VII. Techniques of microscopy, brightfield and beyond
Transmitted and reflected light are the two basic modes for using a microscope. Transmitted light is the mode that most biologists use while materials scientists use reflected.
Transmitted light mode is used when the specimen is transparent. In transmitted light the illuminating light is shown through the specimen by the condenser. Typical applications are histologically prepared specimens and cytologically prepared specimens.
In reflected light the illuminating light is reflected of off a mirror (either half silvered or dichroic) in an intermediate tube and down through the objective. It is then reflected of off the specimen and up through the objective, through the mirror and on up to the eyepieces.
The objective acts as the condenser and objective so that they match perfectly. Reflected light systems can also be set up to do Kohler illumination with both a field and condenser diaphragm. Typically reflected light is used to look at opaque specimens like metals, plastics, rock, coal and semi-conductors.
There are biological applications of reflected light however. Fluorescence is a reflected technique and researchers looking at whole fish have used reflected techniques to see blood moving in blood vessels.
A modern microscope in bright field mode uses a light source located either directly under the field diaphragm or at the rear of the frame. If it is at the rear of the frame there is a mirror under the condenser lens to reflect the light to the condenser. While having the lamp directly under the condenser lens is simpler it restricts the wattage of the lamp that can be used. If the lamp is to big the heat generated will be enough to fry the user.
One fallacy is that adding a module for a technique automatically means that you can't use that microscope for bright field any more. Nothing could be farther from the truth. A phase equipped microscope can be used for bright field with the phase objectives with no apparent degradation. A properly set up fluorescent equipped microscope can do bright field, phase and Normarski with one set of objectives. A phase equipped microscope can do Nomarski and bright field with one set of objectives.
A modern microscope, in other words, will almost always be a bright field instrument. The always refers to some metallurgical microscopes that don't have transmitted bright field capability. The do however have reflected and the above holds true for them.
I really disagree with the practice of replicating objectives on the same nosepiece for phase. Say buying a phase 40X and a bright field 40X. This is a left over from the bad old days. Todays phase objectives work just great for bright field and anything else there performance specs. say they can do.
A. Bright Field
The most common technique in microscopy is bright field. This is the every day use of bright (white light) to view a specimen. With bright field we use a conventional Abbe or Achromatic-Aplanatic condenser. Good bright field is derived from good microscope set up. Please review the sections on Kohler illumination.
B. Polarized light
Polarized light is a fascinating technique that can help determine the chemical make up of the specimen. A typical polarizing module would include a polarizer possibly built into the condenser and an ntermediate piece containing another polarizer, called an analyser, that can be moved in and out of the light path. The polarizer or analyser must be rotatable. In very high performance system both can be rotated and the rotation measured using micrometers.
Polarizing eliminates all of the light except the light vibrating in the direction of the polarizer. Think of it this way, a try walking through a door with a five foot tube held horizontally, now turn it vertically and you can go through the door. A polarizer acts like the door, only thing that fit in the long direction go through.
In a polarized light equipped microscope there are two polarizers. One polarizer, the analyser, is turned ninety degrees to the other. This is the angle of extinction in polarization, no light will get through. If there is nothing to change the polarization of the light none will get though. However many substances, like quartz, DNA strands and uremic acid crystals, can change the polarization of the light. This is called an birefringent material.
Once this change in polarization has occurred we can see the material. To help us see differences in the polarization effect we can add a wave plate to the microscope light path. A wave plate separates the polarized light into two components.
These two components are out of phase with each other. The end result changes in phase angle already induced by the specimen are enhanced. Because of the constructive and destructive action of the light we see colors. These colors are based on the angular difference between the two light components. These colors can tell us a lot about the chemical makeup of the specimen.
Modern polarizers are made of a plastic material sandwiched between two flat glass plates. Wave plates are usually slider plates with a piece of calcite ground flat in it. The plate is rated as to the amount of retardation, such as 1/4 wave length. Don't use loose plastic polarizing material as it distorts the image. Scratches on the material reduces the polarization of the material and can give false results.
Microscopes that are equipped for research polarization have centerable rotatable stages, special de-stressed objectives, centerable nosepieces and polarizing accessories that can be adjusted critically. These accessories are not usually necessary for a clinical instrument. A rotatable polarizer near the field diaphragm lens and a fixed analyser with a slot for a wave plate is usually enough.
When setting up a microscope equipped for research polarizing remove the polarizer and set it up just like a bright field microscope. Then remove the specimen and place the polarizer were it belongs; each manufacturer varies a little in this regard. Look through the binocular tube and turn the polarizer until the light is at its dimmest,the angle of extinction, the light is extinct.
Now replace the specimen. If the specimen is birefringent you will see it. By pushing in a wave plate we can see the effects of subtler polarization differences. Polarizing light accessories can be a very useful addition to a microscope.
To get the most out of polarization you need a microscope with a potent light source. Polarization eats 75% of the light just to operate in theory. In actuality it uses at least 85% so a good light source is a must.
Polarizing microscopes should be kept scrupulously clean. A lot of stuff in the air and around labs is birefringent. These include mineral dust from the soil, most textile fabrics and starch granules. Any of these in the polarized beam path will degrade the polarization. A polarizing microscope should have its condenser and objectives cleaned of dust frequently. Using an air can or an ear syringe when you sit down to use it is a good idea.
