images.1. The surround should be dark2. The specimen can scatter any color light. Therefore the specimen can be any color or

combination of colors.3. Gallery (slides 2-6,2-7)

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III. The polarization microscope.A. Light and polarization:

You saw this diagrammatic illustration of a light ray the first few minutes of the course(slide 2-10). I show it here to remind you that an electromagnetic wave is polarized, thatis, that the wave has an up and down, right and left. Ordinary light has waves with alldifferent orientations and the light is said to be unpolarized. However, it is possible to

create beams of light in which all the waves vibrate in the same plane. Such light is saidto be polarized. There are several ways to polarize light (slide 2-11 to 2-13).B. Polarization and interference.

We have mentioned constructive and destructive interference. Now I can tell you thattwo rays can only interfere with one another when they are vibrating in the same plane(i.e. are polarized). You will remember that our drawings implicitly showed the two raysin this manner. You can get interference in unpolarized light only because the waves ofeach polarity interfere with the others of the same polarity.

C. An aside: How do Polaroid sunglasses work?1. Light reflecting off of water, a car hood etc. is polarized in the transverse direction.2. The Polaroid in the sunglasses absorbs light polarized in that direction. Thus, the

sunglasses absorb most of the reflected light. Unreflected light is unpolarized so theglasses will only absorb a portion (ideally 50%) of that light. The result is that mostof the reflected light is absorbed, but only about half of the unreflected light. Ofcourse, the glasses can also be pigmented to absorb more than the minimumsmentioned above.)

3. Problem: What do you think you would see if you wear Polaroid sunglasses and werelying with your head parallel to the beach looking at the ocean?

D. The effect of two polarizers:Definitions: If two polarizers are placed in series, the polarizer the light enters firstretains the name polarizer while the second is now called an analyzer.1. If the two polarizers have the same orientation, the analyzer has no effect because the

polarizer has already absorbed the light vibrating in the appropriate plane.2. If the two polarizers have an orientation of 90 degrees to one another, little if any, lightpasses through because the polarizer absorbs light of one polarity and the analyzerabsorbs the light of the opposite polarity. Such an arrangement of polarizers iscalled crossed polars.

3. Any substance that rotates the plane of the polarized light can appear darker (if thepolarizer and analyzer have the same orientation, and the substance rotates the planeof polarized light 90 degrees the substance would appear black on a graybackground) or lighter (if the same substance was placed between crossed polars itappears gray on a black background).

E. The polarization microscope:

1. Simply a cross between the situation we have just described and the bright fieldmicroscope. The microscope has polarizer between the light source and thespecimen, and the analyzer between the specimen and eye.

2. Used to determine if objects rotate the plane of polarized light, or to study one or moreof these substances. Objects that rotate the plane of polarized light are generallycomposed of many parallel subunits and are called birefringent (slides 2-14 to 2-19).

F. Polarization in practice.

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B. Mechanism1. A phase annulus (annular diaphragm) is present in the condenser and a matched phase

plate is present in the objective (slide 2-23). The annulus illuminates the specimenwith a hollow cone of light and the phase plate maximizes interference between thediffracted and non-diffracted light in the real (=intermediate image). The objectivemagnifies this real image exactly as it does in the brightfield microscope.

2. The mechanism by which the specimen, the annulus and the phase plate maximizeinterference is not difficult, but, for lack of time, we will not consider it here. Askyou instructor for details if interested.

C. Characteristics of phase contrast:1. The phase contrast microscope emphasizes small differences in refractive indexes

between areas of the specimen.a) Therefore, it works very well for objects that have a refractive index only slightly

different from the surrounding area (phase retarding capability of less than 0.1wavelength).

b) Because it amplifies differences in refractive indexes so dramatically, it does notwork as well with specimens that have a very different refractive index from the

surround.2. Phase contrast creates rings around objects, which can make the image confusing(slide 2-27). These rings are particularly noticeable if the change of refractive indexis relatively severe and becomes annoying if the object retards light more than aboutone half wavelength. It also makes measuring the size of objects inaccurate.

3. Phase contrast does not use the full numerical aperture of the optical system.Therefore resolution is actually worse than brightfield.

4. The image produced by the phase contrast microscope is artificial. It emphasizeschanges in refractive indexes between objects. Usually this involves emphasizingedges of an object rather than the object itself. Further, the same component mightlook light if surrounded by one substance and dark if surrounded by one with a

different refractive index.5. The phase contrast mechanism reduces the image brightness about 98-99% comparedto brightfield. Therefore it is important that the microscope light be much morepowerful than is necessary for brightfield.

6. Phase contrast is considerably more expensive than brightfield, but not nearly asexpensive as DIC.

D. Phase contrast in practice.1. It will only work with phase contrast objectives. (But you can use phase contrast

objectives with other forms of microscopy with only a very slight compromise inoptical quality.)

