FOR IMAGE CAPTURE
any of the following methods, it is a good idea to include
a portion of a millimeter ruler in the field, to serve
as a guide to magnifications and reductions. If a large
number of images are being captured at the same magnification,
the ruler need appear in only a few, widely spaced,
images larger than 15 mm in their longest dimension,
our best results are obtained from scanning the section
directly on a flat-bed desktop scanner (Silverscanner
II from La Cie with transparency adapter). The images
produced are equal in resolution to those produced via
35 mm film and scanning from 35 mm film, and the background
and color fidelity is superior to those made from film
images smaller than 15 mm, we have photographed sections
at any desired magnification, using high-quality lenses
(macroscopic or microscopic) and 35 mm color film. The
images on film are digitized using Kodak Photo CD processing.
At this time the Kodak process yields the cheapest high-quality
scanning available for large numbers of images on film.
better, much faster, and ultimately perhaps cheaper
results can be obtained by photographing specimens directly
with a digital camera.
first digital camera we were able to use, the Kodak
DCS 200, produced images rather better than those obtained
from Photo CD. The images produced from the DCS200 camera
share the advantages of those produced from large fields
by the SilverScanner: clear background, good color fidelity,
resolution as good as that seen in Photo CD, and little
editing needed. Using this digital camera, images are
obtained within 5 seconds; there is no need for film
developing and waiting (up to 1 month lately) for Photo
CD processing. The next camera tested, the newly available
Leaf Lumina, costs about 1/4 less than the Kodak camera,
captures at a higher resolution than the Kodak camera
and yields noticeably better images. Capture time, however,
is about 7 minutes per image. Both cameras are compatible
with Nikon macro lenses, but the Leaf Lumina is not
compatible for use with microscopes, while the Kodak
DCS 200 produces fast, very good images from the microscope.
While the cost of either of the cameras is considerable,
their cost is more than made up by the time they save
in editing, which is the most expensive aspect of image
capture. (In our experience, images captured by digital
camera require about 80% less editing time than those
captured through color film.)
Scanning Large-Format Negative Film
obtained the highest resolution images by scanning black
and white, large format, 4 x 5 in. negative film on
the desktop scanner in Transparency mode. For our purposes,
the production of a catalog of the brain collections,
this is not a practical method for acquiring large numbers
of images at relatively low magnification. But for high-magnification
views of restricted portions of sections, this could
be the most desirable means of electronic rendition
of image data for particular research purposes. It may
be the best means of producing detailed atlases for
research use. The file size of the images (13 to 50
Mb) is not practical for use with currently generally
available equipment; this will probably change in the
future. We have not yet tested large-format color film.
Tabletop Scanning Procedures
transparency mode, we place a section on its slide,
situated so that the section (not the slide) is oriented
as desired relative to the vertical and horizontal margins
of the transparency window on the scanner surface. We
place a transparent millimeter rule somewhere near the
section on the scanner surface. We scan at 800 ppi (pixels
per inch), as a compromise between our scanner's default
400 ppi and its maximum 1600 ppi. The maximum resolution,
for the size sections we usually use, yields a file
of 18 Mb, too large to work with conveniently. The 800
resolution gives a file of 4 Mb, 400 a file of 1 Mb.
The 800 resolution is also a compromise between better
contrast and better detail fidelity in eventual printing
(see section on Prints and Resolutions below). For a
series of related sections, we find the largest section
in the series that we intend to capture. We preview
the section on the scanner, and create the scanning
marquee so that it contains the section, a comfortable
margin around the section, and a segment of the ruler
showing at least 5 mm. We record the size of the marquee
window (this is displayed on the scanner menu). We use
this window for all subsequent sections in the series.
The window can be dragged around to enclose each of
the subsequent sections. This keeps all images in the
series the same relative size (we keep a piece of ruler
in all the images anyway, as a check on keeping all
images the same relative size). We then scan the images.
Some care in placing slides on the scanner surface keeps
the orientation of successive images in the marquee
window (hence on the computer screen) fairly constant,
this saves much work later in getting images into register
with one another. The images appear in the Adobe Photoshop
program; we save them from there as PICT files using
the High Quality JPEG level in the PICT file option
within the Save menu of the Photoshop program.
