Introduction to the Basic Microscopy

8/10/2019 Introduction to the Basic Microscopy

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Introduction to the Basic Tutorial.

Introduction andtypes of microscope stand.

1. Component parts of the microscope.

2. Image-forming light paths through the Khler-illuminated microscope.3. Setting up Khler illumination.4. The specimen.5. Numerical aperture (N.A.).

o 6.1.Diffraction, resolution and image formation.

o 6.2.Refractive Index, angular aperture and N.A.

o 6.3.Magnification and image detail.

6. The oil immersion objective.7. Errors of adjustment: how to recognize and remedy them.8. Settings for comfortable microscopy.

This tutorial is for those whose work involves day-to-day use of the light microscope, as wellas those approaching the instrument for the first time.

It gives an easy to follow procedure for ensuring that the instrument is set up not a hundred

miles from the maker's specification. It is in effect an instruction on how to set up brightfield

Khler illumination. (August Khler, an employee of the Zeiss microscope company,

proposed this method of setting up the microscope around a hundred years ago).

An understanding of this simple and elegant method of illumination is a prime requirement

for both routine and critical microscopy, as it underlies all the commonly encountered forms

of microscope illumination -- darkfield, phase contrast, interference contrast and the various

types of incident illumination -- which are covered in detail by other tutorials.

The instruction is aimed at the user of the laboratory microscope. Instruments below this level

may or may not have an adjustable substage condenser, and since this component is essential

for satisfactory imaging, especially at the higher powers, instruments without one are not

considered here (but are dealt with in the tutorial on minimal microscopy).

The intention is to pass on sufficient information that microscope users may better understand

their instrument, set it up in a way that produces a satisfactory image in a comfortable setting,

and proceed with their work.

Those aspiring to aficionado status in critical microscopy and to the use of the microscope as

a source of pleasure in itself should proceed to the Advanced Tutorials.

Types of Microscope Stand.Even though the outward appearance of the microscope has changed greatly over the last

century or so, the purpose of the design has not. The function of the microscope stand has

always been to hold the same components -- mirror, condenser, specimen, objective and

eyepiece -- on a common optical axis, and to allow finely controlled axial movement between

them.

The stage supports the specimen to be examined and allows specimen movement in a plane

perpendicular to a fixed optical axis.
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Three basic designs of microscope stand of the past 150 years.

In terms of mechanical stability, the end result of this evolutionary process is the modern

design in which the limb/base is a rigid fixture and all focusing actions are applied to the stage

-- seen on the Olympus microscope (right) above -- a construction much better suited to

supporting heavy accessory devices such as zooms, trinoculars and photomicrographic

cameras.

Recent microscopes incorporate the lamp into the base, enabling the instrument to be moved

from one place to another without upsetting the relationship of the components, so the lamp

can now be added to the list of components held in alignment by the stand.

The detatchable lamp unit seen on the Olympus is now rare, except as an optional extra for

less expensive microscopes sold originally with only a mirror.

In the four hundred years of its development, the microscope has been adapted to any number

of specialized tasks. The illustrations below give some idea of the variety in design. Invertedmicroscopes have become standard if somewhat specialized laboratory instruments, and the

aquarium microscope, whilst no longer manufactured, would be sure to have a modest

following if it were offered for sale today.

Some unusual microscope designs of the last hundred years.

The Component Parts of the Microscope.

The Watson "Service" microscope shown here is of a general design developed in the early


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1900s, and microscopes of this type are still in wide use. It is a simple, rugged, well-made

instrument of a kind that, with proper care, will still be functional after hundreds of years of

frequent to occasional use.

Additionally, it has a drawtube -- the collar seen between the eyepiece and the body tube --

which makes it possible to alter the mechanical and optical tubelength of the instrument. The

use of this refinement will be covered in the advanced tutorials.

Component Parts of the Watson Service Microscope (c. 1950).

The optical components are those of all compound microscopes, and even though this model

has no integral lamp, this discussion will begin with the lamp, and proceed in sequence via the

mirror to the eye.

The Microscope Lamp.