2. Usually have several different phase annuli in a turret under the condenser, so you can

match the phase annulus to the objective (slide 2-28).3. The phase annulus and phase plate must be aligned before use.a) Set up Koehler illuminationb) Swing the proper phase annulus into position.c) Align the phase annulus and plate (slides 2-29, 2-30):

(1) Visualize the phase plate and the phase annulus. Their images are at rearaperture of the objective. (This is the same plane at which you view thecondenser diaphragm when you are setting up Kohler illumination.) Tovisualize them do any of the following:

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(a) Insert the Bertrand lens and leave the ocular in place.(b) Or replace the ocular with a phase telescope .(c) Or remove the ocular and look down the barrel.

(2) Move the condenser or the phase annulus centering controls so that theimages of the annulus and the ring of the plate overlap.

(3) Undo step 1 to get back to the image of the specimen.

E. How to identify phase contrast microscopy images.1. The surround is often featureless, but often less bright than brightfield2. Borders are prominent. Areas where there is a marked difference in refractive index

will often have rings.3. Phase contrast microscopy works with specimens composed of very low contrast.

Knowing if a particular specimen meets these criteria can give you a hint.4. Gallery (slides 2-31 to 2-32; also see 2-46 to 2-48 to compare DIC and phase)

III. Differential Interference Contrast (DIC)A. The name:

Differential interference microscopy was invented by a German named Nomarski and hissystem was patented by Zeiss. Therefore, the term Nomarski interference contrast which

is often used incorrectly to cover all types DIC, can only refer to systems made by Zeiss.Many other microscope manufacturers have similar systems that fall under themicroscope manufacturers have similar systems that fall under the generic term DIC.They are sometimes named after the person who was responsible for working out thedetails for that company (i.e. Smith interference contrast).

B. General principle (slide 2-35):Polarized light is split into two rays of polarized in opposite planes by a special modifiedprism. These two rays, travel through the specimen on separate but parallel paths. Thetwo rays are very close together -- in the case of a 100 x objective the rays might be 0.22m. apart. (Note the illustration in 2-35 shows them inches apart this is highlydiagrammatic!) Because they are vibrating in different planes they can fit in nearly the

same space without causing destructive interference. At boundaries one of these rays willtravel through the region of higher refractive index and be slowed more than itscompanion. Thus, there will be a phase shift between the two rays. After passing throughthe objective, the rays enter a second prism and analyzer where they are recombinedwhere constructive and/or destructive interference occur. The real image so produced ismagnified by the ocular to form the final magnified image as in the other forms of lightmicroscopy.

C. Characteristics of DIC:1. Objects appear in raised or sunken relief (slides 2-36, 2-37).2. Traditionally image is presented with shadows as if by the setting sun. However,

this is arbitrary, and shadows can be reversed by adjusting the prisms.

3. The apparent relief is not real, but an optical trick. What seems to be peaks are areasof different refractive index, and may in reality be perfectly flat or even valleys.Must use caution in image interpretation (slides 2-38 to 2-40).

4. Unlike phase contrast, halos are not present, this means DIC is superior for looking atedges of objects.

5. DIC uses the maximum numerical aperture of the condenser and objective. Therefore,the resolution of DIC can be as good as brightfield.

6. DIC allows very fine optical sectioning (see slide 2-41).

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7. Works best with objects of relatively large phase retarding ability (between 0.1 and 1.0wavelengths). Thus is it complementary to phase which works best with objects oflesser phase retarding ability.

8. Works very well with video microscopy.9. The DIC contrast mechanism reduces the image brightness by about 99% compared to

brightfield. Therefore it is important that the microscope light be much more

powerful than is necessary for brightfield.10. Because DIC uses polarized light, it may not be suitable for specimens that rotatepolarized light themselves.

11. The prisms and the strain-free optics are very expensive. This makes DIC muchmore expensive than the typical phase scope.

D. DIC in practice:1. The physical arrangement of the polarizer, analyzer and prisms vary tremendously

from manufacturer to manufacturer. In most, a prism matched to each objective isinstalled in a rotating turret under the condenser. In most systems there is a singleupper prism used for all objectives in the body of the microscope. The analyzermay be combined with the upper prism or separate.

2. Normally the positions of the polarizer, analyzer and first prism are fixed and theposition of the second prism can be varied by the user to obtain the best image.E. How to identify DIC images.

1. Object appears in pseudo 3-dimentional relief.2. The surround is often featureless, but often less bright than brightfield.3. Borders are prominent, but often less so than phase contrast.4. DIC microscopy works with specimens composed of low contrast and moderate phase

retarding ability. Knowing if a particular specimen meets these criteria can give youa hint.