What it means to you
Polarizing is a powerful technique for analyzing the chemical make up of specimens. A wide range of tools are made for polarized light.
To understand fluorescence it is necessary to understand the physics of fluorescence. A fluorescent material called a fluorochrome is stimulated by light at a given wavelength. This light is called the excitation light and its wavelength is the excitation wavelength.
When the excitation light hits the atoms of the fluorochrome electrons in the shell of the fluorochrome shift position driven by the incoming light energy. When they return to their original positions they emit light at a longer wave length than the excitation light. This emitted light will always be at a longer wave length than the excitation light, otherwise the law of conservation of mass and energy would be violated and we wouldn't want to do that.
Fluorescence tend to produce a very small amount of light. The effect itself tends to be inefficient, lots of light in, not a lot out. Fluorescence tends to be used to find very small amount of things like anti-bodies. If there is very little to bond a fluorochrome to then there will be very little fluorescence.
Fluorochromes can exist naturally in the specimen or be bonded to the specimen in preparation. Chlorophyll is a naturally occurring fluorochrome. Different chlorophyls excite at different wavelengths but they all emit in the red area, though at different wave lengths.
FITC is the most commonly used non-natural occurring fluorochrome. This is the fluorescent material used in many conventional clinical tests including ANA's. Since FITC is used so frequently many people think that all fluorochromes act the same way. Nothing could be farther from the truth.
To set up a fluorescence module on a microscope we must first know the excitation and emission characteristics of the fluorochrome. With out these you can't do a thing. This should be supplied by the manufacturer of the stain or kit that you are using. If they don't supply this, call them up and ask them. If they don't know then they are to dumb to do business with.
An excitation - emission specification should contain the range of wavelengths that will excite the fluorochrome. For instance FITC has one excitation point at 485nm - 495nm with a secondary point at 410nm - 420nm. A good specification will point out that the primary excitation point will produce more emitted light.
Emission specifications should contain the wavelengths that the fluorochrome bonded material will emit. This should include the conditions under which it will do it. For instance some fluorochromes will emit different wave length light based on PH. This is quite valuable but you need to know this or you can set up the microscope to ignore this.
Now that we know what the fluorochromes specifications are we can get the right modules to see it. All modern fluorescence instruments use reflected light. In the bad old days dark field was used to generate fluorescence with transmitted light but there is no reason to use this any more.
Let me reiterate; all modern fluorescence microscopes are assumed to have reflected light modules. Modern transmitted fluorescence microscopes are an oxymoron. If there is a use for transmitted fluorescence it is so rare as to be non-existent. If you are using a transmitted fluorescence microscope for anything in the average research or clinical area get rid of it now. Just put the book down and go call some microscope vendors and buy a reflected light fluorescence module for one of your microscopes.
Why this screed about transmitted fluorescence you ask? Well reflected light fluorescence just plain works better. It provides a lot more fluorescence and it is so much easier to set up that transmitted fluorescence pales by comparison.
A fluorescence module must be designed to get the most out of a specimen. The first requirement is for a lot of light. The more light in the more light out. This means that the light source must be very powerful.
Lets look at a modern fluorescence module. The modern reflected fluorescence module was designed by Dr. Ploem for the E. Leitz company. However all main line manufacturers make reflected fluorescence modules. Any clinical instrument can have one installed in a matter of minutes.
The first element of a fluorescence module is the light source. The two most used are the 100W DC mercury and the 50W AC mercury. These are both high pressure environment arc lamps that use mercury as an integral part of the gas mixture in the lamp. They also require a special power supply to start and drive the lamp.
You must take care with these lamps. If they are not changed at the correct time they can explode. This releases metallic mercury into the room. If this happens you will know it. It sounds like a hand grenade going off. Please see the chapter on light sources.
If a lamp explodes you must evacuate the room for an hour. Call your microscope service technician because something is very wrong. If you have been changing the lamp faithfully then the odds are that the power supply or the socket has a problem.
Change 100W DC mercury burners at 200 hr. intervals and 50W AC mercury burners at 100 hr. intervals. Xenon lamps are changed every 400 hours. Some power supplies for the lamps have hours or use meters built in. Every time you change the lamp you reset the hour meter. If you don't have one then you must keep a log.
When you change a mercury high pressure lamp please be careful. The quartz envelopes these lamps use are very easy to break. While this all sound scary in practice mercury lamps are simple enough to use. Just like so many other things in a lab you must be careful with them.
Of these two lamps the 100W DC mercury arc is the preferred. It produces more light, is longer lived and
is more stable. The stability is the result of the direct current that the 100W lamp uses. Alternating
current (AC) goes through a cycle from plus voltage to minus. Somewhere in there the electricity passes
zero, stops and instantly restarts.
This is what is so wearing on an AC lamp and why DC lamps are better. AC lamps are much more prone to flicker than DC because the voltage is constantly changing. The advantage to an AC system is cost. The power supplies are much easier to build than the DC since AC is what the wall socket provides.
Why use mercury at all then? There are other lamp types after all. Well, mercury lamps produce a lot of light efficiently and they provide it in a discontinuous spectrum. While this doesn't look like an advantage lets look at this.
