Introduction to Stereomicroscopy

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References

The first stereoscopic-style microscope having twin eyepieces and matching objectives was
designed and built by Cherubin d'Orleans in 1671, but the instrument was actually a
pseudostereoscopic system that achieved image erection only by the application of supplemental
lenses.

A major drawback of the d'Orleans design was that the left-side image was projected to the right
eyepiece and the right-side image project to the left eyepiece. It wasn't until over 150 years later
when Sir Charles Wheatstone wrote a treatise on binocular vision that enough interest was
stimulated in stereomicroscopy to provide the impetus for further work.
During the mid-nineteenth century, Francis Herbert Wenham of London designed the first truly
successful stereomicroscope. Wenham incorporated a novel approach by utilizing an achromatic
prism to split the light beam at the rear of a single objective. A few years later, John Ware
Stephenson produced a similar instrument (see Figure 1). The Wenham binocular, as the
microscope design became known, suffered from artifacts brought about by the single lens and
did not actually produce a true stereoscopic effect.
In the early 1890's, Horatio S. Greenough, an American instrument designer, introduced a novel
design that was to become the forefather of modern stereomicroscopes. Greenough convinced
the Carl Zeiss Company of Jena to produce the microscope, but instead of incorporating
Greenough's lens erecting system, Zeiss engineers designed inverting prisms to produce an
erect image. This design has withstood the test of time (and a large number of microscopists),
and was a workhorse in medical and biological dissection throughout the twentieth century. The
microscope is still a favorite for many specific applications.
Stereomicroscopes manufactured during the first half of the twentieth century, or dissection
microscopes as they were called, were much like traditional compound microscopes of the era.
They were heavy, constructed mainly from brass, utilized prisms for image erection, and had
simple lens systems consisting of one or two doublets. The working distance was inversely
proportional to the magnification, and was quite short at the highest available magnifications.
These microscopes were employed primarily for dissection, because there were very few
industrial applications involving small assemblies that required a microscope for examination.
Even watchmakers used monocular loupes!
The first modern stereomicroscope was introduced in the United States by the American Optical
Company in 1957. Named the Cycloptic, this breakthrough design featured a die-cast
aluminum housing, a constant working distance (that, at four inches, was the one of the longest
produced), and an internal magnification changer, which allowed the observer to increase the

objective magnification from 0.7x to 2.5x in five steps. In addition, the microscope utilized onepiece glass erecting prisms, was equipped with a variety of accessories including stands, arms,
and illuminators, and conformed to 1950's styling with a two-tone gray paint scheme (see Figure
2). The microscope's name was derived from a single large central objective at the bottom of the
body through which both the left and right channel accumulated light from the specimen.

In later microscopes, the Cycloptic feature was renamed Common Main Objective(CMO). This
design uses a single large objective lens which, when focused on the specimen, forms an image
at infinity. The Cycloptic, unlike most of the early stereomicroscope designs, had a threaded
mount in the lower microscope body to secure the objective into position just beneath a rotatable
drum containing two pairs of afocal Galilean-style telescopes. As the drum rotated, the telescope
lenses were used in both forward and reversed orientations (magnifying and minifying), to yield
four different magnifications. The fifth magnification resulted from an open channel with no glass.
Galilean lens systems have the advantage of a small focal length, a very small field diameter,
and seldom have magnifications exceeding 2x or 3x. A 2x Galilean lens will provide either 2x or
1/2x magnification, depending upon orientation, and matched pairs can be arranged to produce
many variations. The Cycloptic's head contained what is now known as tube lenses, erecting
prisms, and a pair of eyepieces. This microscope quickly became popular with early
semiconductor manufacturers, most notably Western Electric.
Two years later (in 1959), Bausch & Lomb introduced a stereomicroscope to compete with the
Cycloptic, but with a cutting-edge advance: continuously variable, or zoom, magnification.
Named the StereoZoom, this microscope was the first stereomicroscope without erecting
prisms and was fashioned around the basic Greenough design, which will be discussed in detail
below. It was generally the same size and shape as the Cycloptic (Figure 3), and had a
comparable magnification range (0.7x to 3.0x) with similar working distances. The microscope
also featured a new Bausch & Lomb invention: four first-surface mirrors with enhanced aluminum
coatings, which were strategically positioned to perform the function of both inclination prisms
and Porro erecting prisms. In stereomicroscopy erect images are useful because microscopists
often must perform interactive manipulations on the specimen while under observation. Tasks
such as dissection, micro-welding, industrial assembly, or microinjection of oocytes are more
conveniently conducted when the specimen has the same physical orientation on the microscope
stage as it does when viewed through the eyepieces. Also, the study of true spatial relationships
between specimen features is aided by a natural, erect image.
In addition to having a reduced cost when compared to prism-equipped microscopes, the
StereoZoom was also lighter in weight. The basic microscope system or "Power Pod", as it was
called, was complemented by an enormous selection of auxiliary lenses, eyepieces, illuminators,
arms and stands, all produced with a trend-setting style that endured for over 40 years.

Acceptance of the StereoZoom by a rapidly emerging semiconductor industry was immediate

and long-lived. This novel design dominated the stereomicroscope market for many years until
production was halted in 2000 by Leica, which in the 1980's had combined the microscope
resources of American Optical, Bausch & Lomb, Leitz, Reichert, and Wild.