Microscopes require, especially at the highest powers, intense

illumination. The intensity of a light source depends not so much

upon its absolute power as upon the amount of light emitted from a

given area of the source -- lumens per square millimetre rather

than just lumens. To achieve high intensity, various lamp designs have come and gone,

but one of the commonest and most satisfactory is the low-voltage tungsten filament lamp,

with the filament in the form of a tightly-wound flattened grid.

The combination of a suitable quartz-halogen bulb with a concave spherical reflector

works well and is also in wide use.

For more detail on lamp issues:

Closeup of lamp filament.
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Centreing the lamp using the projected lamp filament image.

Considerations in the design of a microscope lamp.

The Microscope Lamp: Performance Requirements.

There is one essential condition that a microscope lamp designed for Khler

illumination must fulfil, and a number of desirable ones. The essential condition is that

the lamp condenser optics must be capable of projecting an image of the source large

enough to fill the substage condenser of the microscope.

Image of lamp filament formed by lamp condenser in the plane of the substage

diaphragm.

The main structural elements of the microscope lamp body are the source, in a housing

which allows movements for centration and focusing, a condenser lens with an iris

diaphragm, and a filter holder.

This is in turn mounted on a stand which allows the lamp body to be oriented andreliably clamped in any position.

Desirable attributes of the lamp condenser are:

It should have as large a diameter as possible to enable illumination of the large fields

covered by low power objectives.

It should be as powerful as possible in order to project the required filament image in

the shortest possible distance.

It should have the highest possible aperture (lowest possible f number) for the efficient

collection of light radiating from the source.

It should be well-corrected for spherical aberration so that all light collected from the

source is focused into a single image plane, and so that even a small source placed at

its focus is capable of filling it evenly with light.
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the substage optics from this distance, the substage condenser cannot deliver an

optimal performance.

This is not particularly important in the case of Abbe condensers, but is important in

the case of condensers which are achromatic and aplanatic.

If the lamp is intended to provide brightfield illumination for a microscope having

objectives from x10 to x100, without the need for readjustment, the most difficult partof the operation is likely to be filling the field of the low power objective.

If it is assumed that the diameter of the diaphragm in the eyepiece is 20mm, the field

seen by an x10 objective will be 2mm in diameter. If the substage condenser in use has

a focal length of 10mm (about average) and the lamp is situated 250mm from the

substage condenser diaphragm, then the magnification of the substage condenser

system is about x25 (250 divided by 10) and the diameter of lamp condenser required

to fill the field seen by the objective is therefore 25 x 2mm = 50mm.

Very few microscope lamps have a condenser of this diameter, and it would be

unusual for a 50mm diam. condenser to have a focal length sufficiently short to fill the

substage condenser at a lamp distance of 250mm, at least with the size of light source

(2-3mm) commonly available. Having discussed some of the issues involved in the design of an optimized Khler

lamp, we now proceed to the discussion of a particular lamp which has arrived at its

own set of compromises.

Fibre Optic Light Guide as Source.

The light source used with the optimized condenser in this example is the Schott

KL1500 cold light source. The lamp is a 150W, 15V tungsten halogen lamp with its

own ellipsoidal reflector, contained in a fan-cooled housing along with light-

attenuating filters and transformer power supply. The unit can make use of light

guides having circular fibre bundles of 3mm to 8mm in diameter.

Microscope lamp with fibre optic light-guide source.

Control of light intensity is achieved by an internal wheel of inconel metal film on

glass filters which reduce the light by approximately one photographic stop (half) for

each click on the 5-step wheel, allowing a stepwise control of light intensity with no

change of colour temperature. Later models of this light source replaced the filter


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wheel with a variable resistor(?) arrangement which gave continuous control of the

light, but with change of colour temperature in the process. The earlier model is

preferable, especially if

photomicrography is the intention.

The light guide is held in an adapterblank drilled to provide a neat

sliding fit for the light guide ferrule,

which is clamped by means of a hex-

socket grub screw tightened with an

Allen key. The filament source and

the light-guide source can be easily

interchanged without upsetting the

microscope lamp if the position of

the light guide is adjusted so that the

end of the guide locates in the same

plane as the filament of the tungstenbulb.