Basically the sun generates a continuous spectrum of light. In the visible range the sun produces all of the wavelength and they are all about the same strength. This is called a dichroic source. A mercury lamp generates a range of wavelengths that is strong is some areas and weak in others. If we look at a graph or the strength of the light at all visual wavelengths the mercury lamp's looks like peaks and valleys.
This makes a mercury lamp self filtering. If the wavelengths that you want are present they will be present in a strong peak. Al that you have to do is isolate that peak and not worry about adjacent wavelengths. It turns out that mercury lamps have peaks that are very useful in real life. A peak is located at just the right place for FITC, another is located at a good place for near-UV stains like DAPI.
What if you need continuous wavelengths? What if the stain you are using excites at a wavelength were mercury lamps are weak? Then you should use a Xenon lamp. These come in 75W DC and 150W DC versions. While they do not have the self filtering of the mercury they do produce a lot of dichroic illumination.
The 75W DC Xenon is the most common system. However the 150W DC short arc Xenon may be the most powerful lamp you can get for fluorescence. The 150W DC short arc Xenon is not all that available from microscope anufacturers but is available from some reputable after market manufacturers.
All manufacturers make a 100W Halogen system. This is not an arc lamp, it is a filament lamp. It produces far less light than any arc system. It is only usable in the high blue excitation range for fluorescence applications with a lot of fluorescence. If this sounds like FITC you are right. I don't recommend these system however. While they are cheaper they produce so little light that I wonder whether they can affect the quality of the fluorescence. They are also not usable for excitation other than in the blue. I feel that the 100W Halogen fluorescence systems are to limiting and way to underpowered.
The light source is contained in a lamp housing preferably with a mirror in it. If the lamp housing is opened to change the lamp a good system has a safety switch to turn off the power supply. However always disconnect the power supply while changing lamps.
Mirrors in lamp housings are parabolic mirrors that collect the light generated by the back side of the lamp and reflect it back into the light path. A mirror should, and usually will, increase the light output of the light source by 40% or so. Fifty percent is theoretically possible but not practical.
So why don't all light sources have mirrors? Primarily complexity. An improperly set up mirror system may not increase the light output at all. In fact an improperly set up mirror can decrease the light output!
The lamp housing will have controls to center and focus the lamp and mirror (if any). It is critical that the lamp be on center. The most powerful lamp will not produce good fluorescence if it is not properly aligned. You will need to check your manual to find out which controls move the lamp and mirror. Each lamp housing design is different.
The lamp housing connects to the illuminator. This fits between the frame of the microscope and the head. The illuminator contains the field and aperture diaphragms (if any) and the filter system. The filter system may have up to four filter modules although two is the most common.
The filter system is the heart of a fluorescence microscope module so lets take a look at it. Modern fluorescence modules use some type of filter module usually referred to as a cube. The cube contains all the necessary filters for a fluorescence technique. The cube contains three types of filters called the exciter, dichroic mirror and the barrier filter.
The exciter filters the light from the illuminator to give just the excitation light that the fluorochrome needs. Interference filters are used for excitation by and large. Sometimes a stained glass filter will be perfectly adequate however. Most users will find a UG 1 stained glass filter adequate for near-UV stains while most people use an interference filter for FITC. Please see the section on filters. Sometimes two discrete filters are used. One filter will be called an edge filter since it is used the narrow the range of the main filter. Most illuminators will have a place for an extra filter in the illuminator. With improved interference filters this complexity is becoming a thing of the past.
The next filter is both a filter and a mirror. This is the neat part of Dr. Ploem's design, the dichroic mirror. The dichroic mirror has a wavelength (called the center point) below which it reflects and above which it transmits. It is set at a forty five degree angle in the filter cube and reflects the exciter light into the objective.
Dichroic mirrors act as a dual filter. They reflect the exciter light and act as a filter by passing any stay high wavelength through them. They then pass the emitted fluorescent light and reflect back toward the lamp any stray light below the center point.
In any microscope system we have a condenser and an objective. In a reflected system the objective is the condenser. They are therefore perfectly matched and in most cases are operating with the maximum NA. This is another really neat feature of the reflected light system.
The condenser (read objective) illuminates the specimen and the specimen fluoresces. The fluorescence light goes into the objective, at least all that the objectives NA will allow. Objectives used with fluorescence should have as much NA as possible. This will produce the most light to the specimen and gather the most light from the specimen.
Most manufacturers have a line of special objectives for fluorescence. These objective have high NA and are as simple in design as possible. The more glass there is in an objective the more losses there are. This is why sometimes Plan Apochromatic objectives do not do a very good job at fluorescence, they are to complex. Sometime an Achromat will do a very good job because it is so simple. Recently several manufacturers have produced Plan Apochromats especially designed for fluorescence that work very well indeed.
Another problem in fluorescence with objectives is auto-fluorescence. This is when parts of the optical train fluoresce at a given wave length. Typically this happens with objectives when the excitation is near the UV. Some materials in the lens fluoresce and cause the image to look veiled and mushy. If you suspect auto-fluorescence change the objective. If the veiling goes away then the objective was auto-fluoresing.
Modern fluorescence objectives are designed not to do this. This is a major design goal of all manufacturers. The problem is that it is more expensive to make objectives free of auto-fluorescence. Some of the materials that do it can help make cheap, good bright field objectives. If you use an auto-fluorescing objective with bright field you will see no alteration in image quality.