5. Gallery (slides 2-41 to 2-44; also see 2-46 to 2-48 to compare DIC and phase)IV. The Fluorescence Microscope

A. Cf. fluorescence and types of microscopy we have talked about up to now:With brightfield, darkfield, polarizing or interference microscopy the specimen is imagedby differences in light passing through the specimen and light that passes through thesurround. However, fluorescence uses light generated by the specimen.

B. Principle of fluorescence.Many substances have the ability to absorb light energy and store it for a brief period oftime. That energy has to go somewhere, and is normally released as heat, light, or acombination of the two. If the object releases light it is said to fluoresce. The photon oflight released by the absorbing molecule cannot have more energy than the originalabsorbed photon, and usually has less, the remainder of the energy lost as heat. Theenergy of the photon is inversely proportional to its wavelength, therefore the

fluorescence emitted by the excited molecule will invariably have a longer wavelengththan the light originally absorbed.C. Fluorescence in biological objects:

Many biological objects fluoresce naturally. Many others can be made to fluoresce by theaddition of certain fluorescent tags (see slide 2-51).

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D. The transmitted fluorescence microscope:Not generally used anymore, but the principles of this microscope are easier to understandthan the indirect fluorescence microscope (slides 2-52 to 2-54).1. Light source:

The light source for fluorescent microscopes generally produces a high intensitywhite light source. In this case it is directed upward towards the specimen and

objective.2. The exciter filter:This filter is designed to filter out all wavelengths except for those that will beabsorbed by the object to be viewed (in this example blue).

3. The specimen:The object is illuminated with light of which it absorbs small amounts. After a shorttime the object fluoresces with the resultant light being of lower energy (in this casered) than the absorbed photon. The intensity of the fluoresced light is almost alwaysorders of magnitude less than that of the exciting beam.

4. The objective:Both the fluoresced light and the stimulating beam not absorbed by the specimen

enter the objective.5. The barrier filter:The barrier filter is chosen to pass light of the wavelength of the fluorescence butabsorb light of the wavelength of the exciting beam.

6. The ocular:As you should know by now, the ocular will further magnify the real image andproduce the final image.

E. The problem with the transmitted fluorescence microscope:Filters can be very good, but they are not perfect. The intensity of the signal is often verymuch smaller than the intensity of the stimulating beam. Thus even if the barrier filtercan stop 99% of the photons of the stimulating beam, enough photons may get by to

overwhelm the much smaller signal produced by the fluorescence (see slide 2-55).F. The epifluorescence microscope:Nowadays fluorescence microscopes are built on a slightly different plan (slide 2-56).1. Basic construction (slides 2-57 to 2-58)

The epifluorescence microscope, directs the exciting beam down onto the specimenthrough the objective. Thus, the majority of the stimulus beam that is not absorbedby the specimen passes the stimulus beam that is not absorbed by the specimenpasses through the specimen and out the other side of the condenser. This means abrighter exciting beam can be used and that a dim signal can be observed withoutbeing overwhelmed by stray light of the excitation wavelength.a) The light source:

The light source is usually a Xenon or Mercury arc because these sources havea wide range of wavelengths and are very bright. For less critical use, othersources are sometimes used. The light source is usually mounted quite highon the microscope.

b) The exciter filter:This is exactly analogous to the exciter filter on the transmitted fluorescentmicroscope.

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c) The dichroic (dichromatic) mirror:This is what makes the epifluorescent microscope possible. A dichroic mirrorpasses light of some wavelengths and reflects light of other wavelengths. Aparticular mirror is chosen for exciter/barrier filter combination such that themirror reflects light of the stimulating wavelength but passes light of thefluorescent wavelength. In our example, this mirror reflects blue light down

through the objective to the specimen.d) The specimen:As we have seen the specimen absorbs a small percentage of the stimulatingbeam, the rest of the beam passing harmlessly through the slide. Some of thestimulating beam is reflected back through the objective and some of thefluoresced light also enters the objective.

e) The dichroic mirror (again):The fluoresced light passes through the mirror, much of the stimulating lightthat has been reflected into the objective is reflected back towards the lightsource.

f) The barrier filter:

The barrier filter passes the fluoresced light, but absorbs the remaining lightfrom the stimulus beam as well as light of other wavelengths that may haveentered the objective.

g) The image:The final image should consist almost entirely of light that has been fluorescedfrom the specimen.

G. Characteristics of the epifluorescence microscope:1. The most difficult part is sample preparation.2. Lenses must be transparent to light of both the stimulating and fluoresced

wavelengths. This is not always the case as the stimulating beam is often in theblue/U.V. range and many glasses and lens cements absorb in those wavelength.