The two images to the right have

been obtained by using the above

lamp and condenser to project an

image of the guide end onto a white

screen. The first is an image at best

focus and full aperture, and the

other is at the same focus and at

much reduced aperture. A fairly

even fill of the active aperture of the guide can be obtained by rotating the other end of

the guide at the lamp housing and clamping it in the optimum position.

Due to slight differences in orientation of individual fibres at each end of the guide,

the fill will never be completely uniform, so the final result is a fairly uniform circular

source of intense light 5mm in diameter which can be varied over a 16:1 brightness

range without change in colour temperature.

The circular shape is ideally suited to the illumination of the circular optics of the

microscope. The ability to substitute light-guides of 3mm to 8mm diameter greatly

increases the freedom to illuminate microscope condenser optics of different diameter

over a much greater range of lamp distance.

In particular, when used with the lowest power objectives, the largest light-guide

allows the lamp to be moved very close to the microscope whilst still filling theaperture of a low power substage condenser. These condensers are not so sensitive to

spherical errors introduced by having the lamp close, and by this means large fields

can be satisfactorily illuminated.

The large diameter of the source also allows a large lamp condenser to be easily filled

with light, minimizing the effects of any spherical errors that the condenser may have.

The light-guides available for the Schott (and other) fibre light sources can greatly

extend the range of lighting possibilities for the microscopist. If for example a three

branch flexible light-guide is used, one of the guides can be used to power a Khler

lamp providing brightfield, darkfield or Rheinberg illumination via the microscope

substage, and the other two used above the stage to provide incident illumination.

Once the lighting balance has been established, all three sources can be made tosimultaneously increase or decrease in brightness by using the single control at the


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lamp power supply.

This is especially useful in complex lighting arrangements when exposures must be

bracketed.

Additionally and importantly, the light provided is effectively filtered of its heat

component which means that the microscope lamp housing does not grow hot. The

heating effect at the specimen is also greatly reduced. Even though the guideattenuates the light by about 50%, the tightly focused spot from the ellipsoidal

reflector lamp ensures an intensity at the exit end of the guide close to that of a normal

tungsten microscope lamp -- but not as bright as the light from a bare quartz-halogen

lamp.

Condenser Aperture Considerations.

Due to considerations of the refractive index of the glass in the individual transmitting

fibres, and total internal and external reflection phenomena occurring at the polished

ends of the fibre bundle, the guide can only accept and emit light over a certain solid

angle. For the Schott guides, this angle is about 88, corresponding to an NA of 0.7,which also happens to be f 0.7. This means that even though aspheric condenser lenses

are made with apertures approaching f 0.6, (acceptance angle 110) there is no point in

using one, as a fibre light-guide placed at its focus is incapable of completely filling it

with light. This means that the present condenser with its aperture of f 0.7 is just large

enough to accept all of the rays emanating from the light guide, and therefore

represents an optimum match.

(There would be some advantage in using the wider aperture lens with a tungsten

filament source, as these are not restricted in the angle over which they emit light).

The construction of the lamp allows for the easy substitution of other light sources,

including white LEDs. These show increasing promise as microscope illuminants, and

some of the issues involved in using them are dealt with on the following page.

The white LED as a Microscope Illuminant.

Since their development in the early nineties, white-light LEDs have made great

progress. They are inherently more efficient than tungsten filament lamps in terms of

the amount of electrical energy which they are able to convert into light. Whereas a

tungsten lamp produces light at a rate of 20-25 Lumens per Watt, presently available

white LEDs operate at 50 Lumens/Watt, and developers believe that with

improvements of the phosphors used in their construction, this figure may be doubled

again in the near future. Over the last three or four years, the light output of commercially available white

LEDs has gone from 1000 to 7-8000 millicandelas. Whilst improvements in output are

certain to continue, currently available white LEDs are more than equal to the task of

replacing tungsten filament lamps in many, even most, light microscope applications.

In the LED types considered here, the diode die itself produces blue light at a

wavelength of about 470nm, and this excites a phosphor which produces yellow. The

mixture of these two colours gives a fair approximation to a daylight which is

unavoidably deficient in red. In spite of this, colour rendition with digital cameras is

surprisingly good (see tests below).