CD and Digital Camera Procedures
images less than 5 mm in maximal width, our best results
were obtained using a 35 mm film camera (for Photo CD)
or the Kodak DCS 200 Digital camera, mounted on a good
quality research microscope. Such microscopes have appropriate
lenses, condensers and light sources to produce excellent
with Macro Lenses
images more than 5 mm, but less than 15 mm in maximal
width, our best results were obtained using the Leaf
Lumina scanning camera with our Leitz series of macro
lenses. Using the same lenses with a 35 mm film camera
yielded good images for processing by Photo CD, but
not as good as those obtained with the Lumina. In contrast
to using a research microscope, using macro lenses requires
careful attention to the light source; and to the optimal
distances between the specimen, the lens, and the camera,
which vary considerably with the image size.
charts on the following pages show the optimal distances,
empirically determined using a 35 mm film camera, for
images with their maximal dimension in a range of sizes.
We used Leitz-Wetzlar Summar lenses of 24, 35, 42 and
80 mm. On the charts, distance from camera refers to
the length of an extension tube or extension bellows
used to move the lens some distance away from the front
of the camera. Focal distance refers to the distance
between the lens and the specimen when the specimen
is in focus. Referring first to a field size chart,
it can be determined that for a specimen of 50 mm width,
the 42 mm or the 80 mm lens can be used; the 42 mm lens
requires 5 mm of extension tube or bellows, the 80 mm
lens requires 50 mm extension. Referring next to the
focal distance chart, it is seen that the 42 mm lens
should be 50 mm from the specimen; the 80 mm lens should
be 250 mm from the specimen.
best results were obtained using a flat-field light
source, eliminating the need for optical condensers.
To provide this illumination, we devised and constructed
a portable Slide Holder and Illuminator for macrophotography,
illustrated on the following pages. Its basic unit is
a commercially available Logan Desktop Light Box (No.
810/920, Logan Electric Spec. Mfg. Co., Chicago, IL
60622). This box has outside dimensions of 12.5 x 9.5
x 2.5 inches. It contains one fluorescent tubular lamp,
color corrected to 5000xK, centered in front of a hemicylindrical
reflector which reflects light through a translucent
plastic top. This top is held in place by retaining
brackets, which can be loosened, or removed and then
replaced, to allow alternative top pieces to be inserted.
There is a wire bracket on the side so that the box
can be hung vertically, as well as set on a surface
horizontally. Over the plastic top, and retained in
the brackets, we placed a sheet of Eastman Kodak Opal
Glass to maximize evenness of illumination. Over this,
and also retained in the brackets we inserted a sheet
of metal (galvanized steel) with a window cut out. In
our most used version, the window was 4 x 6 inches.
Placed on top of this, and not retained in the brackets,
we placed a slide holder made of 1/8 inch Plexiglas,
painted flat black. This also had a window (in the most
used version this window was 3 x 4 inches). Along one
of the 4 inch and one of the three inch sides of this
window, another strip of black-painted plastic was fastened
at right angles to the plane of the window, and a groove
was cut into the window side of the strips 1 inch above
the plane of the window. A third similar strip extended
from the 3-inch strip down the other 4 inch side for
a distance of 1.3 inches. The grooves cut in the three
strips formed a slot into which could be inserted any
glass slide with one dimension of 3 inches; we used
this arrangement for slides of 3 x 1, 3 x 2 and 3 x
4 inches. Similar holders were constructed, with larger
windows, for larger slides. Inexpensive small ceramic
magnets were used to hold the plastic slide holder against
the metal sheet. Magnets can be added or removed to
adjust the tightness of the hold, but they always allow
for gross and fine movements of the holder. These movements
allow fine manual control of the placement of sections
on the slides in the field of view of the camera lenses.
The apparatus permits a full range of rotary and linear
motions restricted to the plane of the slide, and keeps
the sections firmly in place at the end of a movement.