The excitation light goes through the specimen and hopefully is not heard of again. It is a good idea to lower the condenser as this will help get rid of stray reflections. This is the only time it is wise to lower the condenser. Some manufacturers provide simple dark slides that fit into the stage so that the condenser doesn't have to be moved and there will be a black, absorptive area below the slide.
The emitted fluorescent light is now passed through the dichroic mirror. The barrier filter then acts to separate out the fluorescent emitted light and any stray light. Some specimens have both naturally occurring and induced fluorescence, a barrier filter can isolate the desired fluorescence and remove the unwanted light.
Barrier filter can be long pass or band pass. More and more system are using interference band pass filters for barriers. These tend to be the most versatile. However they do have a higher filter factor. This should be taken into account if the fluorescence you are working with is very dim.
Fluorescence modules frequently have a position for a second barrier filter. While this is a convenience sometimes it is not a substitute for a well designed barrier filter.
In the real world the most frequent problems of fluorescence microscopy are light backgrounds, not enough light and finding a dark area for the microscope. Theoretically a fluorescence module should produce an image with a jet black background. However this doesn't happen all the time.
One major problem in getting a black background is filter inefficiency. Filters don't cut the light like a knife but have a slope at both the turn on and turn of sides of the filter curve. While this may not seem like much of a problem, and isn't in bright field, fluorescence produces so little light that small filter inefficiencies can drowned out the weak fluorescent light. Filter inefficiency is as much a barrier problem as an excitation problem. If a well thought out exciter filter is exciting the fluorochrome you want and another you don't want the odds are that the problem can be solved with a band pass barrier filter. The correct one will let you see only the fluorescence that you want and eliminate the fluorescence you don't want.
On the other hand if the exciter not producing the ideal wavelength it might be exciting the desired fluorochrome but not very much while it excites an undesired fluorochrome very efficiently. When a band pass barrier is installed while it only lets through the wave length we want there isn't enough light for us to make a good judgement on the specimen.
There is no substitute for a good filter system. You should always try a fluorescence module with your technique before you buy it. There are several after market filter makers that do just a great job. There expertise and skill are a real boon to all fluorescence users.
The most frequent problem with fluorescence modules is not enough light caused by misalignment of the lamp. It is critical that you center the lamp in the lamp housing. While all lamp housing designs are different in control placement they are similar in intent. Lets see how to center a lamp.
First we need a fluorescent specimen. For convenience I use a histologically prepared specimen stained with H and E. I prefer one with a lot of cartilage. I also like the slide label to be white paper. Why this combination you ask? Well every bit of this, the specimen and the label auto-fluorescence.
Start with a low power, open the field diaphragm and aperture diaphragm (if any) and focus on the paper. White paper has brightener dies in it that fluoresce like mad. Focus the collector lens of the illuminator until you see the image of the arc of the lamp. Now center the arc image just slightly of center if the system has a mirror and dead on center if it doesn't.
If you are really out of wack you will need to remove an objective and find the arc image. When you have it centered in that field then go to a low power objective and center it there. You will find that it is slightly different. For light sources equipped with a mirror you must find the mirror image of the arc. Center it right beside, not on top of, the actual arc image. The ideal is that the center of the field will contain the actual image of the arc and the mirror image side by side. Remember that if you change the actual image the mirror image will shift.
Now move the specimen over and take a look at the tissue. Make sure that the images are still in the center of the field and just side by side. If not adjust. Now adjust the collector lens until the field is fully illuminated.
Change objectives to the one you usually use and make sure that the field is fully illuminated. This will take some fiddling I know but it is the best, heck the only way I know to make sure that the field is properly illuminated. Manufacturers each have there own little weird device to help you center the lamp and they universally don't work.
Well ok, they kinda work but I don't use them for anything but just getting into the ball park. I know this procedure sounds fiddly and not definite. Well that's right it is fiddly. However it works and for good reason. Nothings perfect including the light path in microscopes and the arc position in arc lamps. That all means that fiddling is the only thing that is going to work.
We all know that fluorescence is very dim. That means that the observer must be dark adapted to see the fluorescence very well or at all. So the fluorescence equipped microscope must be in a dark room. Well that's in a perfect world. In the real world you need to be in a dark room too. When you are budgeting for a fluorescence module budget for a place to put it.
Well what about curtained off areas and other kludges like that? They don't work. First of all the user needs dark and lots of it to get their eyes to dark adapt. Next they need to stay dark adapted and not have their pupils contact every time they look up.
The other problem with kludged up solutions to fluorescence equipped microscopes is heat. Arc lamps produce a lot of heat. Some Xenon lamps also produce a lot of ozone. All this needs a well set up ventilation system.
A good environment for a fluorescence equipped microscope is a separate room with its own thermostat. The room should have a light excluding door arrangement, two doors or what ever, so some one can come and go with out disturbing the people using fluorescence. Each microscope should have a dimmable lamp beside it so the user can control the light intensity and still be able to write and draw.
I know that this is expensive but how expensive is bad technique and inefficient to you? If you have to dim half the lab to do fluorescence that is going to cost you in the long run. If you don't dim the lights and you can't see then what good is fluorescence to you? What is it worth to you not to get heat prostration from the light source?