3. Must choose proper exciter and barrier filters and the dichroic mirror (slides 2-60 to 2-63). Ideally, you pick a separate set for every fluorescent tag used so that the exciterfilter passes most of the light of the stimulating wavelength (and little else), and thebarrier filter passes as much fluoresced light as possible (and little else). Thedichroic mirror should be chosen to reflect nearly all light of the stimulatingwavelength but pass nearly all of the fluoresced light. Normally, the two filters andthe mirror are sold in a set that can cost upwards of $1,000. In addition, thenarrower the band of transmission, the more specific the signal, but at a reduction inbrightness. This is particularly vexing because many samples have a very dimfluorescence.

4. For critical work, you must have a very bright light source. This is expensive, and

must be aligned carefully (a non-trivial task). If you ever use one, be aware that theunfiltered light is dangerous to look at; it is very bright and filled with U.V. Mostlight sources should be left on for at least 30 min at a time, and never turned onwhile still warm from their last use. The bulbs are also expensive (about $150.00)and have a limited lifetime.

5. Because the objective acts as both condenser and objective, its numerical aperture isvery important. A high numerical aperture objective lens will not only result inmaximum resolution but also in maximum brightness.

6. There is no need of a condenser.

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8. Is quite expensive.H. Epifluorescence microscopy in practice:

1. Particularly useful in indirect immunofluorescence (slides 2-61, 2-65). Here theinvestigator tags an antibody with a fluorescent component and allows the antibodyto react with the antigen. By observing the pattern of fluorescence the investigatorcan determine the distribution of the original antigen in cells and tissues.

2. Operation of the fluorescence microscope is easy, but whoever is responsible for themicroscope will be very protective of it. Be sure to follow his or her directionsexactly as not to injure yourself or damage the microscope.

E. How to identify fluorescence images.1. Surround is normally black.2. Fluorescent tags usually fluoresce over a narrow range of wavelengths. Therefore

each tag will only be a single color. (Note that there can be multiple tags ofdifferent colors.)

3. Gallery (slides 2-62 to 2-65; also see 2-66 to compare fluorescence and phase)

Recording the image

I. Principle:Simple just replace eye with camera (slide 3-1).

II. General considerationsA. Consider sensitivity of the camera the more sensitive the better. Images are often not

terribly bright, especially if contrasting methods such as phase or DIC are used. In many

cases fluorescent images are very dim.B. The camera must be kept rigid on the microscope.1. If the image is magnified 1,000 times then any vibration will also be magnified 1,000

times.2. Thus if possible use a microscope with a photohead (often called a trinocular head), so

that the camera can be on-axis of the optical train.C. If you are taking color images, you may have to worry about color temperature. Different

light produce different colored white light. (See slide 3-2) Many light sources even willgive different colors depending on how much current flows through them. (Thus turningup the intensity of the light might also cause the image to have a different backgroundcolor.)

III. Digital MicroscopyDigital Still and Video Microscopy (see slide 3-3).A. Advantages:

1. Can use the maximum numerical aperture of both the objective and condenser lenses,which results in the maximum theoretical resolving power for the microscope.

2. Contrast can be raised electronically by the camera so the microscopist does not haveto make optical sacrifices to maintain adequate contrast.

3. In most cases, the highest resolution digital images are monochrome. Software can beused to superimpose several monochrome images.

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4. Digital microscopy allows interfacing with the computer. This allows:a) Subtraction of background image.b) Summation of many images for maximum information content.c) For motility studies, the investigator can electronically compare images at two

times and subtract images of all objects moving.B. Disadvantages:

1. Good cameras can be very expensive ($30,000), although for many uses a much lessexpensive camera can be used.2. For video, the framing rate is fixed because of video technology. It is very difficult to

get more than 30 frames per second. For fast moving objects this is not enough (i.e.Chlamydomonasflagella make a complete beat in 1/60 sec.).

3. Printing out the image can sometimes be difficult.

Specimen Preparation

I. Rationale:Some biological specimens, e.g. unicellular protozoa, can be placed between a coverslip and slideand directly viewed by one of the forms of microscopy just mentioned. However, manyinteresting biological specimens cannot be treated in this manner (an elephant probably wouldntstand for it).

II. Common types of specimen preparation:A. Whole mounts (slide 3-4, 3-5):

This is a common procedure if the specimen is small. In many cases the specimen mustbe first killed and preserved. Staining is optional, and the specimen can be mounted in avariety of different media. Examples of whole mounts include insects, fungi, protozoa,and parts of animals and plants.

B. Squashes (slide 3-6):Frequently, it is desirable to look at cells of a tissue, without worrying about therelationship of cells to one another. The easiest way to do this is to squash the tissuebetween the coverslip and slide after which it may be stained and mounted. Thistechnique is commonly used to study chromosomes.