Power consumption is very low, and very little heat is generated. At an operating

voltage of 3.6 V, the current is 30mA -- a total power rating of around 120mW,producing a light output of about 6000 mcd.


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From data sheet for narrow-beam white LED {below, left).

Another attractive feature of these devices is their 50,000+ hours life expectancy.

Whilst there is some fall-off of light output over time with the currently available

LEDs due mainly to yellowing of the epoxy resin used in the package, this is not

noticeable inside thousands of hours of usage. Experimenters should obtain a full specification for any LED intended for microscope

use, as there are some varieties which generate a blue in the near ultraviolet at 380nm

and could be harmful to the eyes when viewed directly as in a brightfield microscope,

even allowing for the UV absorption of the microscope optics.

Narrow-beam white LED.

..with small source.

Wide-beam white LED.

..with larger source.

The pictures above (taken at the same magnification) show two similar-looking 5mm

white LEDs, one of which is close to ideal as a microscope illuminant, and the other is

all but unusable.

The LED on the left has a small emitting area which is situated sufficiently far from

the strongly curved lens moulded into the clear epoxy package for most of the emitted

light to be concentrated into a beam of about 30.

When such an LED is placed at the principle focus of a wide aperture condenser lens

having an angular aperture of about 90, only the central portion of the lens is filled

with light. Seen from the microscope, a central circle of about half the diameter of the

condenser lens is illuminated.


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The view of the back lens of the objective is equally unsatisfactory, and it is near to

impossible to fill both the lamp condenser and

the aperture of a high-power objective

simultaneously. True Kher illumination is

therefore not achievable.

By contrast, the other LED (right, above) has alarger source placed close to the centre of

curvature of its lens, producing a beam of

remarkably even distrubution over an angle of

90 -- just large enough to fill a lamp condenser

of 0.7 NA.

Since the microscope field is filled with a highly

enlarged portion of the emitting luminous

source, and this source has an uneven

distribution of blue and yellow light (in all

examples of this LED examined), as well as

images of the fine wires connecting the LED dieto the external pins, some unevenness of colour

and intensity is visible at the specimen plane.

It can be reduced but not entirely eliminated by the introduction of some diffusion

immediately in contact with the LED. This has been done in the assembly above,

where the LED is a sliding fit in a metal sleeve which has a plastic film diffuser at its

end.

Colour Rendition.

The following pictures have been shot using a Kodak DC4800 digital camera, the

same optics (a x20 achromatic objective with Abbe condenser in a Khler brightfield)

and specimen (a stained cross-section of a Yew stem), but using LED (top pics) and

tungsten filament (bottom pics) as light source.

In both cases, the pictures were taken at a light level for comfortable viewing, giving

shutter speeds of around 1/20th. sec. at ISO 100.

The two leftmost pictures are with the camera's white-balance setting on "tungsten"

(equivalent to 3200K), and the two on the right were taken at the colour temperature

setting on the camera which gave the most neutral/white backround.

In pictures 3 and 4, the tungsten source was an Olympus/Hosobuchi 6V, 30W grid

filament lamp with some blue filtration and an operating voltage of 3V to give a

comfortable light level for visual use. This explains why the colour cast of picture 3 isso warm, even on the tungsten setting.

No colour manipulations have been applied to any picture.


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1. White LED, Tungsten setting. 2. White LED, 9000K.

3. Tungsten Lamp, Tungsten

setting.4. Tungsten lamp, 3200K.

It can be seen that the picture with the cleanest white background and the most

accurate colour rendition is given by the combination of LED illumination and camera

set to 9000K. The falloff in brightness in the lower right of the picture (more obviousin the picture than in visual use) is due to uneven blue/yellow distribution at the

source.

The accuracy of the green colour, as well as saturation in the reds is better than those

of the tungsten illuminated picture. This is a surprising result given the spectral

distribution of this LED.

Nichia, the first company to make a white LED, has recently produced a warm

white LED having both red and yellow phosphors, and giving a light which has a

colour temperature of 2500 - 3200K. Perhaps they will employ the same technique to

produce an LED which has a better approximation to daylight.