During the early 1960's, zooming stereomicroscopes were introduced by Nikon, Olympus,
Unitron, and other (not so well known) Japanese companies that were beginning to make their
presence known in the United States. Collectively, the Japanese, American, and European
microscope manufacturers continued advancing the development of "bigger and better"
stereomicroscopes having a host of new features. These advances were accelerated by the
invention of high-speed computers, which made it feasible for optical designers to tackle the
complex problem of creating an effective variable magnification zoom lens system with wellcorrected optical aberrations.
Today's stereomicroscope designs feature high numerical aperture objectives that produce high
contrast images, which have a minimum amount of flare and geometrical distortion. The
observation tubes will accommodate high-eyepoint eyepieces having a field of view up to 26
millimeters, with a diopter adjustment that allows the image and reticle to be merged into focus
simultaneously. In addition, many models sport high zoom ratios (up to 12x-15x) that provide a
wide magnification range (between 2x and 540x) and reduce the necessity to change objectives.
Ergonomic features incorporated into the microscope designs help to reduce fatigue during long
hours of operation, and new accessories enable modern stereomicroscopes to image specimens
that were impractical just a few years ago.
The human eyes and brain function together to produce what is referred to asstereoscopic
vision, which provides spatial, three-dimensional images of the objects surrounding us. This is
because of the brain's interpretation of the two slightly different images received from each of the
retinas. The average human eyes are separated by a distance of approximately 64-65
millimeters, and each eye perceives an object from a somewhat different viewpoint that differs by
a few degrees from the other. When transmitted to the brain, the images are fused together, but
still retain a high degree of depth perception, which is truly remarkable. The stereomicroscope
takes advantage of this ability to perceive depth by transmitting twin images that are inclined by a
small angle (usually between 10 and 12 degrees) to yield a true stereoscopic effect.
Stereomicroscope Designs
In some stereomicroscope systems, specimens are imaged utilizing two separate compound
microscope optical trains, each consisting of an eyepiece, an objective, and intermediate lens
elements. Other designs employ a common objective shared between two individual optical
channels. Two distinct images, originating from slightly different viewing angles, are projected
onto the microscopist's retinas, where they stimulate nerve endings to transfer the information to
the brain for processing. The result is a single three-dimensional image of the specimen whose

resolution is limited by the microscope optical system parameters and the frequency of nerve
endings in the retina, much like the limiting grain size in photographic film or the pixel density in a
charged coupled device (CCD) digital camera.
Stereomicroscopes can be roughly divided into two basic families, each of which has both
positive and negative characteristics. The oldest stereomicroscopic system, named after the
inventor Greenough, utilizes twin body tubes that are inclined to produce the stereo effect. A
newer system, termed the common main objective (introduced above), utilizes a single large
objective that is shared between a pair of eyepiece tubes and lens systems. Either type of
microscope can be equipped with step-type individual lenses to change magnification, or a
continuously variable zoom-type magnification system. The following discussion addresses the
advantages and disadvantages of both the Greenough and common main objective
stereomicroscope designs.

The Greenough design, introduced by Zeiss at the turn of the twentieth century, consists of two
identical (and symmetrical) optical systems each containing a separate eyepiece and objective
arranged in accurate alignment within a single housing (Figure 4). A major advantage of this
design is the high numerical apertures that can be obtained because the objectives are very
similar in design to those utilized in classical compound microscopes. In general, the lower
portions of the body tubes, containing the slender objectives, are tapered and converge at the
best focus of the object plane. The upper end of the body tubes project a pair of images into the
observer's eyes, normally with a pair of standard eyepieces. The size, focus, rotation, and
centering of the two images must be held constant within very tight tolerances, so that the eyes
view essentially the same scene. The one departure from sameness is the slightly different
viewing angle at which each image is projected onto the retina. Because of the convergence
angle, typically ranging from 10 to 12 degrees in modern designs, the left eye views the object
from the left side while the right eye views the same object from a slightly different perspective on
the right side.
A pair of erecting prisms or mirror system is utilized to de-rotate and invert the magnified image
received from the objectives and present it to the observer as it would appear without a
microscope. The body tubes are built to provide a straight line-of-sight in some designs, while
others enlist the aid of additional prisms to allow inclination of the tubes and a more natural
viewing position for the microscopist. Because the image-forming light rays pass through the
complex lens system on center, the quality of the image is symmetrical about its center, as is the
case with most compound microscopes. In addition, correction for optical aberrations in
Greenough-type microscopes is less difficult than with common main objective designs, because
the lenses are smaller, axially symmetrical, and do not rely heavily on light rays passing through
the objective periphery.

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Nikon SMZ1500 Stereomicroscope

Explore zoom magnification, focus, and changes in illumination intensity on a variety of specimens with a virtual
Nikon SMZ1500 stereomicroscope.

A distortion artifact arises in the Greenough microscope design due to the oblique separation of
each body tube from a common axis. Termed the Keystone effect, this distortion causes the
area on the left side of the right eye to appear slightly smaller than that on the right-hand side of
the same image, and of course the reverse is true for the left eye's image (see Figure 5).
Keystone distortion arises from the fact that the intermediate images produced by each body
tube are inclined with respect to the specimen plane, and tilted relative to each other, so that only
the central regions are in simultaneous focus at identical magnifications. The result is that
peripheral portions of the viewing field are focused either slightly above or below the actual
specimen plane and have very small differences in magnification, although the eyes usually
compensate for this effect and it is often not noticeable to the microscopist. During prolonged
observation periods, however, fatigue and eyestrain can be accelerated by the Keystone effect.
The small change in magnification and focus across the field of view in Greenough
stereomicroscopes might be noticed in a photograph or video image produced through one side
of the instrument, especially if the object is primarily flat and rectilinear. In photomicrography,
focus discontinuities brought on by the inclination angle are easily compensated by tilting either
the specimen or one of the beam paths so that the microscope optical axis is perpendicular to
the lateral specimen plane. When undertaking measurements with a reticle, the linear eyepiece
grid should be positioned in a vertical direction to minimize the Keystone effect. Another solution
is to tip the specimen or the microscope five or six degrees and negate the convergence.
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Chromatic Aberration

Chromatic aberrations are important wavelength-dependent artifacts that occur because the refractive index of
every optical glass formulation varies as a function of wavelength.

Common main objective stereomicroscope designs center on the refracting action of a single,
large diameter objective lens, through which both the left and right channels view the object .
Each channel operates as an independent optical train parallel to the other (this is the reason
they are also known as parallel microscopes; Figure 4), and there is collimated light between the
individual channels and the objective (the image is projected to infinity). This arrangement
guarantees that convergence of the left and right optical axes coincide with the focal point in the
specimen plane. Because this parallel axis arrangement is usually extended to include the
eyepieces, the left and right images are viewed by the microscopist's eyes with little or no
convergence. A major advantage of the common main objective system is that the optical axis of
the objective is normal to the specimen plane, and there is no inherent tilt of the image at the
eyepiece focal plane.
Although in most situations there are the usual 10 to 12 degrees of convergence at the
specimen, the brain is not used to interpreting three-dimensional images without convergence,
leading to a unique anomaly that is specific to CMO stereomicroscopes. When viewing
specimens through this type of microscope, the center portions of the specimen appear to be
slightly elevated, so that a flat specimen now appears to have a convex shape. For example, a
coin will have the appearance of being thicker in the center, so it would rock from side to side
when inverted on a flat surface. This artifact is referred to as a perspective distortion, but
should not cause concern unless the microscope is utilized to judge flatness or height (see
Figure 5). Specimens with complex or rounded shapes, while displaying a certain amount of
perspective distortion, often do not appear to be distorted when viewed through the
stereomicroscope.