C. Smears (slide 3-6, 3-7):A smear is simply a means of spreading blood or similar bodily fluid over the slide, where

it could be examined as is or stained and mounted.D. Sections (slides 3-8 to 3-10):

This is probably the most general and useful technique for studying portions of complexorganisms. The sample must be fixed, embedded, sectioned, stained and mounted. Thereis a bewildering array of specialized fixations, embeddings, and staining procedures; infact entire courses are often taught covering only this subject. We will go over use ofsome of these steps as examples of specimen preparation. Remembers that considerationson fixation, dehydration and staining will apply to the other methods of specimenpreparation as well.

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label is usually an enzyme that makes a colored produce or a fluorescentmolecule.

E. A recent modification of the above fixation, embedding, sectioning and staining It isalso possible to freeze a tissue and to section it in its frozen state. This is especiallyuseful if the investigator wants to do enzyme or antibody localization on the tissue,because this procedure is less likely to destroy enzyme activity or antigenicity than

the traditional fixation and embedding steps.

Introduction to Electron Microscopy

I. Disadvantages with electron microscopyA. Complicated (see slide 4-1):

1. Mechanically2. Electrically3. Operationally

4. Result: needs much more operator training, the microscope is considerably less reliable, ittakes much longer to look at a specimen etc.

B. Expensive:Both to buy ($100,000-300,000) and to maintain ($4-15,000 yearly, not including routinemaintenance)

C. Electrons do not penetrate well:

Thus the specimen must be very thin. Almost all cells are too thick for electrons topenetrate. In addition, electrons will not even penetrate very far into air. This requiresthe specimen to be placed in a vacuum.

D. Only non-living cells can be easily examined:Some experimental systems have been designed to examine living cells, but the best thatcan be done is to view dying cells or organisms.

E. Therefore, one cannot show dynamic biological events. This is a very important limitationbecause most of the important biological questions involve changes over time. Suchchanges must be inferred from a series of static images (see slide 4-2). There is a certainamount of danger in this as sometimes there is no way to really check this inference.

F. Specimen preparation is vastly more difficult:

Due to the features a mentioned above, it usually takes days to weeks to preparespecimens for viewing.

G. Very time intensive:Generally at least one person must do routine maintenance, cleaning, changing filaments,etc. In some places a person is hired full-time to take care of one or two microscopes. Inaddition, the microscopist using the microscope will generally have to spend considerabletime to obtain good images.

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H. Limited field of view:This is a consequence of the higher magnification. At 100,000 magnification, the typicalfield of view will contain only about 0.01% of a typical cell cross-section. (And becausethe cell might be sliced into 200 sections, you are actually viewing less than one millionthof the cell at a time.)

I. Electrons do not have color:

Many of the pretty SEM pictures have been painted with one of those Hi-lighter pens orphotoshopped (see slide 4-3). However, electrons do loose energy as they pass throughthe specimen and thus it is possible to generate pseudo-color in expensive analyticalelectron microscopes (slide 4-4, 4-5).

II. The advantage of electron microscopy:A. Resolution (cf. slides 4-6, 4-7):

Eye = 0.1 mmLight microscope = 0.2 um (a 500x improvement)Electron microscope = 0.1 nm (a 1,000,000 improvement on the eye and a 2,000ximprovement on the light microscope.

B. Theory behind the increased resolution:

1. Particles can be thought of as waves and waves as particles (slide 4-8). Thewavelength of a particle can be statedwavelength = h(Planks constant)/mass*velocity

2. Substituting the known values for Planks constant and the mass of the electron andrestating the velocity of the electron as a function of the accelerating voltage, theequation becomes:wavelength = 12/(accelerating voltage)1/2where the wavelength is in nm, and theaccelerating voltage is in V.

3. An example:For a typical accelerating voltage of 100,000 volts, the wavelength of the electronswould be 0.04 nm or about 20,000 times smaller than light.

4. As we saw in the formula for the theoretical resolving power of the light microscope,the smaller the wavelength the greater the resolving power. The NA of the presentday electron microscopes is not high enough to take full advantage of the lowerwavelength of the electron, but the effective resolution is still about 2000x better.

The Electron Microscope

I. The electron gun:

A. Purpose:The electron gun is designed to produce a bright focused electron beam with all of theelectrons of the same energy (therefore the same wavelength).

B. Design (slide 4-10, 4-11):A small amount of current (but large voltage) is passed through the filament. Theelectrons come off of the filament and are focused by the wehneld. At this point theyhave very low velocity, but are accelerated by the large potential difference between thewehneld and the anode.

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C. Adjustment:The operator must be very careful in adjusting the current through the filament. At first,brightness increases with increasing current as more electrons are released from thefilament. However, at some point (the saturation point), a further increase of current nolonger results in increased brightness but does markedly shorten filament life. Themicroscope should be operated at, or just below, the saturation point. Changing the

filament is expensive and takes hours of an instructors time.II. Electron lenses:A. Purpose:

To focus the electron beam as glass lenses do for the light microscope.B. Design (slide 4-12):

An electron lens uses a magnetic field to redirect electrons. They are basically composedof an electromagnet wrapped around a hole. Unlike glass lenses, the focal length (hencepower), is changed by changing current. Thus, magnification and focusing are doneelectronically rather than mechanically as in the light microscope. All electron lenses areconverging; thus diverging lenses cannot be used to correct for aberrations etc. as in glasslenses.