Luminance Considerations.

The x20 brightfield setup described above was used to determine the brightness of the

white LED relative to that of the Olympus 30W/6V tungsten filament lamp.

The meter used was a Gossen Lunasix 3 with the

microscope attatchment illustrated on the right. The

microscope eyepiece was replaced with the meter, and each

illuminant was used in turn (with necessary lamp refocus

adjustments) and an intensity reading made. The results

showed that the filament lamp (at 6V) provided an

illumination 4.5 photographic stops more intense than the

LED (at 3.6V), or 16 - 32 times brighter.

Also of interest is the relative brightness of the white LED

in a Khler lamp, and a 60 Watt mains frosted light bulb

sometimes used (in the absence of anything better) to light a

laboratory microscope. Measurement showed that the LED

in the Khler lamp was 3.5 photographic stops brighter than

the bulb.

With the 60W bulb, a x40 power darkfield using an Abbe condenser with a substage

stop is just bright enough for good observation. Since high-power darkfields of good

saturation are probably the most light-demanding of microscope systems, it would
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seem that LEDs as microscope illuminants have well and truly arrived, but do not as

yet offer as bright illumination as tungsten filament lamps designed for the job.

With a few tweaks to the packaging of these devices, an LED ideally suited to

microscopes could easily be produced.

An LED Optimized for Microscopy.

Within the constraints of a 5mm diameter package, the LED pictured above on the

right is very close to ideal as a Khler microscope illuminant.

If the diameter of the indented bowl of phosphor surrounding the die could be

increased a millimetre or so to 3.5 or 4mm, and situated just beyond the centre of

curvature of the lens such that a slight degree of magnification of the source is

achieved, and the angle of the emitted beam kept to 90, the only necessary refinement

would be a slight degree of opalescent (textureless) diffusion introduced into the

epoxy casing to even out the distribution of blue and yellow (and red) light at the

source. Barium sulphate crystals/particles of an appropriate size suspended in clear

epoxy would be my guess at the ideal diffusing arrangement. There is no reason why a larger package should not be used, as long as the real or

apparent size of the source viewed from any point in the collecting area of the

condenser is around 4mm -- this being a source size which will comfortably fill the

microscope substage condenser optics (say 30mm) at a lamp distance of 250mm.

If any company would consider marketing such an LED in volume, Micrographia

could guarantee to buy at least a dozen.

A microscope lamp utilizing electronic flash.

Historical background: Microscope lamps of the past.

The Mirror.

The mirror is used only to fold the optical path of the microscope into a convenient

space. It also introduces another source of potential maladjustment into the system,

and another surface to collect dust. Having said this, the mirror does not need to be of

the highest optical quality to do its job, nor does a small amount of dust on the mirror

make much difference to the quality of the image. A slight film of fine dust (such as

remains after dusting the mirror with a blower brush) can actually be useful in locating

the beam of light from the lamp when setting up the instrument.

Always use the flat side of the mirror in combination with a substage condenser.

The Substage Condenser.

The substage condenser fitted to most microscopes is of a design

originated by Ernst Abbe in the late 1800s and is usually referred to as the

Abbe condenser. Whilst condensers of higher correction are available, the

Abbe condenser has proven to be quite satisfactory for routine

microscopy.

A substage condenser of some kind is an absolute requirement for serious -- or at least

satisfactory -- microscopy. The objectives from x20 upwards require the subject to be

illuminated evenly over quite a large angle, and neither a concave mirror nor

(especially) a flat mirror is capable of achieving this. If no condenser is used with a

high power objective, the result is an image which is dark, coarse, contrasty and

lacking in detail -- described by earlier microscopists as "a rotten image". Lowerpower objectives however, can give acceptable, even quite pleasing images without a
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condenser if the mirror is directed toward a close, well frosted lightbulb or a bright

white cloud.

An important point to note here is that the substage condenser diaphragm is used to

control the solid angle of the light emerging from the condenser, illuminating the

specimen, and filling the objective -- not for adjusting the brightness of the image.Brightness adjustment can be achieved by removing or placing a filter in the substage

stop-carrier or by dimming the lightbulb, locating a brighter or a greyer cloud etc.