Perspective distortion is sometimes referred to as doming or the globular effect, and results
from a combination of keystone and pincushion distortion. As an example, presented in Figure 5
is a slightly exaggerated illustration of how a United States Lincoln penny, a disc-shaped flat
coin, would appear in a stereomicroscope with severe perspective distortion. The original penny
is shown at the top of the illustration to have a flat surface. Just beneath are the images
projected simultaneously by the microscope to both the left and right eyes, which demonstrate an
asymmetrical pincushion distortion directed toward the central axis of the microscope. The final
result is perception of a dome- or globe-shaped object when the images from both eyepieces are
projected onto the retinas and fused together in the brain. Most high-end research grade
common main objective stereomicroscopes produced by the major manufacturers have virtually
eliminated this artifact, but it still occurs in some less expensive microscopes.
Another artifact often encountered with common main objective stereomicroscopes is that small
amounts of off-axis aberrations such as astigmatism, coma, and lateral chromatic aberration
appear in the center of each image. This occurs because each optical channel is receiving light
rays from an off-center region of the large objective instead of directly from the center, where
aberrations (especially those occurring off-axis) are at a minimum or practically non-existent in
lenses with the best optical corrections. The effect is generally not noticed when both eyes are
employed to view the specimen, but a photomicrograph or digital image may have asymmetric
geometry across the field.
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Distortion Optical Aberrations

Distortion is an aberration commonly seen in stereoscopic microscopy, which is manifested by changes in the
shape of an image rather than the sharpness or color spectrum.

In general, the chromatic aberrations are difficult and expensive to correct, especially considering
the large size and volumes of glass used in manufacture of the objectives. Some CMO
stereomicroscope designs have made this a non-issue by providing the facility to offset the large
central objective, positioning it on the axis of either the left or right side channel. Other
microscope designs even provide a means for replacing the large objective with a conventional
infinity-corrected objective that can be utilized to view and photograph specimens at high
magnifications (and numerical apertures).
The greatest design feature and practical advantage of a common main objective
stereomicroscope, as with most modern microscopes, is the infinity optical system. A collimated
light pathway, with two parallel axes for the channels, exists between the objective and
removable head/observation tube assembly (labeled infinity space in Figure 6). This allows the
effortless introduction of accessories, such as beamsplitters, coaxial episcopic illuminators, photo
or digital video intermediate tubes, drawing tubes, eyelevel risers, and image transfer tubes into

the space between the microscope body and head. It is also possible to place these accessories
in the space between the objective and zoom body, although this is rarely done in practice.
Because the optical system produces a parallel bundle of light rays between the body and
microscope head, the added accessories do not introduce significant aberrations or shift the
position of images observed in the microscope. Such versatility is not available in
stereomicroscopes designed around the Greenough principles.

It is a difficult task to determine which of the two designs (CMO or Greenough) is superior,
because there are no universally accepted criteria for comparing performance between the
stereomicroscope systems. Common main objective microscopes, in general, have a greater
light-gathering power than the Greenough-design and are often more highly corrected for optical
aberration. Some observations and photomicrography might best be conducted utilizing a CMO
microscope, while other situations may call for features exclusive to the Greenough design. As a
consequence, each microscopist must make the determination whether one design will be more
appropriate for the task at hand and use this information to develop a strategy for
stereomicroscopy investigations.
In most circumstances, the choice between Greenough or common main objective
stereomicroscopes is usually based on the application, and not whether one design is superior to
the other. Greenough microscopes are typically employed for "workhorse" applications, such as
soldering miniature electronic components, dissecting biological specimens, and similar routine
tasks. These microscopes are relatively small, inexpensive, very rugged, simple to use, and easy
to maintain. Common main objective microscopes are generally utilized for more complex
applications requiring high resolution with advanced optical and illumination accessories. The
wide spectrum of accessories available for these microscopes lends to their strength in the
research arena. In many industrial situations, Greenough microscopes are likely to be found in
production lines, while common main objective microscopes are limited to the research and
development laboratories. Another consideration is the economics of microscope purchase,
especially on a large scale. Common main objective stereomicroscopes can cost several times
more than a Greenough microscope, which is a chief consideration for manufacturers who may
require tens to hundreds of microscopes. However, there are exceptions. If a common main
objective microscope is the better tool for a job, the true cost of ownership may be lower in the
end.
Magnification in Stereomicroscopy: Objectives and Eyepieces
The total magnification achieved in a stereomicroscope is the product of the objective and
eyepiece magnifications, plus that contributed by any intermediate or external auxiliary
magnifying lens systems. Over the years, a number of independent methods have been
developed to change (increase or decrease) the magnification factor of stereomicroscopes. In

the simplest microscopes, the objectives (or single objective in a CMO design) are permanently
mounted in the lower body housing, and magnification can only be altered by introducing
eyepieces of varying power. Slightly more complex microscopes have interchangeable objectives
that allow total magnification factors to be adjusted either by using a higher or lower power
objective or by substituting eyepieces of differing magnification. Objectives in these models are
mounted by screw threads or clamps, which enable relatively quick changeover to a new
magnification.
Mid-level stereomicroscopes are equipped with either a sliding objective housing or a rotating
turret containing several matched sets of objectives to produce varying magnification factors. In
order to adjust the microscope magnification, the operator simply twists the turret to position a
new auxiliary paired set of objectives beneath the channel tubes. Microscopes having this design
were once very popular, but are rarely manufactured today.
The highest quality stereomicroscopes are equipped with a zoom lens system or arotating
drum containing Galilean telescopes that are utilized to increase and decrease overall
magnification. The rotating drum system functions as an integral intermediate tube (or piece)
containing paired sets of lenses that can be installed into the optical pathway by rotating the
drum. In most models, positive dtentes are employed to act as "click stops" to secure the lens
mounts into correct alignment, and are marked to notify the operator of the new magnification
factor. The drum usually has a pair of empty lens mounts that are devoid of auxiliary lenses and
can be positioned into the optical path to allow use of the objective and eyepiece combination
without additional magnification.