C. Aberrations:Electron lenses, just like glass lenses have aberrations.1. Chromatic aberration:

The wavelength of the electron is determined by its energy. Therefore, electronsthat acquire a different energy level due to loss of energy with the specimen or to aninsufficiently regulated power supply will focus at a different plant than those of themain beam. This is a particular problem for thick specimens, because electrons willlose energy unevenly as they interact with the specimen.

2. Spherical aberration:This is a severe limitation on the resolution of the TEM because it cannot becorrected for by convex lenses, nor by making the lens aspherical.

3. Astigmatism: (slides 4-14 to 4-16)Astigmatism in the EM like astigmatism in regular optics means that the focallength of the lens is different in the different directions. It thus prevents a singlefocus point, and blurs the final image (slide 4-14a and b). Astigmatism is a constantworry, because astigmatism changes as the apertures (TEM equivalent ofdiaphragms) get dirty and as parts of the microscope age. Luckily, it is possible toelectronically correct astigmatism. A stigmator is a ring of electromagnets whichcan be adjusted independently (slide 4-15). By varying the current to each magnetone can remove almost all stigmatism from an electron beam. The easiest way to dothis is to focus on a hole and adjust the stigmators to give an even freshnel fringe(slide 4-15).

4. Other aberrations:Other aberrations such as curvature of field, distortion, and coma are not majorproblems in modern, well-designed and aligned microscopes.

III. Image formation:The electron source and lenses function much like an upside-down light microscope (slide 4-18,4-19).

A. Electrons are produced by the electron gun and are accelerated into a beam by the anode.B. The electron beam is focused onto the specimen by means of the condenser lenses. This

slide shows a single condenser lens; however modern microscopes almost always have

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two lenses to allow for satisfactory brightness and resolution. Not shown in this diagramare the condenser apertures that are similar in function to the condenser diaphragm of thelight microscope.

C. The object is put into the electron path immediately above the objective lens.D. After interacting with the specimen, the electrons enter the objective lens. Unlike the light

microscope, the electron microscope has only a single objective whose focal length is

electronically changed to change magnifications.E. The intermediate lens has no real counterpart in the light microscope, but further magnifiesthe image produced by the objective. This lens is usually turned off when using theelectron microscope at low magnifications.

F. The projector lens is similar to the ocular in the light microscope. It magnifies the imageproduced by the objective and, if present, the intermediate lens.

G. The final image. There are three ways in which the image can be formed:1. The viewing screen. The human eye cannot see electrons directly, so the electrons are

directed to a screen coated with cadmium and/or zinc sulfides. When an electronhits this screen, it emits a flash of yellow-green light that the operator can seethrough a glass panel.

2. Recording the image. Most modern microscopes have a hi-resolution digital camera.The image is recorded as a digital file and can be further processed on themicroscope or using conventional software. Note: The image is your DATA. Anyprocessing that one does beyond cropping, adjusting brightness and contrast must bespelled out and must not change the fundamental truth of the image. Anything elseis academic fraud.

H. Focusing (slides 4-21, 4-22):This is considerably more difficult in the TEM than the light microscope. The problem isthat what looks good to the eye is usually not in the focus that will yield the mostinformation. Underfocus gives the image with the most contrast and looks crisper thantrue focus, but results in less well-resolved objects. Most microscopists like to take

pictures a little underfocused. The easiest way to do this is by finding a hole, adjusting itto find the underfocus ring, and go towards true focus until the ring almost disappears.I. A longitudinal section through a TEM (slide 4-23):

This is what a cutaway view of the real microscope is like. Note the positions of thevarious lenses, specimen, screen and photographic plates.

IV. The vacuum system:A. Purpose:

Remember that electrons have very poor penetrating power, so an electron beam cannottravel far through air. Another reason for a high vacuum is more subtle; if an electroncollides with an air molecule, the electron will scatter. Thus an electron beam will looseits focus and ability to for a good image. The vacuum system thus removes air from the

microscope column at several points (slide 4-23).

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B. The mechanical pump (fore pump) (slide 20):The mechanical pump can rapidly move a lot of air out of the microscope column by themethod shown. However, such pumps cannot give the high vacuum necessary forsatisfactory performance.