In practice, once the microscope has been set up, the condenser and its diaphragm

setting can be largely forgotten until the objective is changed for one of higher or

lower power.

More Information on Condensers.

1. Notes on Construction and Use.2. Dismantling and Cleaning the Condenser.

The Specimen.

The specimen is usually supported by a slide and, essentially at the higher powers of

the biological microscope, covered by a coverglass. Thus introduced into the image-

forming light path, slide and coverglass become part of the optical system. The

thickness of the slide is important to the correction of the substage condenser, and the

thickness of the coverglass is critical to the performance of the objective, especially those of

higher power (x20 and greater).

Most substage condensers are corrected to work with a slide thickness of 1.0mm, and most

microscope objectives of x20 or greater power are designed to work with a coverglass

thickness of 0.17mm (thickness no. 1).

The Objective.

The objective is the most important component of the optical system in terms

of the quality of the final image. For over a hundred years, dating from Ernst

Abbe's introduction (in the 1870's) of apochromatic corrections, the best

objectives have been capable of resolving the finest detail predicted by theory.

Since then, great improvements have been made in field size, field flatness and image quality

toward the edges of the field. The modern microscope objective probably represents thehighest degree of optical perfection and precision engineering which is manufactured in

volume for public consumption. The diagram shows a construction (not to scale) typical of a

x40 achromatic objective standard on most laboratory microscopes.

The screw thread of microscope objectives has been a standard across the industry since 1858,

when it was first proposed by the Royal Microscopical Society. Here is adiagram of the RMS

standard objective thread.

More recently, larger diameter threads have appeared to accommodate the needs of modern

objective design.

More Information on Objectives.1. Objective Markings: What they mean.
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2. Aperture and Resolution in the Microscope Objective.

The Eyepiece.

The eyepiece relays to the eye an image projected by the objective into the

plane of the eyepiece diaphragm, further magnifying it in the process. In older

microscopes, the eyepiece also corrected residual colour errors remaining inthe objective. Modern infinity-tubelength objectives are fully corrected in

themselves, but still require additional focusing optics and appropriate eyepieces to produce

their image. The matching of older objectives to a suitable eyepiece is also discussed in the

advanced tutorials.

In short, all objectives manufactured before the arrival of infinity-correction required

"compensation" of varying degrees. There was no industry-wide standard on the matter, so

each manufacturer produced eyepieces which compensated the lateral colour errors of their

own objectives. The degree of compensation of a compensating eyepiece can be roughly

gauged by the intensity of the red fringe seen inside the eyepiece diaphragm when used on

brightfield. The brighter the fringe, the greater the degree of compensation.All of the apochromatic objectives of this (almost hundred year) period, and many of the

higher power achromats, required compensating eyepieces.

The other variety of eyepiece in common use was the Huyghenian -- best suited to achromatic

objectives in general, and particularly to low power achromats which often require little or no

correction. These eyepieces are distinguished by a blue fringe around their field diaphragm

when the eyepiece is used in brightfield.

The Eye.

The cornea and the eye lens are the final optical components in the image-

forming path to the retina. In a person with normal vision, the eyelens will be

relaxed as though the eye is forming an image of a very distant object, and the

focusing controls on the microscope used to achieve image sharpness. The

optics of the eyepiece are such that all image-forming rays pass through a circle (called the

Ramsden disc) a few millimetres exterior to the eyepiece lens and just smaller than the

diameter of the pupil. The eye is bought close enough to the eyepiece for the ramsden disc

and the pupil to coincide, at which point the full circular field of the microscope is seen.

Learning to hold the head still in this optimum position, especially with a binocular

instrument, is one of many skills acquired by the microscopist.

Click for adiagram of the human eye.

Having briefly covered the components of the microscope and their function, the next step is

to refer to diagrams of the Khler setup to see the optical relationship between the

components prior to the setup procedure itself.

Numerical Aperture.

This topic is covered in three pages.
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1. Diffraction,

Resolution.

2. R.I. and

N.A.

3.