Zoom systems (illustrated in Figure 7) provide a continuously variable magnification range that
can be adjusted by turning a knob located either on the periphery of the microscope body or
integrated within the body itself. This design eliminates the blank-out that occurs with possible
visual loss of spatial relationships between specimen features when magnification is changed in
discrete, stepped settings. In some of the older literature, zoom systems are often referred to
as pancratic systems after the Greek words pan for "each" and kratos for "power". Zoom ratios
vary between 4:1 and 15:1, depending upon the microscope age, manufacturer, and model. In
general, a zoom lens system contains a minimum of three lens groups, enlisting two or more
elements for each group, which are strategically positioned with respect to each other. One
element is fixed within the channel tube, while the other two are smoothly translated up and
down within the channel by precision cams. The system is designed to allow rapid and
continuous changes in magnification while simultaneously keeping the microscope in focus.

Following the zoom system, additional lens elements are utilized to relay and/or erect the image
before projecting it into the eyepieces. Several of the newer stereomicroscope models employ a
positive click-stop that alerts the microscopist at selected magnification positions in the zoom
range. This distinction is essential for calibration of the magnification level at a given power step,
a feature often found useful when performing linear measurements.
Early stereomicroscope zoom lens systems had a magnification range of approximately 7x to
30x. The magnification factors slowly grew as optical performance improved in this class of
microscopes, and more recent student microscopes now feature zoom ranges between 2x and
70x. Mid-level stereomicroscopes have zoom magnification factors with an upper magnification
limit between 250x and 400x, while high-end research microscopes sport zoom systems that can
reach over 500x in magnification. This wide magnification range is complemented by a depth of
field and working distances that are much larger than are found in compound microscopes
having equivalent magnifications. The working distance on modern stereomicroscopes varies
between 20 and 140 millimeters, depending upon the objective magnification and zoom ratio.
With the addition of specialized auxiliary attachment lenses, working distances of 300 millimeters
or more can be achieved. Field diameters are also much wider than those attainable with
compound microscopes.
Auxiliary attachment lenses can be fitted to the objective barrel on specially designed
stereomicroscopes (Figure 8). In general, the attachment lenses are threaded to rotate into a
matching thread set on the front of the objective barrel. Other versions attach to the barrel with a
clamping device. These lenses enable the microscopist to either increase or decrease the
magnification of the primary objective.
Attachment lenses are useful when image quality is not the overriding factor, because optical
corrections cannot be as accurately performed due to the fact that the lens is not mounted in the
identical position each time it is attached. In addition, attachment lenses modify the objective
working distance (the distance between the specimen and the objective front lens element). A
lens that increases the microscope magnification will also simultaneously render a short working
distance, while an attachment lens that serves to decrease magnification produces a
corresponding increase in working distance.

Modern stereomicroscopes are equipped with standardized widefield high-eyepoint eyepieces

that are available in magnifications ranging from 5x to 30x in approximately 5x increments. Most
of these eyepieces can be utilized with or without eyeglasses, and protective rubber cups are
available to avoid contact between a microscopist's eyeglasses and the eyepiece eyelens.
Eyepieces generally are equipped with a diopter adjustment to allow simultaneous focusing of
the specimen and measuring reticles, and binocular microscope observation tube mounts
(heads) now have moveable tubes that enable the operator to vary the interpupillary distance
between eyepieces over a range of 55 to 75 millimeters. The interpupillary adjustment is often
accomplished by rotating the prism bodies with respect to their optical axes. Because the
objectives are fixed in their relationship to the prisms, the adjustment does not alter the
stereoscopic effect. This convenience reduces fatigue during extended observation periods, but
requires re-adjustment when the instrument is used by more than one operator. Note that
microscopists who wear eyeglasses to correct for shortsightedness and differences in vision
between eyes should also wear their glasses for microscopy. Eyeglasses worn only for close-up
work should be removed during observation because the microscope produces the image at
some distance.
The field of view (sometimes abbreviated FOV), which is visible and in focus when observing
specimens in a microscope, is determined by the objective magnification and the size of the fixed

field diaphragm in the eyepiece. When the magnification is increased in either a conventional or
stereomicroscope, the field of view size is decreased if the eyepiece diaphragm diameter is held
constant. Conversely, when magnification is decreased, the field of view is increased at fixed
eyepiece diaphragm diameters. Changing the size of the eyepiece diaphragm opening (this must
be done during manufacture) will either increase the field of view at fixed magnification (for a
larger diaphragm size), or decrease the field of view (smaller diaphragm size).
In most compound and stereomicroscope eyepieces, the physical diameter of the field
diaphragm (located either in front or behind the eyepiece field lens) is measured in millimeters
and called the field number, which is often abbreviated and referred to simply as FN. The actual
physical size of the field diaphragm and apparent optical field size can vary in eyepiece designs
having a field lens below the diaphragm. Measuring and photomicrography reticles are placed in
the plane of the eyepiece field diaphragm, so as to appear in the same optically conjugate plane
as the specimen.
The field number of the eyepiece, usually inscribed on the housing exterior, is divided by the
magnification power of the objective to quantitatively determine the field of view size. Included in
the calculation should also be the zoom setting and any additional accessories inserted into the
optical path that may have a magnification factor. However, the eyepiece magnification is not
included, which is a relatively common mistake made by novices in microscopy. When a wider
field of view is desired, the microscopist should choose eyepieces with a higher field number. In
the lower magnification ranges, stereomicroscopes have substantially larger fields of view than
classical laboratory compound microscopes. The typical field size with a 10x eyepiece and a low
power objective (0.5x) is around 65 to 80 millimeters (depending upon the zoom factor), which
greatly exceeds the size observed (about 40 millimeters) with a compound microscope at
comparable magnification. These large field sizes require a high degree of illumination, and it is
often difficult to provide a continuous level of illumination across the entire viewfield.
Resolution and Depth of Field in Stereomicroscopy
Resolution in stereomicroscopy is determined by the wavelength of illumination and the
numerical aperture of the objective, just as it is with any other form of optical microscopy. The
numerical aperture is a measure of the resolving power of the objective and is defined as onehalf the angular aperture of the objective multiplied by the refractive index of the imaging
medium, which is usually air in stereomicroscopy. By dividing the illumination wavelength (in
microns) by the numerical aperture, the smallest distance discernible between two specimen
points is given by the equation (the Raleigh Criterion):
Resolution (d) = 0.61 / (n sin())

