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Due to the fact that a large number of microscope users rely upon direct
observation of the specimen, it is critically important to understand the
relationship between the microscope and the human eye. Regardless of
technical advancement, the human eye as a visual detector (in combination
with the brain) is the most efficient image-processing system that has ever
been encountered. There are no man-made devices that can match the
abilities of the human eye in regards to imaging speed and resolution. The
principles of operation underlying modern cameras, however, are strongly
related to the structure and operation of the eye (see the anatomical description in Figure 1). Together with the muscle-adjusted lens,
the curved surface of the cornea projects an optical image onto the retina (the detector). The level of incident brightness is controlled
via the variable diameter of an iris (much like an optical diaphragm) under the control of specific muscles. A sharp image is produced
by the flexible lens, the focal length of which is changed by another set of muscles so that focusing is possible on any object at a
distance between approximately 20 centimeters and infinity. The image itself is detected on the retina by approximately 130 million
photoreceptor rod cells (responsible for recognition of grey levels) and 7 million photoreceptor cone cells (color recognition), and is
then transferred to the brain along the shortest possible path through the optic nerve.
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screen. Such images are termed virtual images and they appear upright, not inverted. In a compound microscope, the image appears
to be floating in space just below the top of the observation tube (at the level of the fixed diaphragm of the eyepiece) where the
eyepiece is inserted. What you are observing is not tangible; it cannot be grasped. Rather, it is a map or representation of the
specimen in various colors and/or shades of gray from black to white.
More than an 8-fold or 10-fold magnification is not very useful with a simple
bi-convex lens because of the resulting small field of view and the fact that
the lens must be brought into very close proximity to the eye. In order to
achieve higher magnifications we must use the compound microscope,
which was originally developed by the Janssen brothers in the Netherlands
and Galileo in Italy around the beginning of the 1600s. In its simplest form,
the instrument is composed of two convex lenses aligned in series: An
object glass (more commonly referred to as an objective) closer to the object
or specimen, and an eyepiece (ocular) lens closer to the observer's eye (with means of adjusting the position of the specimen and the
microscope lenses). The compound microscope achieves a two-stage magnification where the objective projects a magnified image
into the body tube of the microscope and the eyepiece further magnifies the image projected. The total magnification equals the
magnification of the objective multiplied by the magnification of the eyepiece:
The earliest compound microscopes were hindered by optical aberrations (both chromatic and spherical). Such defects result from the
fact that white light is composed of numerous wavelengths, and when light waves pass through the periphery of a lens, they are not
brought into focus with those passing through the center. The images that early microscopes produced were often blurred with colorful
halos until lens makers in the mid-1700s discovered that by combining two lenses made of glass with different color dispersions, much
of the chromatic aberration could be reduced or eliminated. Modern microscopes are often modular with interchangeable parts for
different purposes, and can have several lenses arranged one behind the other, thus allowing magnifications of up to 2000x and
higher, and the capability of producing images with remarkable clarity and contrast.
Illustrated in Figure 5 is the infinity color-corrected optical system (ICS) principle used with a modern microscope featuring a tube lens
as added support for the objective. In the microscope beam path (Figure 5(a)), the object or specimen is recorded by the objective and
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is first projected at infinity with a parallel bundle of wavefronts or rays. In effect, the light rays originating from one point of the
specimen travel in straight, parallel lines behind the objective. The tube lens then functions in a similar way to a camera to focus the
parallel ray bundles, producing a magnified intermediate image located inside the eyepiece at its front focal plane. The eyepiece,
acting as a second magnifier, translates the dimension of the intermediate image into parallel rays. The resulting viewing angle of the
sophisticated compound microscope system is much larger than results from direct observation (Figure 5(b)), where the object is seen
directly from a distance of approximately 25 centimeters. The region where these parallel bundles intersect is termed the eye point,
and that is where the iris of the eye should be located. The cornea and lens of the eye focus these parallel rays onto the retina. As
described above, the total magnification equals the objective magnification multiplied by the eyepiece magnification. In this illustrative
example, the overall magnification of the microscope is 100x (10x objective with a 10x eyepiece).
These basic principles of magnification underlie the operation and construction of the compound microscope. The elaboration of these
principles has led to the development, over the past several hundred years, of today's sophisticated instruments capable of producing
high-quality images from low to high magnification.
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Contributing Authors
Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.
Erin E. Wilson and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State
University, Tallahassee, Florida, 32310.
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