C. The diffusion pump (slides 22-25):This is the most common mechanism for obtaining the ultimate vacuum. In this pump,

hot oil is boiled and moves up into the chimney of the pump. There it is cooled,condenses and moves outward and down. The key to the pumps effectiveness is that thisdownward stream is very rapid and carries air molecules down with it. The air moleculesare thus concentrated at the base of the pump and removed by the mechanical pump. Thistype of pump works very well, but it is very sensitive to mistreatment. If the coolingwater stops, hot oil is spewed into the interior of the super clean microscope, a processcalled backstreaming. Backstreaming can also occur if the mechanical pump shuts downor if the valve between the mechanical and diffusion pumps is closed. Such a catastropherequires weeks of work to clean up. Dont ever let it happen to you.

D. The tubomolecular pump (slide 4-32).This is basically a series of very high speed fans that whaps the molecules of air from the

column. It is very fast, although it does not remove water vapor as well as most othermolecules. Because the blades rotate so rapidly, the bearings must be very good (bearingfailures have been known to occur with messy results). This is the kind of pump we haveon the SEM. This pump is considered clean (no oil contamination), but still has to betreated carefully. It is more expensive than the diff pump and needs maintenance.

EM Specimen Preparation

I. Whole mounts:

A. Specimen support:A specimen thin enough to be electron lucent, is normally small enough to fall throughthe holes in the copper grid. Therefore, a very thin, electron transparent film is normallyapplied first. Usually this film is composed of a plastic film (formvar or colloidon) whichhas been cast onto a glass slide and floated off onto water. For critical work, a layer of Cis placed over the plastic. C is electron transparent and has the added advantage of beingconductive so it will conduct electrons away from the specimen and prevent charging.For more critical work, the plastic can be dissolved away after C coating. For theultimate in resolution, you can make a holey C film and look through the holes.

B. Types of specimens:The specimens must be thin, and lack water. Generally these specimens consist of parts

of cells (eg chromosomes or diatom frustules, see slides 4-36, 4-37). Some cells haveexceptionally thin edges that can be imaged in the scope, but very few cells can beexamined in their entirety (slide 4-38).

II. Positive stain:A. Principle (slide 4-40)

Here the specimen absorbs some substance that blocks electrons.B. Uses:

The positive stain is rarely intentionally used but some images of positively stainedmolecules and subcellular particles can be found in the literature.

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III. Negative stain:A. Principle (slide 4-40):

Here the object is surrounded by stain. The object itself is clear on a dark background.This is an easy technique and it will give high quality images. As a consequence it is verycommon.

B. Procedure:

Simply place specimen on grid, cover with an aqueous solution of a heavy metal salt,remove most of the stain solution, and allow the rest to dry around the specimen.C. Uses:

Negative stain is extensively for microorganisms (slide 4-41), viruses (slide 4-42),subcellular particles, and molecules (slide 4-43).

IV. Shadowing:A. Principle (slide 4-45):

An electron dense layer is evaporated onto the specimen, usually from one side. Themetal layer piles up against objects and is absent from their shadow. Under themicroscope, the heavy metal absorbs electrons, the sample being effectively electrontransparent. This procedure normally results in dark objects and white shadows, but some

people reverse the contrast to give an image that is more normal looking.B. Procedure (slides 4-46 to 4-48):The specimen is placed in a vacuum evaporator, and electrodes are set up in such a wayas to allow a current to heat the heavy metal. After the specimen and metal source areplaced in the correct geometrical arrangement, the vacuum chamber is evacuated and thecurrent applied. After the heavy metal has been evaporated, air is readmitted into thechamber and the finished grids are removed.

C. Uses:Used for microorganisms (slide 4-49), subcellular particles and molecules (slide 4-50). Itis extensively used to image nucleic acids (slide 4-51).

D. A modification: low angle rotary shadowing. Here the specimen is rotated as the metal is

evaporated. This doesnt result in a shadow, but the metal does pile up at the specimen.The metal atoms tend to move after deposition to form granular aggregates. This is usedto show the structure of nucleic acid and protein molecules (slides 4-53, 4-54).

V. Freeze fracture:A. Principles (slide 4-55, 4-56):

This is really a specialized form of shadowing, but it is important enough to be consideredby itself. Here the specimen is frozen, and then placed in a freeze-fracture, freeze-etchmachine. This machine is similar to the vacuum evaporator with the added feature ofbeing above to keep the specimen at about -100 to -150 degrees C. The sample is thencracked with a cold knife while under vacuum. The cracks tend to go between the leafletsof any membranes close to the path of the crack. The specimen is immediately shadowed

with a heavy metal. It is this step that will provide the contrast under the TEM. Arelatively thick carbon layer is applied next; this carbon layer is necessary to hold theresultant replica together through subsequent processing. Carbon is chosen for this rolebecause it is nearly electron transparent. After creation of the replica, the sample isremoved from the machine, and the specimen itself is dissolved. Lastly, the cleanedreplica is picked up on a grid and placed into the microscope to be viewed.

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B. Procedure:The exact procedure is complex and very dependent on the specimen and the type (anage) of freeze-fracture machine. If you were to contemplate using this technique, gettraining from someone who uses it regularly.