where d is the smallest resolvable distance, is the illuminating wavelength (usually a mixture
centered around 550 nanometers in stereomicroscopy), n is the refractive index of the medium
between the objective and specimen, and is the objective one-half angular aperture. As an
example, a Nikon SMZ1500 stereomicroscope equipped with a 1.6x apochromatic objective
having a numerical aperture of 0.21, will have a maximum resolution of approximately 1.6
micrometers when the specimen is illuminated with white light having an average wavelength of
550 nanometers. Note that the resolution calculated for the 1.6x objective assumes the imaging
medium between the specimen and the objective is air. Objective lenses manufactured for
common main objective stereomicroscopes typically vary in magnification from 0.5x to 2.0x, with
three or four intermediate values.
The magnification, working distance, and numerical aperture of typical stereomicroscope
objectives at varying magnification are presented in Table 1. In the past, several manufacturers
have assigned color codes to their stereomicroscope objective magnification values. Table 1 also
lists the color code assignment for a series of Nikon stereomicroscope objectives having this
identifying information. Note that many manufacturers do not assign a specific color code to
stereomicroscope objectives, and the codes listed in Table 1 are intended only to alert readers
that some objectives may display this and other specialized proprietary nomenclature.
Stereomicroscope Objective Specifications

Objective
Magnification

Color Code

Numerical
Aperture

Working
Distance
(Millimeters)

ED Plan 0.5x

Red

0.045

155

ED Plan 0.75x

Yellow

0.68

117

ED Plan 1x

White

0.09

84

ED Plan 1.5x

Green

0.14

50.5

ED Plan 2x

Blue

0.18

40

Plan Apo 0.5x

N/A

0.066

136

Plan Apo 1x

N/A

0.13

54

Plan Apo 1.6x

N/A

0.21

24

Table 1
The resolving power of stereomicroscope objectives is determined solely by the objective
numerical aperture and is not influenced by optical parameters of the eyepiece. Overall
resolution will not be affected when exchanging 10x eyepieces for 20x or higher magnification
eyepieces, although specimen detail that is not visible at the lower magnification will often be
revealed when the eyepiece magnification is increased. The highest power eyepieces (30x or
higher) may approach empty magnification, especially when the total microscope magnification
exceeds that available from the objective numerical aperture. In order to gauge and compare the
performance of one microscope to another, the resolution value is often expressed in terms of
line pairs per millimeter (lp/mm). In the case of the Nikon 1.6x objective discussed above, the
resolution approaches 630 line pairs per millimeter under optimum conditions.
Auxiliary attachment lenses, which range in power from 0.3x to 2.0x, can alter the working
distance and resolving power of a stereomicroscope optical system. In general, the resolving
power influence is proportional to the magnification factor of the attachment lens. The field
diameter is inversely proportional to the magnification factor, while the depth of field is inversely
proportional to the magnification factor squared. Changes in working distance are also inversely
proportional to the magnification factor, but are difficult to compute because the function is not
linear. In addition, use of these auxiliary lenses will not have significant impact on image
brightness in most cases.
Numerical Aperture and Equivalent f-Number Values
Numerical Aperture

f-Number

0.023

21.7

0.029

17.2

0.052

9.6

0.085

5.9

0.104

4.8

0.118

4.2

0.128

3.9

0.131

3.8

Table 2
Lenses designed for general photography are rated with a system that is based on fnumbers (abbreviated f), rather than numerical aperture (Table 2). In fact, these two values
appear different, but actually express the same quantity: the light gathering ability of a
photography lens or microscope objective. F-numbers can be easily converted to numerical
aperture (and vice versa) by taking the reciprocal of twice the other's value:
f-Number (f) = 1 / (2 x NA) and NA = 1 / (2 x f)

Numerical aperture (in microscopy) is equal to the refractive index of the imaging medium
multiplied by the angular aperture of the objective. The f-number is calculated by dividing the
focal length of the lens system by the aperture diameter. If a 50-millimeter focal length lens has
the same aperture diameter as a 100-millimeter lens, the shorter lens has twice the f-number as
the longer. In cases where the maximum diameter is the same in both lenses, the size is f/2 for
the 50-millimeter lens and f/4 for the 100 millimeter lens.
The aperture diameter is fixed in a stereomicroscope objective, similar to the situation with
conventional compound microscope objectives. As the microscope magnification is increased or
decreased by changing the zoom factor, the focal length is also altered accordingly. At higher
magnifications, the ratio of the aperture diameter to focal length increases, and the opposite is
true as magnification is decreased.
The focal length of a 2.0x stereomicroscope objective is half that of a 1.0x objective, which in
turn, is half that of a 0.5x objective. In some of the Nikon SMZ series stereomicroscopes (U, 10a,
800, and 1000), the 0.5x objective has a focal length of 200 millimeters, while the 1.0x is 100
millimeters, and the 2.0x objective focal length is 50 millimeters. The relative size of the zoom
system aperture (as compared to that of the objective) functions to control the f-number (and
numerical aperture) of the entire microscope system. In late model microscopes, such as the
SMZ1500, objective focal lengths have been reduced in order to increase the total system
numerical aperture. Thus, a 0.5x objective designed for the SMZ1500 has a 160-millimeter focal
length, with the 1.0x and 2.0x objectives having focal lengths equal to one-half and one-quarter
that of the 0.5x lens, respectively.
Some manufacturers supply adapter rings that allow objectives designed for a specific
microscope to be used on other (usually earlier model) stereomicroscopes. In several cases, two
objectives having the same magnification can have different focal lengths due to variations in
tube lens and zoom channel aperture specifications. As an example, the Nikon SMZ-U
stereomicroscope 1.0x objective has a focal length of 100 millimeters, while the later model
SMZ1500 microscope employs a focal length of 80 millimeters for an objective having similar
magnification and optical corrections. The difference between the two microscope designs is the
size of the zoom system aperture, which results in shorter focal lengths for the SMZ1500 series
objectives. When interchanging objectives having the same magnification but different focal
lengths, an additional factor must be introduced into total magnification calculations to correct for
the focal length differences.
Depth of Field in Stereomicroscope Objectives
Objective