Fluorescent light output is obviously increased by more NA in the objectives. It is also increased by lower magnification in the eyepieces. Ten power eye pieces are the most that should be used for most fluorescence. I don't see the need for 15X or 20X eyepieces.
What this means to you
Fluorescence produces light from fluorochromes by exciting the fluorochrome with one color of light so it emits at another color. While not the simplest technique manufacturers have developed tools to simplify this powerful technique. Fluorescence does not produce a lot of light.
Contrast Enhancement or Optical Staining in general.
Dark field was the first contrast enhancement or optical stain technique that was invented. Through out the 19th century and into the twentieth dark field was the technique of choice for increasing the contrast of specimens. Though supplanted in popularity by phase contrast, dark field is still used.
What optical stains do is to increase the contrast of a specimen with out the potentially invasive problem of staining the specimen. Optical stains are very useful were ever the specimen is unstained, partially stained or very weakly stained. Conventional staining techniques can usually produce a contrast range of 6 to 1 or as high as 16 to 1. If the specimen is unstained the range can be as low as 1 to 1 or as high as 1 to 4. Optical stains work very well with unstained specimens to bring out more contrast in the specimen. A typical example is the use of phase contrast for looking at unstained urinalysis specimens.
A typical dark field module for a microscope includes a oil dark field condenser and a 100X objective with a diaphragm. The dark field condenser produces illumination at an N.A. that is greater than the N.A. of the objective and that has a central stop in the condenser. What happens is that the illuminating light, zero order light, never reaches the objective.
What does reach the objective is the diffracted orders. The elimination of zero order light produces a great increase in contrast. However the elimination of the zero order decreases resolution. Also high N.A. objectives can't be used since they will pick up the zero order light of the condenser. This is why 100X objectives for dark field have diaphragms to reduce N.A.. With out them the dark field effect doesn't exist.
Dark field has other problems. High performance dark field condensers have to use oil. Since the N.A. of the condenser has to be high enough to exceed the objective N.A. oil is a must for all but the lowest powers. Setting up a dark field oil condenser is just like setting up a bright field condenser with out a condenser diaphragm except messier and you can't see anything until it is right. A very difficult task!
A simple dark field condenser can be made with just a central stop inserted in an Abbe condenser. A coin on the field diaphragm lens can make a low power dark field condenser. However this design eats light. At 100X it takes a lot of light to make one of these work. All manufacturers make an Ultra dark field condenser. This uses a partial spherical mirror to deflect the light headed to the center of the condenser out to the edges. These condensers are complex and expensive.
Ultra type condensers use oil. There are dry dark field condensers with reasonable efficiency but they will not usually work above 40X (.70 NA). Since Ultra condensers don't work well for low power objectives a dedicated dark field microscopist will have to have two condensers to cover the full range of magnifications.
All manufacturers make universal condensers with dark field included. With out exception these are simple central stops that rotate into place in the condenser. These do not perform as well as dedicated dark field condensers. For occasional use they are OK but for any real use they use to much light to be practical.
Dark field has the advantage that while it lacks resolution it has very good ability to detect the presence of objects it can't resolve. This fact was used at the turn of the century to develop some very exotic techniques. While this is little used today it does explain why a dark field microscope and specimen should be kept clean. The smallest dust particle on the slide will show up as a point of light.
Dark field has been supplanted by phase contrast and D.I.C. Nomarski as the preferred optical staining technique for all of these reasons. Except for some exotic techniques like some silver stains and some types of autoradiographs I can't see why it would be used. Certainly I have to question it's use as a daily technique.
What it means to you
Dark field is an antique technique that produces great contrast and low resolution with great difficulty. Not recommended for routine use.
Phase microscopy was developed by Dr. Zernicke earning him the the Nobel Prize for Physics. Learn to use this technique correctly and receive the plaudits of your co-workers! Phase microscopy is also an optical stain.
Phase operates by reducing the zero order light and shifting it a quarter wave length, producing constructive and destructive interference. So what does that all mean?
A phase microscopy module contains a phase condenser or condenser add on and a phase contrast objective. The condenser contains a ring that only lets light pass through it. The specimen is illuminated by this light passing through the condenser phase ring.
The objective has a conjugate phase ring in it made of two layers. One layer reduces the intensity of the light, a neutral density filter, and the other shifts the light by a quarter of a wave length of light. The un-refracted, zero order, light will pass through this ring. The phase ring in the condenser is conjugate to the phase ring in the objective.
In any microscope most of the light after the specimen plane is non-diffracted, zero order light. When this light passes through the ring in a phase objective the intensity of the zero order light is reduced. Then the phase angle of the light is changed by a quarter wave length. This sets up constructive - destructive interference between the zero order light and the diffracted orders. Contrast is enhanced in two ways, from the reduction of the intensity of the zero order and from the interference between the zero order and the diffracted orders.
A good phase system can increase contrast markedly from very low to quite usable. However the phase ring in the condenser does have a haloing effect on the specimen. If you are looking at unstained cells with a phase equipped microscope you will see a halo around each cell. This is an effect generated by the phase ring in the condenser.