Magnification.

Page 1: Diffraction, Resolution and Image Formation.

The resolving power of the light microscope depends upon two factors:

1. The absolute limit to resolution imposed by the wavelength of the light illuminating the

specimen. No instrument which forms its image by wave interference can resolve detail which

is smaller than about half the wavelength of the wave energy (light in the case of the

microscope) being used to examine the specimen. This is as true of the accoustic and the

(transmission) electron microscope as it is of the light microscope.

2. The Numerical Aperture (N.A.) of the objective in use. To explain this term, it is necessary

to show why the amount of detail that can be seen with the microscope depends not only on

wavelength, but on the angle over which the objective is capable of receiving light from the

specimen. This is in turn dependent upon the refractive indices of the media in the light path.

N.A. is the lens specification which takes these factors into account, and is effectively an

index of the objective's ability to resolve fine detail.

An explanation of N.A. must necessarily deal with the optical phenomenon of diffraction.

The following account of image formation by a microscope objective is essentially a

simplified version of the diffraction theory (minus the mathematics) put forward by Ernst

Abbe in 1873.

Diffraction and Subject Detail.

Consider a subject under a brightfield microscope which has a pattern of detail in which very

small opaque objects are separated from one another by a distance equal to their owndiameter. The diagram below represents the diffraction which occurs at a single narrow slit,

and is used here to illustrate what happens when light passes through the space separating the

opaque objects of the above example.

Given the approximation that the wavefront of light arriving at this slit from a very distant

point source is planar, Huyghens' principle states that along the imaginary line b which

represents the wavefront momentarily present between the edges of the slit, each point on b

could itself be considered a secondary source of wavelets which radiate from that point. This

provides a basis for

determining the distribution

of the light energy passingthrough the slit, which, due to

interference between the rays,

is neither even nor random.

The point P1 on the screen is

so situated that the light

wavelet emanating from a

point very close to the upper

edge of the slit, and another

emanating from a point very

close to the lower edge of theslit have a path difference of
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one wavelength. Whilst these two rays interfere constructively, they are only two rays of the

infinite number of ray pairs along the line b.

To determine the net effect of the interaction of ray pairs across the entire aperture, consider

the ray passing very close to the upper edge of the slit, and the ray immediately adjacent to

and below it. Between these two rays, the path difference at P1 is extremely small, and the

rays are very close to a condition of constructive interference -- but not quite.As second rays emanating from points further from the uppermost ray are considered, the path

difference steadily increases until the ray from the centre of the slit is reached. With this ray,

the path difference is half a wavelength, and total destructive interference occurs. A condition

very close to total destructive interference also occurs with rays very close to the central ray.

The degree of destructive interference gradually diminishes as second rays approaching the

remote edge of the slit are selected and the interference is once more constructive. The result

of this at the screen P is a zone of darkness on either side of the point P1, with P1 at the

position of maximum darkness.

Similarly, at the point P2, the path difference between the upper and lower rays is 1

wavelengths, and P2 marks the centre of a zone of constructive interference -- called the firstorder diffraction maximum. And similarly on the other side of the axis.

Second, third and higher order diffraction maxima are formed at points where the path

difference is an odd number of half-wavelengths, and the intervening minima where the path

difference is an even number of half-wavelengths.

The diagram on the left shows the

condition for the formation of the

second order diffraction minimum

(P3 above), where the path difference

between the upper and lower rays is

two wavelengths (four half

wavelengths).

For an objective to form an image of

the object detail represented by these

diffracted rays, it must be capable of

accepting them.

As a minimum requirement, the

objective must be capable of

accepting both the axial rays (centred on P0) and at least part of the first-order diffractionmaximum. The clarity with which any detail is rendered depends upon the percentage capture

of the diffracted rays it generates. Complete capture of the first-order diffracted rays will

produce an image which is capable of revealing detail close to the diffraction limit for that

aperture. Second and third order maxima, being decreasingly intense, make less contribution

to image detail.