C. Uses:The freeze-fracture technique is used extensively to study membranes structure.

membrane fusion, etc. (slides 4-56, 4-57), because the cleavage plane normally passesthrough the centers of the membrane. It is rarely used for studies that do not involvemembranes. Freeze fracture and thin-section are compared in slide 4-58.

D. Caution: This procedure gives a three-dimensional image. However, whether the imageprojects towards or away from the observer is largely determined by the brain processingthe image, rather than by real information present in that image. Simply turning theimage over is likely to give the impression of the opposite orientation (slide 4-59).Therefore, as a microscopist you must be very careful in presenting the image to give theright topographic impression, and, as a viewer, you must be skeptical in accepting suchimages as a representation of reality.

VI. Freeze-fracture, freeze-etch:

A. Principle (slide 4-60):The procedure is in all respects similar to freeze fracture, except that after fracture thespecimen is warmed to about -100 C for a few min. Under a good vacuum, some of theice will sublime (etch) away from the specimen, allowing biological objects to projectout of the ice. After this the replica is made in the normal way. Where freeze-fracturegives images of the inside of hydrophobic objects such as membranes, freeze-fracture,freeze-etch gives images of any non-etchable structures.

B. Procedure:Even more difficult than freeze-fracture. Get help before trying.

C. Uses:Gives three-dimensional images of subcellular organelles, particles cytoskeletal proteins,

etc. in situ (slides 4-61, 4-62).VII. The Thin-Section Technique.A. Introduction:

Thin-sectioning is by far the most widely used specimen preparation procedure intransmission electron microscopy. We will, therefore, spend more time on this subjectthan the others. I would remind you that some sections of this section also are relevant toother techniques previously discussed; for example, the specimens might be fixed beforefreeze-fracture with fixatives commonly used for thin-section.

B. Overview (slide 4-65):C. Fixation:

1. Purpose:

Like fixation for the light microscope, the fixation procedure is designed to preservestructure as close as possible to the structure present in the living organism. It isalso important the fixation procedure stabilized the specimen through subsequentsteps in processing.

2. Contents of fixative solutions:Solutions used for fixation contain a fixative agent which cross-linksmacromolecules, a buffer to stabilize the pH, and one or more substances to ensurethe proper osmotic balance.

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3. General comment on fixation protocols:The optimal fixation procedures are strongly dependent on the organism and thegoal of the investigator. There is an unbelievable amount of black magic inchoosing the proper pH, time, temperature, fixative agent(s), type of buffer, osmoticstrength, etc. As an investigator, the first thing to do when choosing a protocol is tolook in the literature for good images of samples similar to those you wish to

prepare, and follow the method that seemed to work best. This is not guaranteed togive you good results, but it is a starting point. Also, it is important to use as small asample as possible to ensure adequate penetration of the fixative.

4. Common fixatives:a) Glutaraldehyde (HCO-CH2-CH2-CH2-HCO):

(1) Advantages:(a) Results in good preservation, especially of microtubules, vacuoles,

vesicles, ribosomes, etc.(b) Is a rapid penetrator.(c) Does not always denature enzymes and epitopes.

(2) Disadvantages:

(a) Does not stabilize lipids or carbohydrates.(b) Denatures some epitopes and enzymes.(3) Comments:

Glutaraldehyde is the universal first fixative. It makes very strong(essentially irreversible) cross-links between protein components. Payclose attention to pH as glutaraldehyde will polymerize above pH 8.

b) Formaldehyde (HCO-CH3):(1) Advantages:

(a) Faster penetrater than glutaraldehyde.(b) Less likely to denature sensitive epitopes and enzymes.

(2) Disadvantages:

(a) Does not cross-link as well as glutaraldehyde.(b) Cross-links are reversible.(3) Comments:Formaldehyde is extensively used where speed of penetration is important,often in a mixture with glutaraldehyde. In addition it is used inimmunolocalization or enzyme localization where the activity of a non-denatured protein is important.

c) Osmium tetroxide (OsO4):(1) Advantages:

(a) Fixes lipids and fatty acids.(b) Imparts some contrast to the tissue.

(2) Disadvantages:(a) Dangerous.(b) Will not fix carbohydrates, nucleic acids(c) Will not by itself fix many important cellular structures such as

microtubules etc. and can even break down previously stabilizedactin filaments.

(d) Denatures everything.(e) Is a relatively slow penetrator.(f) Expensive.

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Sectioning itself destroys the knife edge. Therefore, even under thebest conditions, you can only get a few good sections before youhave to move the knife and start over (slide 4-70).

b) Diamond knives (slide 4-71):(1) Advantages:

(a) Can cut consistently good sections.

(b) Are only very slowly dulled by proper cutting of the block.Therefore you can cut thousands of sections from th

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