HR Plan
Apo 1x

Zoom Factor

Numerical
Aperture

Depth of Field
(Micrometers)
10x

15x

20x

30x

0.75

0.023

1,348

1,072

934

796

0.029

820

655

573

491

0.052

239

193

170

147

0.085

80

66

59

52

0.104

48

41

37

33

0.118

35

30

27

25

10

0.128

28

24

22

21

11.25

0.131

26

21

21

19

Table 3
Depth of field is an important concept in stereomicroscopy (perhaps even more so than with
other common forms of optical microscopy), and is strongly influenced by the total magnification
of the instrument, including the contribution from both the objective and auxiliary attachment
lenses. At a magnification of 50x, using a 1x objective (numerical aperture 0.10), 10x eyepieces,
and a zoom factor of 5, the depth of field exhibited by a typical stereomicroscope is
approximately 55 micrometers. If a 2x attachment lens is added to the microscope when it is
configured for operation at 50x, the new magnification will be 100x, but the depth of field drops to
about 14 micrometers, a substantial decrease from the value (55 micrometers) without the

auxiliary lens. In this situation, it is wiser to change the eyepiece magnification from 10x to 20x to
achieve the added magnification so as to retain the larger depth of field value (see Table 3).
Increasing the objective numerical aperture through enhanced optical correction (for instance,
from achromat to apochromat) will also produce a modest decrease in field depth.
Depth of field values for a Nikon plan apochromatic 1x objective are presented in Table 3, where
they are listed as a function of zoom magnification factor and eyepiece magnification. It is clear
from the data in the table that numerical aperture increases with increasing zoom magnification,
while the depth of field decreases with increasing eyepiece and zoom magnification factors.
Interactive Flash Tutorial
Nikon SMZ1500 Light Pathways

Examine the optical system, lightpath, and aperture diaphragm operation in Nikon's SMZ1500 stereoscopic
microscope with this interactive Flash tutorial.

Reducing the size of the double iris diaphragm positioned between the objective and the
eyepieces can enhance depth of field. This diaphragm is opened and closed using a wheel or
lever in the microscope body housing. There are actually two diaphragms, one for each of the
channels, in the common main objective stereomicroscope design. The role of these diaphragms
is to produce an increase in field depth while simultaneously improving specimen contrast
observed in the eyepieces. Depth of field and numerical aperture variations, as a function of
diaphragm opening size, are presented in Table 4 for the Nikon plan apochromatic 1x objective at
the highest zoom magnification factor (11.25). As the diaphragm size is ramped down, the depth
of field utilizing a 10x eyepiece increases from 26 to 89 millimeters, approximately a 200 percent
increase. Simultaneously, the numerical aperture drops from a value of 0.131 to 0.063, or almost
100 percent. Similar effects are observed at higher eyepiece magnifications.
Depth of Field and Numerical Aperture
versus
Iris Diaphragm Opening Size
Numerical
Aperture

Depth of Field
(Micrometers)
10x

15x

20x

30x

0.131

26

22

21

19

0.095

44

39

37

35

0.063

89

83

79

76

Table 4
Closing the iris diaphragms will also produce a decrease in overall light intensity, increasing
exposure times for both digital and film camera systems. In most cases, the optimum setting for
the diaphragms is determined by experimentation. As the diaphragms are slowly closed, the
image begins to display more contrast as illumination intensity slowly fades. At some point,
depending upon the optical configuration of the microscope, the image begins to degrade and
specimen details exhibit diffraction phenomena while minute structural details disappear. The
best setting is a balance between maximum specimen detail and maximum contrast as seen in
the eyepieces, on film, or in digital images.
Photomicrography and Digital Imaging
Both Greenough and common main objective stereomicroscopes are readily adaptable to image
capture utilizing traditional photomicrography techniques (film) or through advanced digital
imaging. Often photomicrography is employed as a tool for recording the spatial distribution of
specimen details prior to observation and imaging with a higher-power compound microscope.
This technique is often necessary for biological specimens, where dissecting, staining, and
selective mounts are performed.
The principal concern with digital imaging and photomicrography in stereomicroscopy is the low
numerical aperture of the objectives, and the inability to capture on film (or in a digital image) the
tremendous depth of field observed through the eyepieces. There are also several limiting factors
that should be considered when photographing specimens through a single body tube utilizing a

Greenough-style stereomicroscope. Because the microscope objective is positioned at a slight

angle to the specimen, depth and resolution seen in the microscope eyepieces is not recorded
on film. Some manufacturers once provided accessories that help to alleviate these problems,
but many of the older microscopes have spare and accessory parts inventories that are
exhausted, limiting the choices for photomicrographers.
Older stereomicroscopes can be equipped with a digital or film camera using attachments that
are available over the Internet or through optics and science supply houses. These attachments
exist for almost every conceivable camera system, and many will fit the camera directly onto an
observation tube with the eyepiece left in place. Newer stereomicroscopes have trinocular heads
or photographic intermediate tubes (sometimes requiring a projection eyepiece) as an option, but
these are often limited in use to the camera systems specified by the microscope manufacturer.

The microscope presented in Figure 9 is a state-of-the-art Nikon research-level

stereomicroscope equipped for both traditional imaging with Polaroid film and with a digital video
camera. The camera systems are coupled to the microscope through a beamsplitter attachment
that is attached as an intermediate piece between the microscope body and the binocular head.
Both single and double-port beamsplitters are available from Nikon for use with either one or two
camera systems. The optical path is directed into the camera ports with a selection lever located
on the front portion of the intermediate piece. Standard c-mount, f-mount, and proprietary
coupling systems are available to support a wide variety of camera systems. In addition, Nikon
offers projection lenses of varying magnification that can be utilized to vary the image size on film
or in digital images.
Interactive Java Tutorial
Photomask Reticle Operation

Practice adjustment of the photomask reticle mounted in a focusing eyepiece using this interactive tutorial.