In unstained tissue you may not be able to see the phase haloing since the image is much more complex, but it is there. In some applications this haloing can be quite distracting, in others it is of little importance when weighted against the gains in contrast. In fact in some image analysis applications the phase halo can be used to advantage to distinguish a cell from the background.
Phase contrast has a number of advantages over other optical staining techniques. It is simple to produce. Phase only requires an Abbe condenser as its basic condenser optics. Just about any type of objective can have a phase ring installed by the manufacturer, achromat to apochromat, plan or not.
Most phase modules sold are sold with a rotating condenser. A rotating condenser will have phase rings for most if not all of the manufactures phase objectives. To change from one phase objective to another simply change the objective and rotate the correct phase ring into place on the phase condenser.
The first step in using phase in the real world is to set up correct Kohler illumination. With out this all bets are off. Then you need to align the phase rings. The one in the condenser is the only one that can be changed. This is referred to as phase centering. To center the phase we first need to see the phase ring in the objective and the ring in the condenser. To do this we need a phase centering telescope or a Bertrand lens. A phase telescope is put in place of an eyepiece and a Bertrand lens is built in to the microscope but they do the same thing, allow us to see both phase rings. Remember the phase rings are conjugate.
To use a phase telescope, remove an eyepiece and insert the phase telescope in the eyepiece tube. Focus the phase telescope by pulling (lengthening) it or pushing (shortening) it. When it is in focus you will see two rings, a light one and a dark one. The light one is in the condenser and the dark one is in the objective.
If you have a built in Bertrand lens the procedure is similar. Swing in the Bertrand lens and focus. These controls are unique to each manufacturer so consult your manual. Now it is time to center the phase rings.
Centering controls come in two types, one centers all and separate centering for each ring. The best is one centers all. The only reason a manufacturer produces a separate centering system is that they feel they can't control their manufacturing process well enough to produce a one centers all system. Every manufacturer except the remnants of A.O. produces a one centers all system.
Read your manual and find out were the phase centering controls are. With the phase rings in focus center the light ring over the dark ring. When you look at the specimen you will see good phase. I you have a separate centering system you will need to do this for each phase objective.
Some manufacturers produce "simple phase" or "clinical phase" systems. These usually use just the 40X objective and one phase ring. Some of them, such as the old A.O. system and the Nikon clinical phase system, are very elegant and work quite well. Some others are kludgey and just about don't work. Try any of these before you buy and consider the advantages of a real phase system; ease of use and flexibility.
The manufacturer can control the contrast enhancement effect by varying the amount of neutral density in the objective and whether the quarter wave material is on top of the neutral density filter or not. Some manufacturers make a range of phase contrast objective but for commercial reasons only consistently sell only one kind.
This is to bad. Sometimes a change in phase type can be a real help. However the cost to stock the different objective and the lack of understanding about phase made these objectives to expensive for manufacturers to market effectively. When you are buying a phase module ask to see the different type of phase the manufacturer has, at least in pictures.
What this means to you
Phase is the most used contrast enhancement technique. It does a good job at a low price. The only draw back is the phase halo that surrounds the specimen.
E. Differential Interference Contrast After Nomarski
Differential Interference Contrast After Nomarski is a type of Differential Interference Contrast developed by Dr. Nomarski. For simplicities sake we will refer to it as Nomarski. During the fifties and sixties there were many type of interference contrast systems manufactured however Dr. Nomarski showed how to manufacture a system with excellent performance and ease or use for relatively little money. At present only Nomarski is manufactured.
What Nomarski does is to render differences in specimen refractive index as differences in apparent height. This gives the specimen a three dimensional appearance. The optical stain effect is striking. The lack of a phase halo and the hills and valleys effect of the three dimensional appearance makes working with an unstained specimen much easier.
A Nomarski microscope must be equipped with Plan objectives. Some makers allow the use of Apochromatic objectives while others use only Achromats. Since Nomarski uses polarizers a very powerful light source such as a 12V-100W halogen is necessary.
The first thing then light beam in a Nomarski microscope encounters is a polarizer. Now that the light is all vibrating the same direction it passes through a Nomarski prism located in a rotating condenser. This is the neat part of the Nomarski system.
The Nomarski prism phase delays half the light by an amount known as the shear angle. The light then passes through the specimen. The shear angle is less than the resolution limit of the microscopes so the shear angle will not effect resolution. If the sheered light or the ordinary (non-sheered) light goes through a longer path then there will be a phase angle shift between the two beams and contrast will be enhanced.
Now the light goes through a second Nomarski prism and polarizer. The second Nomarski prism rejoins the light beams and sets up constructive and destructive interference that enhances the specimen contrast based on the refractive index or path length difference in the specimen. The difference between high and low refractive or path length differences are now seen as differences in height as the image takes on the appearances of hills and valleys.
A typical application of Nomarski would be to increase the contrast of an unstained Ameoba. The sheered light passes though the mounting medium just at the very edge of the critter and the ordinary light passes through the membrane. The difference in path length or view it as refractive index means that the edge will have its contrast increased from 1-1 to 1-5 or better.
This will make the Amoeba appear to rise out of the medium or sink in to it depending on the set up of the Nomarski. The second Nomarski prism is always adjustable to control the amount of Nomarski effect we see. The prism is either located in an intermediate piece or built into a collar attached to the objective. If it is in the intermediate piece then one second prism will be used for all objectives and the second polarizer will be built into it. If the second prism is in a collar then the polarizer will be in an intermediate piece.