It should be clear from the diagram that the finer the detail in the specimen (the smaller the

value of b), the greater the angle which must be assumed by the rays forming the various

maxima in order to achieve the necessary path differences -- requiring the use of an objective

of correspondingly larger angular aperture to capture them. Very fine detail will have maxima

passing outside the aperture of the objective and will not be imaged.


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The ability of the objective to accept diffracted rays of a given angle is however strongly

dependent on the refractive indices of the media between the objective and the specimen --

usually some combination of air, water, glass and oil.

At this point the concept of Numerical Aperture becomes useful, and is dealt with in the next

section.

The Light Path through the Microscope.

Rather than attempting to illustrate all ray paths using a single optical diagram, the

distribution of light in a Khler-illuminated microscope is here represented as four light

paths of practical importance to the microscopist:

1. The path taken by light emanating from the lamp filament,2. The conjugate image positions of the lamp field diaphragm,3. The conjugate image positions of the substage condenser diaphragm, and4. The image forming rays from the specimen to the eye.

...and presented as four parallel diagrams (below). The lens systems shown are not to

scale, and the ray paths are representational only -- they are not the result of calculation

or ray-tracing. In the real world, a differing choice of condensers, objectives andeyepieces will give slightly differing ray paths.

These diagrams (conforming to the convention that light rays should pass from left to

right) extend out of the page to the right for two or three screens (depending upon your

screen resolution) enabling a detailed examination of the four ray paths.

It is then possible to form a complete impression of what is happening in any section of

the microscope by scrolling vertically between the diagrams to compare the ray paths.

Images of a given point are formed in the optical train wherever the rays originating from

that point cross over. The first image formed will be upside-down; the second will be

right way up -- and so on in alternation throughout the system.

Path 4 shows that the first (inverted) image of the specimen is formed in the plane of the

eyepiece, and the second (erect) on the retina. The retina normally receives inverted

images of everyday objects, so the microscope image therefore appears upside down.

Adjusting to this in order to follow a moving specimen is probably the first major skill

required of the beginner.

1. Illumination Light Path.


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(with notes on the major components).

scope Lamp.

with a tightly wound grid

denser system and focussed

ment in the plane of the

must be large enough to fill

ocal plane of the objective

with light.

trolled by means of an iris

diately in front of the lamp

The Substage Consenser.

The condenser accepts light coming

from the lamp and focuses it onto

the specimen at a far greater

angular aperture than the lamp

alone could achieve.

It is focused to form an image of the

lamp diaphragm blades in the plane

of the specimen, which means that

the microscope field is filled with an

image of the lamp condenser lens

filled with light. (See next light

path).

.

S

pe

c

i

m

e

n

The Objective.

This is the most critical component in the

system with regard to the quality of the

image produced by the instrument. The

lens system shown here is a

representation of a typical X40

achromat, and in practice has a working

distance of about 1 mm.

The back focal plane of the objective is

shown external to the lens system. Whilst

this is true for lower power objectives,

the BFP of a real x40 objective is usually

within the thickness of the rear elements.

The E

The second most

component in the

system. It further m

the image produced

objective, and on

microscopes,

compensates for

residual ch

difference of magn

remaining in the ob

The additional optic

modern infinity-co

systems are not sho

2. Conjugate Images of the Lamp Diaphragm.

3. Conjugate Images of the Substage Condenser Diaphragm.


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4.Image-forming Rays from the Specimen to the Eye.

The Optimized Microscope.

The term "Khler-illuminated microscope" describes a system in which all

the optical components are design-optimized to work together and aremutually focused upon one another. In this configuration, certain image

planes will always coincide.

The reader will have noticed that ray path 3 is contained within ray path 1,

and similarly, ray path 4 is contained within 2. This demonstrates the fact

that in a Khler setup, the images of the lamp diaphragm and the specimen

always coincide, as do the images of the lamp filament and the substage

condenser diaphragm.

These conditions define the principle and practice of Khler illumination

and give the microscopist control over aperture of illumination (andtherefore image resolution, contrast and depth of focus) and over area of

field illuminated (giving additional contrast control by reducing glare

caused by extraneous light).

To the extent that the setup of the microscope departs from the Khler

condition, the above controls are less precise in their effect. If the


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Documents

Introduction to the Basic Microscopy