A photo reticle can be inserted into one of the eyepieces for composing images for capture, or
the focus finder in the exposure monitoring system can be utilized for the same purpose.
Magnification in photomicrographs or digital images is calculated by the product of the projection
lens magnification (if used) times the zoom magnification and the objective magnification. Some
beamsplitter ports also introduce a fourth magnification factor, usually 0.5x to 2.5x that must be
included in the calculation. Other microscope manufacturers offer similar camera systems
designed exclusively for their stereomicroscope product line-ups.
A unique aspect of photomicrography in stereomicroscopy is the ability to compose images that
are stereo pairs, by employing specimens having significant three-dimensional spatial
relationships among structural details. The first step is to photograph the specimen using the left
eyepiece, followed by another photograph through the right eyepiece. An alternative procedure
that can also be utilized with common main objective stereomicroscopes involves tilting the
specimen on the horizontal (stage) axis by an angle of seven to eight degrees to the left of the
microscope optical axis. After capturing a photomicrograph or digital image, the specimen is tilted
an identical amount to the right of the optical axis and another photomicrograph (digital image) is
recorded. This maneuver produces the same effect as taking two sequential photographs with a
Greenough-style stereomicroscope.
After printing (or digital image processing) the photomicrographs, they can be mounted (or
displayed on a computer monitor) side-by-side and viewed with a stereo viewer, rendering
specimen details in striking three-dimensional displays. It is important that the orientation and
alignment of the stereo pairs coincides with the requirements of the stereo viewer.
Conclusion
Magnification is often thought of as the most important criteria for judging the performance of an
optical microscope. This is far from true, because the correct magnification is the one sufficient
for the task at hand and should not be unnecessarily exceeded. Many classical investigations
into the basis of cellular structure and function, and the minute details of semiconductor anatomy,
are best conducted with classical transmitted and reflected compound optical microscopes.
Magnifications in the 400x to 1000x range are required for these studies, which usually do not
rely heavily on large depths of field for successful observation. On the other hand, a wide variety
of specimens must be examined at smaller magnifications, but require a larger depth of field with
a high degree of contrast.
Stereomicroscopes have characteristics that are valuable in situations where three-dimensional
observation and perception of depth and contrast is critical to the interpretation of specimen
structure. These instruments are also essential when micromanipulation of the specimen is
required in a large and comfortable working space. The wide field of view and variable
magnification displayed by stereomicroscopes is also useful for construction of miniature
industrial assemblies, or for biological research that requires careful manipulation of delicate and
sensitive living organisms.
Considering the wide range of accessories currently available for stereomicroscope systems, this
class of microscopes is extremely useful in a multitude of applications. Stands and illuminating
bases are available from all of the manufacturers, and can be adapted to virtually any working
situation. There are a wide choice of objectives and eyepieces, enhanced with attachment lenses
and coaxial illuminators that are fitted to the microscope as an intermediate tube. Working
distances can range from 3-5 centimeters to as much as 20 centimeters in some models,
allowing for a considerable amount of working room between the objective and specimen.
Modern stereomicroscopes are designed with ergonomic issues in mind, and most of the optical
assemblies are sealed pods that are protected against dust and tampering, and contain lens
shields to protect the optical elements from environmental hazards. Antireflective coatings
vaporized onto the surface of large objective front lenses serve to protect these delicate parts
from attack by corrosive liquids or gasses, or from abrasive particles that might cause chips and
scratches.
The utility of stereomicroscopes is limited only by their resolving power. These microscopes are
enjoying widespread use in a variety of disciplines that have tasks requiring the features found in
modern instruments of this class. Among them are education (biology, chemistry, botany,
geology, and zoology), medicine and pathology, the semiconductor industry, metallurgy, textiles,
and other industries that require assembly and inspection of miniature components.

Cricket inspired leap for bionic sensors [Print

to PDF] [Print to RTF]
The CICADA project brought together scientists interested in bridging the gap between
advanced engineering and complex biological systems. They were able to transfer the
knowledge they obtained from the cricket to create a prototype in sensor design, one
which, in the future, may even provide a replacement for hair cells in human hearing.

The cricket has tiny hairs which are so sensitive that they alert the insect to the possibility of
imminent danger. Scientists at the university of Twente in the Netherlands, inspired by the crickets,
have been able to create super-sensitive sensor technology capable of detecting acoustic signals at
thermal noise levels. Based on drag-force mediated rotations of membranes, the scientists created
a prototype sensor, achieving a feat in mechanical design.
The project partners began by quantifying the chain of events in the cricket's response reaction. In
particular they examined the way that crickets use hair-based sensors to measure small changes in
air flow and detect approaching predators. In order to do this they mapped the motion of a single
hair, as well as the array of hairs found on the cricket's cerci.
The next phase of the project, taken up by the scientists at Twente University, was to fabricate the
flow sensing electro-mechanical elements. They achieved this by means of silicon micro-machining
technology. These sensors were integrated into arrays. An entire surface filled with these 'hairs'
created the possibility of detecting patterns that come near to the 'sense of direction' the cricket
has.
This innovative sensor, inspired by nature, may be applied to solving hearing problems in humans.
It is the ageing and deterioration of hair cells which cause bad hearing and these 'artificial hairs'
may lead the way in designing replacement hair cells.

Biology of the Night Crawler (Lumbricus terrestris)

By Doug Collicutt

Description

Click for more images!

The Night Crawler (Lumbricus terrestris) is a large worm, measuring up to
25 cm in length and up to 1 cm in diameter. They have a distinct, darker
coloured "head" end which does contain the primitive "brain" of the animal,
and this tends to be the end of the worm that travels "forward" the most.
The "tail" end of the worm tends to be more flattened than the head and
lighter in colour. Common garden worms (Aporrectodea spp. - I couldn't
track down which species we have here) are smaller and don't have a dark
coloured head end.
Worms do have a proper top (dorsal) and bottom (ventral) surface, they are
not just symmetrical tube-like organisms. The surface of the worm's skin is
smooth and slimy, but also has many tiny bristles or "setae" (pronounced
set-ay) protruding from it. These help the worm move and serve to anchor
it in its burrows for self defense. The setae are part of the reason that
robins have such a hard time pulling worms out of the ground. If you place
a big Night Crawler on a piece of cardboard or paper, you can hear the setae
scraping as the worm crawls!

Night Crawlers are the biggest worms around here, but they pale in comparison to
Australia's giant earth worm that may exceed 3 metres in length!

