Chapter 6 Image Optical Path
Chapter 6 Image Optical Path
Chapter 6
Image Optical Path
© C. Robert Bagnell, Jr., Ph.D., 2012
Image Forming Light Path and Image Set
The objective lens forms its own light path and
image set that incorporates the ocular, and the
microscopist’s eye lens. This light path is illustrated in
figure 6.1. Figure 6.5 illustrates the ray diagram of
image formation.
Figure 6.1 Image Path
Specimen Plane
Each point in the specimen causes some light to
leave in the form of diverging rays. This light is
referred to as diffracted light. Some of the light striking
the specimen remains unaffected and passes on as
nearly parallel rays. This light is referred to as direct
Eye Lens
Image Plane
Back Focal Plane
Specimen Plane
Objective Back Focal Plane
The objective lens will focus rays of parallel
light in its back focal plane as illustrated in figure 6.2.
Important optical elements are placed in the
condenser’s front focal plane where their image will be
passed on toward the objective lens as parallel rays.
These include the lamp’s filament image, the
condenser iris, the dark field annulus, the phase
annulus, and the lower Wollaston prism of DIC. In
fact, any object placed in the condenser’s front focal
plane will form a real image in the objective lenses
back focal plane. Light from the specimen does not form a
recognizable image at this principal plane. Instead, the objective
lens forms a diffraction image of the specimen in its back focal
plane. This image is referred to as the Fraunhofer diffraction
pattern and the optical Fourier transform of the specimen
image. The distribution of light in space at this plane makes all
the contrast mechanisms of light microscopy possible.
Condenser Iris
Field Iris
Figure 6.2
To see the images in the objective’s back focal plane you
must look directly down the tube without an eyepiece. If your microscope has a
Bertrand lens, use it to focus on the objective’s back focal plane. To use the Bertrand
lens, leave the eyepieces in place. Rotate the Bertrand lens into position and focus it
using its focus mechanism (usually a ring or lever associated with the lens) while looking
through the eyepieces. If you have no Bertrand lens, perhaps you have a phase telescope.
This is a special eyepiece that comes with phase contrast equipment. Replace a regular
Pathology 464 – Light Microscopy
Chapter 6 Image Optical Path
eyepiece with the phase telescope and slide the inner part of the telescope in and out to
focus. If you have neither of these, remove an eyepiece, cover the hole with some
aluminum foil and punch a pinhole in the center. This will make a crude, but effective,
pinhole camera that will magnify and focus the objective’s back focal plane when you
look through the pinhole.
Intermediate Image Plane
The intermediate image plane exists
Figure 6.3
inside the microscope at a location that is less
than one focal length from the eyepiece (fig.
6.5). A real, magnified image of the specimen
exists in the intermediate image plane. This
image is used as an object for further
magnification by the eyepiece lens. As shown
in figure 6.3, when the specimen is between one
and two focal lengths from the objective lens a Figure 6.4
magnified intermediate image of the specimen
is formed in the image plane. The exact
distance form the objective lens to the specimen
is the lenses working distance. This distance
must be far enough to place the intermediate
image at the microscope’s tube length. As
shown in figure 6.4, when the object is at
exactly one focal length the image focuses at infinity. Many modern objective lenses use
this technique called infinity corrected optics. These lenses require that an auxiliary
lens be placed before the eyepiece to create the intermediate image. All other types of
objective lenses require that the object be between one and two focal lengths away from
the objective lens. Because the intermediate image is closer than one focal length from
the eyepiece, a virtual image of the specimen is formed by the eyepiece outside the
microscope. This image requires the lens in the microscopist’s eye plus her retina plus
her brain to be seen.
Eyepoint Image Plane
The eyepoint is near the back focal plane of the eyepiece. It is outside the
microscope. It is the point where the microscopist’s eye lens is placed. All the real
images that occur at the objective’s back focal plane also occur at the eyepoint: the
filament image, the condenser aperture image, the phase annulus image, and the image of
any other object placed in the condenser’s front focal plane or the objective’s back focal
plane. When the microscopist looks into the microscope and focuses on the image of the
specimen, the eyepoint is less than one focal length from the microscopist’s eye lens. Any
object that forms a real image at the eyepoint will be completely out of focus on the
microscopist’s retina. In this way, a light source that consists of a small coiled
inhomogeneous filament can completely and evenly illuminate the specimen by Köhler
Pathology 464 – Light Microscopy
Chapter 6 Image Optical Path
Retinal Image Plane
The lens in the eye of the microscopist is also part of the microscope’s imaging
system. The eyepiece produces a virtual image requiring an additional lens. The
microscopist’s eye lens serves this purpose (figure 6.5). The virtual image “exists”
outside the microscope at the location of the specimen. The lens in the microscopist’s eye
converts this image into a real image on the retina.
Figure 6.5 Schematic of image formation in the light microscope
Eye Lens
Virtual Image
1) Examine the intermediate image of a specimen in your microscope by focusing on a
specimen at low power, removing the eyepiece and placing a screen made from a piece of
lens tissue into the tube. Over what distance is this image in focus?
2) Using Köhler illumination, observe a familiar specimen under high power. Close the
condenser aperture from fully open to fully closed. What happens to the depth of field; to
the contrast; to the intensity of the illumination?
Pathology 464 – Light Microscopy
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