Introduction to the Basic Tutorial.

Introduction and types 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 well
as 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.

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. Inverted
microscopes 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

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.

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 and
reliably 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.

Its diaphragm should be close to the condenser field lens, and should be capable of
closing down to a very small aperture -- small enough to encroach upon the field of
the highest power objective.
Desirable attributes of the light source are:
It must have the highest possible intensity (luminous flux emitted per unit area),
distributed uniformly across its area.
Its intensity should be continuously variable over the entire range required by the
microscope without change in colour, uniformity or effective size.
Desirable attributes of the lamp construction are:
Lamp tilt, rotate, and distance from the microscope must be easily adjustable, and
once set, should be sufficiently rigid to resist accidental misalignment.
There should be provision for centreing the source relative to the lamp
condenser/diaphragm.
Filters etc. should be easily removeable and replaceable, and preferably located well
forward of the lamp condenser.
The condenser/diaphragm assembly should be easily removeable, and its components
easily separated for cleaning.
Focusing of the source should be possible without altering the distance of the lamp
condenser from the microscope.
The lamp should not become too hot for comfortable handling.
If the microscope requires different light sources, they should be easily
interchangeable, ideally without upsetting the lamp alignment.
Not all of these conditions are met in any one microscope lamp. The nature of the
compromises which must be made are dealt with next.

Reconciling the Requirements.

The difficulty of reconciling points 1-4 is underlined by the following observations.

Powerful condensers have strongly curved elements which, by the geometry of
spherical surfaces, restricts them to a smaller maximum diameter than less powerful
condensers.
Less powerful condensers, which can be larger in diameter, produce a smaller image
of the source at any given projection distance, and must therefore be moved further
from the microscope in order to fill the substage condenser. The further away they are
moved, the smaller field they illuminate.
Lenses suffering from spherical aberration project a less intense image of the source,
as light from different zones of the lens comes to a focus in widely separated planes in
the region of the main focus. Lenses of smaller aperture suffer less from spherical
aberration, but are less efficient at collecting light from the source.
Lenses of large aperture, efficient at collecting light from the source, are difficult to
correct for spherical aberration.
For lenses of any given power (focal length), larger aperture (smaller f number)
translates into larger diameter.
In addition to these generalizations on condenser lenses, there are some specific
conditions which must be met regarding the requirements of the microscope.
Microscope substage condensers are (or used to be) spherically corrected for a lamp
distance of 250mm, and a slide thickness of 1mm. Unless the lamp is capable of filling

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 part
of 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 lightattenuating 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

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 adapter

blank drilled to provide a neat
sliding fit for the light guide ferrule,
which is clamped by means of a hexsocket 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 tungsten
bulb.
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 the
aperture 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 to
simultaneously increase or decrease in brightness by using the single control at the

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 guide
attenuates 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.

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
..with small source.

LED.Wide-beam
white
..with larger source.

LED.

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.

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 a
larger 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 die
to 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 is
so warm, even on the tungsten setting.
No colour manipulations have been applied to any picture.

1. White LED, Tungsten setting.

2. White LED, 9000K.

3. Tungsten Lamp, Tungsten

4. Tungsten lamp, 3200K.
setting.

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 obvious
in 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

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". Lower
power objectives however, can give acceptable, even quite pleasing images without a

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 imageforming 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 the
highest 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 a diagram 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.

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 in
the 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 imageforming 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 a diagram 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.

1.
Diffraction, 2. R.I. and 3.
Resolution.
N.A.
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 own
diameter. 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 passing
through 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 the
slit have a path difference of

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 first
order 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 diffraction
maximum. 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.

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, and
4. 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 and
eyepieces
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.

(with

Microscope
Lamp.
bulb with a tightly wound grid
condenser system and focussed
filament in the plane of the
age must be large enough to fill
ack focal plane of the objective
led
with
light.
controlled by means of an iris
mmediately in front of the lamp

notes

on

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).

2. Conjugate Images of the Lamp Diaphragm.

3. Conjugate Images of the Substage Condenser Diaphragm.

the

.
S
p
e
c
i
m
e
n

major

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.

components).

The
Ey
The second most
component in the
system. It further ma
the image produced
objective, and on
microscopes,
compensates
for
residual
chr
difference of magnif
remaining in the ob
The additional optics
modern infinity-co
systems are not show

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 are
mutually 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 (and
therefore 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

diaphragms and their images are not correctly located, diaphragm use
produces vignetting with its attendant effects on resolution and evenness
of
illumination.
The next step is the hands-on business of configuring the microscope to the
Khler system.