If a prism is used for each objective each objective used for Nomarski will have to have a Nomarski prism designed for it. The design and manufacture of Nomarski prisms is based on the NA of the objective. While similar NA objectives may sort of work with prisms they will not work as well as a matched prism-objective pair.
To set up a Nomarski equipped microscope first set up Kohler illumination. This is critical to the performance of the effect. Pay particular attention to the setting of the condenser diaphragm. Set the condenser for the correct prism for the objective you want to use and insert the second prism.
Make sure the second polarizer is in place. You now need to check the polarizers to make sure they are at extinction (crossed). To do this remove an eye piece and observe the back focal plane of the objective.
You will see a black line across the illuminated field (a fringe). The manufacturer will have only one adjustable polarizer in the system. Turn this until the fringe is at its darkest. If your microscope is equipped with a Bertrand lens you can use it to see the fringe.
Now adjust the second prism so that the image is at its darkest. Adjust the prism slightly ahead and then behind this point. What did you see?
Well at its darkest the image didn't look very impressive. When the prism was moved then you could see the hills and valleys, three-D, effect of Nomarski. On one side of the darkest point nucleuses were hills and on the other side they become valleys. Either is correct, the prism should be set just slightly to one side or other of the darkest point.
Some Nomarski systems allow the Nomarski prism to be moved far enough to develop a colored background and image highlights. This is called Nomarski colors. This is caused by the second Nomarski prism recombining the light with a large phase angle difference.
This is a really pretty effect. Usually this is not of any practical value however every once in a while this effect will make thing more veiwable. It's also real valuable for winning microscope photo contests because it's very pretty and judges just love it.
A limit to the Nomarski effect is its directionality. The 3-D effect will appear to go only in one direction, the shadowing direction. This is controlled by the alignment angle of the Nomarski prisms. It is not affected by the adjustment of the prisms.If you look closely the effect will be much stronger in on direction than in another.
Since there is nothing you can do about the prism alignment you must move the specimen to get the best effect. A rotating stage is an excellent accessory for a Nomarski system. You can rotate the specimen to find the best angle for it. This may not be necessary for routine viewing but for photography it is a must.
The advantages of the Nomarski system are many. The resolution of the microscope is not affected, the contrast enhancement effect is very high and it is not that difficult to operate. In fact since the light in a Nomarski system is sheered there is theoretically possible that Nomarski increases the resolution of a microscope. This effect is very slight if at all.
The disadvantages of Nomarski are cost and increased complexity. The cost is substantial, around $3,000 however a Nomarski system should last the full life of the microscope. The complexity increase is not that bad but it is real. You must understand the parts of the Nomarski module and how to set them up or you will get mush.
Presently almost all Nomarski modules are sold for industrial reflected light work or research. It is sad that clinical people haven't used this system more. Its applications for parasitology and mycology are, I feel, extensive. However since it has not been used in the past it is not used in the present. Go figure.
What this means to you
Nomarski is a powerful contrast enhancement technique. It produces a 3-D looking image with high contrast and excellent resolution. If you are using unstained of low contrast stained specimens take a look at Nomarski.
Co-bright field is when the transmitted bright field illumination system is turned on when the fluorescence is being used. You will have to balance the intensity of the bright field illumination to match the fluorescence. Sometimes a neutral density filter will be necessary to reduce the bright field intensity. This allows you to see the whole specimen. The fluorescing specimen will be its usual color and the rest of the specimen will be the color of the barrier filter. This is one problem, the specimen will be filtered through the barrier filter. With FITC this leaves a of-red colored specimen.
Another problem is that most fluorescent specimens are not counter stained. The image will have very low contrast. Because of this co-bright field is usually used to help find the specimen and then is turned of. The specimen is then observed by the fluorescent illumination.
If the specimen is of low contrast a good co-technique is co-phase. Transmitted light phase is used to view the non-fluorescing parts of the specimen and you can still see the fluorescing parts of the specimen. You still have to be careful to adjust the transmitted light intensity.
This technique can work quite well. The advantages are that phase objectives work reasonably well for most fluorescent techniques and phase systems really increase the contrast of unstained specimens. What looked like cell outlines in bright field look like whole cells with co-phase. The specimen will show the distinctive phase halo.
There are draw backs however. The primary one is the objectives. While a lot of different types of objectives are made with phase rings not all objectives are made for phase. It may turn out that the objective you want to use is not made for phase. Fluorescent light output is slightly lower with phase objective than non-phase objectives since some of the light has to go through the phase ring in the objective. This will only be a problem if the fluorescence is very dim.
Co-Nomarski is possible but the draw backs are large, however the payoffs can be big to. In co-Nomarski the specimen is viewed with both fluorescence and Nomarski. The non-fluorescing parts are imaged by Nomarski. This provides very good contrast and no phase halo.
The big draw back to co-Nomarski is light loss. The returning fluorescent light must pass through a Wollaston prism and a polarizer along with the transmitted light. This will decrease the intensity of the fluorescence by 65% or so. If you have a lot of fluorescent light then it will work. If the specimen is dim then all bets are off.
