Download Capturing Microscope Images (continued)

Use of microscopes
prepared by
Jennifer Gose
DDDI Project Coordinator
Consortium for Internet Imaging and Database Systems
College of Agricultural and Environmental Sciences
The University of Georgia
Using microscopes to capture images for
diagnosing agricultural problems requires:
- appropriate specimen preparation
- proper microscope setup
- correct use of microscopes
- proper camera use
Specimen preparation is a very important aspect of capturing
microscope images, and varies greatly with subjects. Although
specific microscopes vary in features and operation, principles
of setup and manipulation are usually similar. Video cameras
require a different approach to image capture than hand-held
still-picture cameras, but again, principles of using cameras are
similar.
Setting up a trinocular dissecting
microscope for best viewing
“Trinocular” means three oculars or
eyepieces
Ocular tube for
mounting a camera
Oculars
for viewing
by eye
A stereo “dissecting” scope has two complete
lens systems, producing two separate images
which are combined in the mind of the observer to
produce a three-dimensional image that allows
easier manipulation of
specimens.
Two
independent
lens systems
Specimens can be illuminated from two light sources; one
below and one above, depending on needs of viewing a
particular specimen
Upper
illuminator
for “incident
light”
viewing
Lower
illuminator
for
“transmitted
light”
viewing
Illuminating specimens from below provides for “transmitted”
light viewing and from above for “incident” light viewing. One
control switch selects the light source and the other varies
light intensity. To preserve bulb life, IT IS IMPORTANT
to reduce current surge by
turning the dimmer all the way
down before operating the lightselection switch.
Light source
selection switch
Dimmer switch
for lights
Focus and magnification can be varied to
produce the best view of a specimen
Focus
control
Magnification
zoom-control
Specimens for incident-light viewing can be placed against
various backgrounds to improve contrast and feature
identification
Light-colored
stage for dark
specimens and
dark-colored
stage for light
ones
As with any images used for diagnoses, relative size
is extremely important.
Whenever possible, a comparative scale should be
included with an image, or at least the approximate
magnification indicated.
cm.
approx. 100X
To send an image from the dissecting scope to the ocular tube
for mounting a camera, a diverter prism is moved out of the
field of view on one lens system. This process eliminates the
image from one of the viewing eyepieces, and may require
repositioning the specimen for best viewing.
For viewing
through
eyepiece
For
capturing
with
camera
A stereo dissecting microscope is normally
used to view relatively large specimens at
magnifications from about 5X to about 50X.
The scope provides a very large range of
adjustment to accommodate a variety of
specimen sizes and to allow many options for
specimen illumination.
To accommodate large specimens, the entire focusing head of
the scope can be raised on the support column.
Small specimens can be accommodated by lowering the
focusing head on the support column. The illuminator can be
moved to provide light as needed for best viewing.
Focusing
head
Support
column
Adjust the microscope eyepieces so you can
comfortably see a well-focused image through both.
Space eyepieces so you can comfortably see
through both at the same time.
If one eyepiece tube is fixed focus, close the
opposite eye and, using the microscope focusing
knob, carefully focus an image of the sample for
the open eye. (If both eyepiece tubes are variable
focus, either one will do.) Now close the other eye
and use the variable focus eyepiece tube to bring
the image into focus for the open eye.
Focus on the specimen at the lowest Zoom setting and be
sure the feature of interest is in the center of the field of view.
There will be a much larger field of view but much less detail
visible than at higher Zoom settings.
Zoom to the desired magnification level and refocus if
necessary. The field of view will be much reduced, but the
feature of interest will be much magnified.
When the microscope has been adjusted to
produce the best image of a specimen, it is
ready for capturing with the camera. Now the
diverter-prism must be pulled into position to
direct light up the third ocular tube to the
camera mounted on it.
REMEMBER
What you see in the camera or on the imagecapture viewer is what will be saved to a digital
file. NOT NECESSARILY what you see by eye
through the eyepieces. BE SURE a final focus
is made in the camera viewer or on the imagecapture viewer.
REVIEW
1) Be sure illuminator dimmer is turned all the way down before
selecting a light source on the scope.
2) Once the light source is selected, turn dimmer control up
slowly
3) Move focusing head to a position on the support column that
allows appropriate focusing
4) Focus on specimen at desired magnification
5) Adjust eyepieces for best viewing
6) Adjust illuminator for best lighting
7) “Zoom” to a different magnification if desired, refocus and
readjust light
8) Divert image to camera, reposition specimen and fine focus on
camera or image-capture viewer.
9) Capture desired image
Setting up a trinocular compound
microscope for best viewing
“Trinocular” means three oculars, or
eyepieces
Ocular tube for
mounting a
camera
Oculars for
viewing by
eye
“Compound” means having multiple
objective (lower) lenses mounted on
a rotating turret (lens holder)
Rotating turret
to hold
objective
lenses
Objective
lenses
Focusing the microscope on specimen slides can be
accomplished with a coarse adjustment control for rapid
adjustment, and a fine control for small adjustments, particularly
necessary at high magnification
levels.
Coarse control
Fine control
The “mechanical stage” holds specimen slides and
allows them to be moved precisely under objective
lenses to focus on any area of interest.