The raised slots which hold the section keep the specimen
one inch above the surface of the opal glass. This insures
that any specks or other defects in that surface will
be out of the focal plane and will not mar the resulting
pictures after photography. The apparatus can be used
horizontally with an overhead camera, or vertically
placed in front of a camera. It is eminently portable,
and can be carried in a tote bag, backpack, or briefcase.
Henry Wieferich, technician in the Psychology Department,
Michigan State University, and Dr. Robert C. Switzer
III of Neuroscience Associates, Knoxville, TN, provided
much valuable input in the design and construction of
of the methods described above can produce images of
various sizes and resolutions. In selecting among these,
balances must be achieved among such factors as storage
space, image quality, and the capacity of user equipment
to deal with image files. Images that are too large,
in resolution or size, cannot be effectively stored,
transmitted, or effectively used: disk capacity, computer
memory, and retrieval time are limited. Images that
are too small are not good images. Our goal is to produce
good, usable images. Our definition of usable is an
image that looks good on-screen, can be accommodated
in generally available equipment, and from which good
prints can be made. To look good on-screen, an image
should have a resolution of 72 pixels per inch (ppi)
and a size of 640 x 420 pixels. Images viewed at less
than their true size are distorted (this is best seen
looking at print on an image at different sizes in different
viewing programs), and images viewed at larger than
their true size are rapidly and obviously distorted
("pixelized"). Such optimally sized images
are about 0.8 Mb in size, and are readily managed by
generally available computers. Good prints, however,
require higher resolution, larger images. Our best results
have been obtained with images of 200 ppi over 8 x 10
inches (725 X 576 pixels), with image size 8 Mb. To
compromise between these image sizes, our tests have
shown a size of 2.1 Mb on screen, with a resolution
of 150 ppi over 5 x 7 inches (1050 x 700 pixels) yields
good on-screen images and good prints (after expanding
the file by interpolation to 8 Mb using the standard
printing software - Adobe Photoshop).
findings are based on examination of high quality color
prints, printed using a dye-sublimation printer (Kodak
XL 7700) by DLM Imaging, Madison, Wisconsin. Images
were captured from a given sample of tissue at various
resolutions using two capture mechanisms: 1) scanning
the tissue directly in a good quality desktop scanner
(SilverScanner II from LaCie) in transparency mode,
and 2) Kodak Photo CD from film images. The scanner
scans at 1600, 800, and 400 (the default level) pixels
per inch (ppi), as well as a number of lower resolutions
(300, 150, etc.) We made scans at the three highest
levels. For our size of image, about 1.5 x 2.5 inches,
the highest resolution, 1600 ppi, yields files of 18
MB, 800 ppi yields 4 Mb files, and 400 ppi yields 1
Mb files. The Photo CD process yields an "image
pack" from a given film image, the same image in
five different resolutions. For our test we used the
three highest-resolution images:
x 2048 pixels, 18Mb file , the MAXimum PCD resolution
1536 x 1024 pixels, 4 Mb file, the Higher Resolution
(HR) PCD resolution
768 x 512 pixels, 1 Mb file, the Base Image (BI) PCD
resolution (the higher resolutions are obtained by
interpolating into the Base Image)
768 x 512 pixels, 1 Mb file, the Base Image (BI) PCD
resolution (the higher resolutions are obtained by
interpolating into the Base Image).
prints on the dye sublimation printer, files of 8 Mb
are required, and the printer has a resolution of 210
dpi (dots per inch). Whatever their initial size and
resolution, files were resized in Adobe Photoshop to
conform to this 8 Mb, 210 dpi format. The question being
tested was, what difference does it make what the original
resolution was, before conversion to the printer resolution?
The answer obtained held for both the Photo CD and the
scanned images. Starting with higher resolution files
yielded prints with less contrast, but finer shadings
of detail and color. With some images this is advantageous;
for other images it was better to have the greater contrast
and better outlines obtained from lower resolution initial
files. Starting with lower resolution files produced
prints which looked very similar to what is produced
on-screen by applying a sharpening filter (e.g. Unsharp
Mask in Photoshop) to the higher resolution image.
digitizing by direct scanning, digital camera, or Photo
CD, we retrieve the image, edit it (using Adobe Photoshop),
and apply any desired labeling. In the editing process,
contrast, color balance, background, and position of
the image in the screen frame are optimized. Editing
time various according to capture method and computer
speed, ranging from 40 minutes when captured from film
and edited with a Macintosh IIci computer, to less than
3 minutes when captured directly from tissue by a scanner
or digital camera and edited with a Power Mac 8100 computer.