Until recently it was very difficult, and a bit of a kludge, to put both fluorescence and Nomarski modules on a bench top microscope. In fact not all research microscopes could do it! Before you commit to this technique check with your microscope representative to see if it is feasible for your instrument. Oddly enough for a period of a year or so Olympus's bench top microscope could do co-Nomarski while its research microscope couldn't.
Co-techniques are two of the above techniques used at once. In practice fluorescence will be one of them. The problem with fluorescence is that if it doesn't fluoresce then you can't see it. By using co-bright field, phase or Nomarski you can see the non-fluoresing parts of the specimen.
What this means to you
Co-techniques pair a transmitted light technique with fluorescence to allow you to see both fluorescing and non- fluorescing parts of the specimen.
Unusual, over looked and weird techniques
Hoffman Modulation Contrast
Designed by Dr. Hoffman, a really great guy, this contrast enhancement system uses a slit rather than a ring to reduce zero order light. This system produces an image that looks like Nomarski but doesn't quite equal Nomarski for performance.
Hoffman has been called "poor mans Nomarski" because they both give a pseudo-three D effect to the image. Nomarski does it by shearing the light and Hoffman does it by illuminating the specimen from one side with a slit.
The slit in a Hoffman system is conjugate to a slit in the objective that, like phase, reduces the intensity and shifts the light by a quarter wave. It is very important when using a Hoffman system to get all of this aligned. In later Hoffman systems there is a polarizer located before the slit to help adjust contrast.
The advantages of the Hoffman system are good contrast enhancement, low cost versus Nomarski, no phase halo, long working distances, and it can be used with plastic tissue culture vessels. Nomarski can't really be used with plastic containers since the residual strain in plastic containers shows up in the polarized light system in Nomarski and will obscure the specimen.
Hoffman has had its greatest success in the tissue culture market were its combination of features provides real advantages for users. There are drawbacks however. Hoffman is a directional technique like Nomarski. The "shadowing" of 3-D effect is from one direction and that is controlled by the Hoffman optics, not the specimen.
Hoffman systems are produced by Hoffman and not by microscope manufacturers. This means that you must use Hoffman objectives and condensers on your microscope. If Hoffman made really good condensers and objectives this would be OK however they make at best mediocre equipment. In the past they would modify manufacturers parts for Hoffman but they have stopped this practice.
This is what makes Hoffman an out of the way technique. The objective selection is small and may not work well with objectives all ready on your microscope. Historically they have had quality and delivery problems that were embarrassing to them and to the dealers that sold their equipment.
Hoffman can be difficult, read daunting, to set up. Usually Hoffman condensers don't have positive mechanical ways to move the slit. This means that you have to get you fingers into were ever the slit is and move this assembly around until it lines up. This can really be a pain.
Hoffman is normally sold by dealers. Before you buy a Hoffman system have the dealer set it up and see if they can make it work with your specimens. Since just about all microscope dealers will sell Hoffman if one dealer can't do it try another brand or dealer. Hoffman is tricky enough that a dealer with well trained people is preferable to a lower priced product that isn't well supported.
One thing I will say is that I have had training with the Hoffman folks and they know what they are doing. They give very thorough training sessions. They seem quite willing to provide training for dealer staff. They also provide very good support and sales aids. This means that if your dealer person doesn't know the Hoffman it probably isn't Hoffman's fault.
What this means to you
If you are doing tissue culture take a look at Hoffman but have it demonstrated first.
While not produced commercially any more oblique illumination is found on some older and usually high performing instruments. The Olympus Vanox I is an example. In Oblique illumination the condenser is set for Kohler illumination and then the condenser diaphragm is pulled to one side and rotated until the desired effect is achieved.
The result is that the specimen is illuminated from one side. This produces a shadowing effect in one direction. While scenic as all get out it is of dubious practical value. It is neat to use if you want to win photo competitions. The effects that it gives are stunning. The real problem with Oblique is that it is so obscure that if you set it up on your microscope and some one else comes along... they will think its in bad shape! My hunch is that it was killed of as a technique because if you don't know its there and engage it accidentally it will cost you a repair call to get it straightened out.
What it means to you
Neat old technique not produced any more. Use it at your risk but a sure photo contest winner.
In the realm of photo contest winners Rheinberg stands alone. This technique produces different colors in the foreground than in the background. Its real easy to do, although more in the nature of a Mr. Science experiment than a serious technique.
To get Rheinberg illumination you cut out a circle of colored cellophane or other colored, transparent material the size of the back opening of your condenser. Now cut out another circle of cellophane of a contrasting collar about 1/3rd that size. Cut a concentric hole in the first piece and place the second piece in it and tape this all together.
Now you have a "bulls eye" of two colors of cellophane. Place this in the back of the condenser and set up the microscope for bright field. What you see is the background in the outer color and everything else in the center color. Neat huh?
The size of the two pieces of cellophane, the two colors and the condenser diaphragm setting controls the contrast and color results. To change anything you need to bring out the scissors. This technique has never been produced commercially.
No one has ever found a real use for Rheinberg but every photo contest has Rheinberg entries and they win. I wonder whether we are overlooking this technique. Give this a try with your unstained specimens and see what it does. Let me know what you find.
What it means to you
This provides a two color enhanced image. Is this a trick or an overlooked technique? Try it and find out. Real inexpensive.