The microscope illuminator, or light source, provides
light for transmission through the specimen for viewing
its structure. The amount of light is modified with a
shutter or diaphragm using a
control knob to open or close it
for more or less light.
Diaphragm control
Illuminator
The condenser focuses light from the illuminator onto the
specimen slide to provide the best lighting for viewing. It can be
raised or lowered and centered for precise focusing as necessary.
It has its own shutter diaphragm too,
for varying the amount of light
reaching the specimen slide.
Centering
controls
Diaphragm
control
Focusing
knobs
To send light from the
microscope to a camera
mounted on the third ocular
tube, a diverter-prism is moved
into the field of view by pulling
out its control arm.
Setting up a microscope for best
viewing involves adjusting the parts of
the scope to the viewer, focusing the
scope on the specimen slide and
adjusting the illuminator or light source
to the slide being viewed.
Turn on microscope illuminator by rotating dimmer knob
slowly clockwise. Fast turn-on shortens bulb life.
Dimmer knob
Place a sample slide on the microscope
stage under the lowest power objective lens.
Adjust the microscope eyepieces so you can comfortably
see a well-focused image through both
Space eyepieces so you can comfortably see
through both at the same time.
If one eyepiece tube is fixed focus, close the
opposite eye and, using the microscope focusing
knob, carefully focus an image of the sample for the
open eye. (If both eyepiece tubes are variable focus,
either one will do.) Now close the other eye and use
the variable focus eyepiece tube to bring the image
into focus for the open eye.
Focus on the specimen slide (e.g. these wood cells from a
piece of paper). Using the 4X objective lens, there will be a
much larger field of view but much less detail visible than
with the 40X lens.
4X
40X
Rotate the illuminator diaphragm control until the
diaphragm is closed all the way. Using the focusing
knob on the condenser, bring the diaphragm image into
as sharp focus as possible. Using the centering
controls, be sure the diaphragm image is positioned in
the center of the field of view.
4X
40X
Adjust the condenser diaphragm so that the specimen
features are as clearly visible as desired.
Condenser diaphragm
wide open
Condenser diaphragm closed
down to provide correct
contrast for clear viewing
The nature of light and lenses results in a plane of focus which
becomes thinner as magnification increases. That means for a
particular specimen slide, it becomes possible to focus through
the thickness of a specimen.
Some parts will be in focus
while others will not, as in these
wood cells focused at different
“depths” through the slide.
As with any images used for diagnoses, relative size
is extremely important. Whenever possible, a
comparative scale should be included with an image,
or at least the approximate magnification indicated.
cm.
approx. 100X
When the microscope has been adjusted to
produce the best image of a specimen, it is
ready for capturing with the camera. Now the
diverter-prism must be pulled into position to
direct light up the third ocular tube to the
camera mounted on it.
REMEMBER
What you see in the camera or on the imagecapture viewer is what will be saved to a digital
file. NOT NECESSARILY what you see by eye
through the eyepieces. BE SURE a final focus is
made in the camera viewer or on the imagecapture viewer.
REVIEW
1)Turn on microscope illuminator slowly.
2) Place specimen slide on mechanical stage
3) Focus on specimen at desired magnification
4) Adjust eyepieces for best viewing
5) Adjust condenser for best lighting
6) Adjust condenser diaphragm for best contrast
7) Refine focus for depth of field
8) Refine focus for image capture
9) Capture image
Capturing
Microscope Images
An analog video camera
can be adapted to a
microscope and
individual video frames
captured and converted
to digital image files.
Images at least 640x480
pixels in size are best,
saved as JPG files.
(Digital video cameras
are now available too,
with USB output.)
Capturing
Microscope Images
(continued)
The same camera
may be used on
compound as well as
dissecting scopes
with the proper
adapter. The system
can be adjusted so
that the viewed image
is par focal on either
scope.
Capturing Microscope Images (continued)
An image-capture device is
required when using an
analog camera.
Capturing Microscope
Images (continued)
Many hand-held digital
cameras can be adapted to
capture images from
microscopes. Very high
resolution images produce
unnecessarily large image
files, so the camera should
be set to a lower resolution
and to JPG file type for this
process.
REVIEW
1)A video camera can be used to capture images
from microscopes
2) With the proper adapter, the same camera can be
used on more than one microscope
3) When using an analog video camera, an imagecapture device must be used to get an image into
the computer
4) Many hand-held digital cameras can be adapted
to capture images from microscopes too
5) A final fine focus must be made on the imagecapture screen before capturing and image
“Wireless” digitally assisted diagnoses
For certain virulent diseases or for especially valuable
crops, time may be of the essence. In such cases it
may be safer or more cost effective to transmit
information and images of problem symptoms from the
field. Wireless
connectivity is now
possible, although
transmission is slow
and expensive and
coverage is often poor
and/or inconsistent.
Also portable equipment for microscopic
images is expensive.
REVIEW
1) Wireless transmission of information and images may
be important in certain circumstances
2) Wireless Internet connectivity is becoming more
consistent, but still relatively slow and expensive
3) Slow transmission speeds make short Web forms and
small image sizes necessary for wireless digitally
assisted diagnoses
4) Portable microscope equipment is often expensive
and less capable then desktop equivalents
5) Specimen preparation in the field is difficult
6) On-call diagnostic support is required