Editing the same image with the Macintosh IIci and the
Power Mac 8100 shows that the difference in computer
speed cuts editing time for a particular image by more
followed in the editing procedure: 1.
Draw a rectangular marquee around a segment of the ruler
containing 3, 5, or 10 mm, selecting it. Move it as
close as possible to the edge of the section. Keep it
visible next to the section through all the editing
steps below, until the last one. If necessary, while
selected, rotate it (moving it away from the section
if necessary to avoid overlapping the section, but put
or keep it close to the edge of the section) so that
it is parallel to a horizontal edge of the screen window.
Then, while still selected, copy it to the clipboard.
If you should lose it in the editing process, it can
be retrieved by pasting from the clipboard.
Lasso the section and the ruler segment, choose inverse
from the Select menu, use the eyedropper tool to put
the background color as the window background color
(option - click on the background next to the section.
Press the delete key: this will turn everything outside
the lassoed window to the background color.
If necessary, crop the window to provide a pleasing
margin around the image of the section. Show Rulers
from the Windows menu, and note the length of the horizontal
and vertical edges (this will be needed to crop the
remaining images to the same size, even if the original
image was not cropped.)
Resize the image, under Image Size in the Image Menu,
to 1050 pixels wide and 150 ppi resolution. Then resize
the Canvas, under Canvas Size in the Image Menu, to
1050 pixels wide and 650 pixels high.
Do any editing of Levels (contrast, command L), Color
Balance (command Y), or hue/saturation (command U) [these
can also be accessed under Adjust in the Image menu.
Lasso the section (and ruler) and move it to the center
of the window. Note the ruler positions of any landmarks
to be used in bringing other sections into register.
Keep sections in different files in register:
Using Photoshop, show rulers (in Window menu, or command-R).
Lasso the desired image in one of the files
Put the cursor over a structure present in both sections
(files). Drag the cursor a bit, note the position
of the cursor on the rulers.
Move to the other image, lasso the image, put the
cursor on the same structure, drag it to the same
coordinates on the rulers. This will be tricky if
some rotation is necessary: two corresponding points
will be needed, but it can be done.
Type in any identification labels. The text-writing
module in Photoshop allows the same text to be inserted
in many successive images, without re-writing.
Lasso or marquee the ruler segment, rotate it if necessary
to bring it parallel to the horizontal margin, then
move it to a position below the desired location of
the final scale bar.
Draw a rectangular marquee above the ruler segment,
and draw a neat new ruler, using the line tool and the
marquee borders and the image of the original ruler
segment as guides. Then draw a rectangular marquee around
the original ruler and delete it; then draw another
rectangular marquee above the new drawn ruler such that
it cuts into the teeth of the rule; delete it to even
the ends of the teeth. Type in the scale bar label (
e.g. "5 mm").
Save the completed image as High Quality JPEG PICT file.
Repeat the process for the rest of the images in the
series. Photomatic should be able to do all of this
except the cropping, lassoing and marquees. Photomatic
is a program, from Daystar, which will record your actions
during editing, and then replay them to work on additional
images. It will faithfully repeat most editing commands,
BUT NOT THOSE INVOLVING MOUSE-DRAGGING, which somewhat
limits its usefulness.
File Compression and Storage
capture and editing, the resulting 2.1 Mb files are
stored on disks as PICT files, using the "high-quality"
level of JPEG compression available in the Photoshop
PICT file saving menu. Unlike "lower quality"
JPEG compressed files, our files do not undergo noticeable
degradation with repeated "high quality" compression
and decompression. When compressed, the image file occupies
about 100 kb (0.1 Mb) of disk storage space. Thus thousands
of such images can be accommodated on a single 680 Mb