Basic Worm Biology

Without going into a lot of details, here's a few tidbits of worm biology, just
a view things that came to mind and seem to be the kinds of things people
most often ask about.
The body plan of an earth worm is basically a segmented tube. Each
segment is a separate fluid-filled compartment surrounding the digestive
tract (gut) which runs the length of the worm's body. Many of the worm's

internal organs are also segmented, occurring as separate units in each

segment, but there is considerable specialization in the head end of the
worm. The "brain", "hearts" and other organs are clustered in the head end.
Earth worms have no eyes, but they do have cells which are sensitive to
light. They do not have ears, but can feel vibrations in the ground. Earth
worms don't have lungs, they absorb oxygen directly through their moist
skin, which is kept moist by mucous secreting cells. If a worm dries out, it
will suffocate.
Worms move by a process known as "peristaltic contraction". A worm's body
is a fluid filled tube divided into separate segments. There are circular
muscles that surround (ring) each segment and longitudinal muscles
running from segment to segment for the length of the worm. Contraction
of the longitudinal muscles shortens and widens the segments of worms
body. Circular muscle contraction lengthens and narrows the segments. By
alternating these processes in waves down it's entire body length the worms
crawls forward or backward. Inside its tunnel the widening of several
segments serves to anchor that part of the body against the tunnel walls.
The "leading end" segments are then elongated by circular muscle
contraction (squeezing), pushing that end forward, and the "trailing end" is
drawn up by longitudinal muscle contraction.
Worms can survive being cut in half! Well, for a little while, at least. It is
usually only the head end that will regenerate some segments in the lower
end and become a viable worm again. The lower end cannot regenerate a
head. However, most often, when cut in half, worms die.

Classification
The taxonomic classification of the Night Crawler is as follows. (Warning:
Don't try to pronounce these names while chewing gum, serious lingual
damage may occur.)
King

Anim

dom
:
Phyl
um:
Clas
s:
Ord
er:
Sub
orde
r:
Fam
ily:

alia
Annel
ida
Oligo
chaet
a
Haplo
taxid
a
Lumb
ricina
Lumb
ricida

Gen

e
Lumb

us:
Spe

ricus
terres

cies:

tris

Name
Night Crawlers get their common name because they do crawl around on
top of the ground at night. They are also know as "dew worms", probably
because they are found more commonly on nights when the ground is moist
from a dew or rain.
The scientific name for Night Crawlers derives as follows:
Lumbricus: A Latin word for earthworm.
terrestris: Another Latin term, meaning "of or belonging to the earth".
So, Lumbricus terrestris is "earthworm of the earth". (I know that's a bit
redundant, but at least it makes sense!)

Habitat and Range

The Night Crawler is not native to Manitoba, nor to North America. It is a
European species that was introduced to the new world with the advent of
European settlement. It is most prevalent in the southwestern third of
Manitoba, the agricultural region. Actually, it seems that all of Manitoba's
earthworms are exotic species! There are very few native North American

species of worms, and none of them are thought to have made it into
Manitoba after the retreat of the glaciers with the end of the last ice-age. (I
still find it hard to believe that Manitoba has no native earth worms! I'll
keep checking around to see if I can verify this fact, and post an update
later on.) I haven't been able to track down how many species of worms
there are in Manitoba, yet. Ontario boasts 19 different species and North
Dakota has 10.
Moist soils that are rich in organic matter are the preferred habitat of Night
Crawlers. Proximity to human habitation is a major factor in the distribution
and numbers of Night Crawlers. Remember, this is an introduced species, so
it is most likely to occur where people have been active in working or
altering the soil. Golf courses and farm fields near cities are some of the
best places to find Night Crawlers. Forested lands along major waterways
are also good places to find them.

Populations
The populations of Night Crawlers will vary dramatically with soil conditions.
It's thought that they require about 1500 cubic centimetres of soil each in
order to thrive, that's equivalent to a cube of soil 12 cm on a side. If you
laid such blocks out on a lawn, you'd have about 70 of them per square
metre, so populations of Night Crawlers could be as high as 70 per square
metre of lawn! However, published estimates tend to put their populations
at a more modest 10-15 per square metre, still a lot of worms! The
populations probably show a trend towards increasing numbers from spring
until late fall. Presumably the hardships of winter take their toll on worm
populations. How Night Crawler populations relate to the populations of
other earth worm species is uncertain. Each species probably has its
preferences for soil conditions and may dominate the overall worm
population in its preferred habitat.

Life Cycle

Click for more images!

Night Crawlers, and most other worms, are hermaphrodites. That is, each
individual worm contains both male and female reproductive organs.
However, the worms must still mate with another of their species in order to
reproduce. When two worms mate, they lie alongside one another, and both
transfer sperm to the other. Each will lay one or more capsules (like a
cocoon for the eggs), from which will emerge one or two fully formed tiny
worms. The familiar thickened "band" near the front end of most worm
species is a structure called the clitellum (see above, also). It secretes the
mucous and other substances that form the capsule containing the fertilized
eggs.

The term "hermaphrodite" derives from the combination of the names of two Greek gods:
Hermes, a male and Aphrodite, a female. So, something with both male and female
characteristics is a "herm-aphrodite".

It's thought that Night Crawlers mate and lay eggs mainly in the spring and
fall, when soil moisture levels tend to be higher. Each worm may mate and
lay eggs several times each year, but they produce relatively few offspring
per year, perhaps only 10-15 for each adult worm. It may take the tiny
worms up to a year to reach full size and sexual maturity. How long they
live after this in the wild isn't certain, best guesses are anywhere from 3-8
years, but captive worms have been know to live for 10 years!
2.

membedah burung merpati atau ayam

a.

mematikan terlebih dahulu burung merpati atau ayam dengan membiusnya. Pembius yang
biasa digunakan adalah kloroform. Basahi kapas dengan kloroform, masukkan kepala burung
ke dalam kantong plastic dan mengusahakan agar uap kloroform tidak keluar.

b.

Setelah 15 menit burung/ayam telah mati, membasahi bulu-bulu di bagian perut agar bersih
dan tidak berhamburan saat dibelah.

c.

Memotong daging di sisi kiri dan kanan otot dada hingga tulang rusuknya terputus,
mengangkat potongan tersebut sehingga organ-organ dalam terlihat.

d.

Mengamati organ dalam seperti jantung, paru-paru, usus dan lain-lain.

http://ilmuwanmuda.page.tl/Biology.htm akses 8 Sept Utomo, Pristiadi (-)