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Photographic Techniques in Veterinary Pathology
APPENDIX Photographic Techniques in Veterinary Pathology M. Donald McGavin Gross Specimen Photography Judged by the photographs presented at seminars and conferences, requirements for gross specimen photography are not fully appreciated. Because of space limitations in the sixth edition, the techniques used to obtain these types of images are discussed in detail at www.expertconsult.com, including descriptions of the techniques of gross specimen photography, camera stand and photographic lamps, backgrounds, flash photography, photomicrography, and evaluation of photomicrographs.
Studio Lighting The optimal arrangement for the photography of isolated organs is a studio setup as depicted in Fig. 1, with a main light to the left to produce shadows and a fill light on the right to illuminate those shadow sufficiently to show detail in them without being so intense as to erase them. To achieve this, the fill light is 1.5 to 3 times the distance of the main light from the subject. A good default is 1.5 times the distance from the average specimen. To cast shadows downward, the axis of the main light should be approximately 30 degrees above the plane of the specimen and at the 10:30 o’clock position (315 degrees) on a clock face around the axis of the camera lens (see Fig. 1). This means that for an anatomically and correctly oriented specimen, the main source of light comes from the left and above. This seems natural to us because for millions of years man has been programmed to interpret images based on the assumption that there is one light source (the sun), and it comes from above. Lighting from below casts shadows upward and appears unnatural or eerie; it is called “spook” lighting and for that reason is used for movies of that name. To emphasize surface “hills and valleys” (i.e., texture) in a flat surface such as intestinal mucosa or skin, the main light is lowered to 15 to 25 degrees above the plane of the specimen to produce a “skimming light.” With a digital camera it is simple to try different angles of the main light and immediately review the images in the view finder.
Flash Photography If studio lighting is not available or if the specimen is too large to fit on the background or cannot be moved, then flash photography is the alternative. Point-and-shoot cameras produce photographs with excellent exposure and color balance, but the built-in flash unit produces axial lighting (i.e., light on the same axis as the axis of the camera lens). Therefore it casts no shadows, and it is shadows on the surface that are responsible for the depiction of three-dimensional shapes (modeling) of organs and the texture of their surface. The only answer is to move the axis of the light beam away from the axis of the camera lens. There are two main ways to do this. For small
specimens (a maximum dimension of approximately 9 inches), the flash unit can be mounted on a side bracket (Fig. 2), but for larger specimens the flash will have to be held by hand—either by the photographer or an assistant (Figs. 3 and 4). A black background from a glass-topped black box, painted blackboard, or black construction paper is used. These may become blotchy when wet by fluids, but this is easily corrected by Adobe Photoshop. The requirements for a photograph of a gross specimen include the following: • Appropriately dissected specimen • Correctly anatomically oriented specimen • Suitable framing so that anatomic landmarks are included and can be recognized and used for orientation by the viewer • Correctly focused with adequate depth of field • Lighting from above that renders surface modeling and texture • Interesting and aesthetically pleasing composition, if possible • Unobtrusive background—no stainless steel tables, tiled floors, floor drains, or colored backgrounds
Photomicrography Automatic exposure determination, automatic color balancing, and on-the-monitor focusing have made digital photomicrography easier, but some steps to obtain optimal photomicrographs (Fig. 5) are still the responsibility of the operator. Koehler illumination is particularly important and has two steps that need to be carried out after the microscope has been focused on the specimen: (1) focusing the image of the field diaphragm in the microscope’s field of view (easier to do than describe) and (2) setting the position of the aperture diaphragm. The aperture diaphragm controls three important features of the image: (1) resolution, defined as the ability of the lens to separate adjacent points in the image; (2) contrast, the differences between the light and dark tones (gray scale) of colors; and (3) depth of field (i.e., the thickness of the specimen in focus). Maximum resolution occurs when the aperture diaphragm is open so its circle of light in the rear focal plane (RFP) of the objective (visible only when the ocular has been removed) is the same diameter as the circle of light of the objective (Fig. 6). This arrangement is called 100% cone and is an expression of the relative diameters of the illuminated circles from the objective and condenser as viewed in the RFP. Closing the aperture diaphragm increases contrast (similar to “lowering” the condenser), but if the aperture diaphragm is closed too far, diffraction occurs and resolution is markedly reduced (see Fig. 5, B). Thus the choice is a balance between resolution and contrast. There are some rules of thumb for obtaining maximum resolution and good contrast. With thin specimens, such as blood smears and thin tissue sections (3 to 4 µm in thickness),
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APPENDIX Photographic Techniques in Veterinary Pathology
45°
30°
10:30 12 9
3 6
Figure 1 Studio Lighting Arrangement. Note that the axis of main light (left) is 30 degrees above the plane of the specimen to produce shadows that will show the topography of the surface. Also, the main light is at the 10:30 o’clock position to ensure that the light comes from the top left. The fill light on the right is at the 3 o’clock position, but further away (1.5 to 3 times) from the specimen than the main light, to ensure that the shadows cast by the main light are not obliterated. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.)
Figure 2 Camera, Bracket, and Flash Unit. The bracket holds the flash unit approximately 6 inches to the side of the camera with the result that its light produces shadows and thus modeling for small specimens less than 9 inches in length. At greater distances the flash is so close to the axis of the camera lens that it does not produce shadows large enough to depict modeling. Note also that the axis of the camera lens is directed onto the specimen from approximately the 10:30 o’clock position. The handle is extremely handy when holding the camera in an autopsy laboratory. (Courtesy Mr. P.D. Snow, College of Veterinary Medicine, University of Tennessee.)
Figure 3 Flash, Off the Camera. To obtain modeling for specimens whose dimensions are greater than approximately 9 inches, the main light has to be moved further to the left than is possible using a camera bracket. Thus the flash unit is held by hand at the 10:30 o’clock position and approximately 30 degrees above the specimen plane by the photographer, but for larger specimens an assistant is necessary. The background in this case is a glasscovered black box. (Courtesy Dr. C. O’Muireagain, Regional Veterinary Laboratory, Sligo, Ireland.)
Figure 4 Thoracic Viscera, Lungs, Badger, Tuberculosis. The flash unit was held by hand off the camera at the 10:30 o’clock position, as is evident from the shadows cast down by the heart. Note the excellent rendition of modeling of the lung lobes, tuberculosis tubercles in the top left lobe (right cranial) and heart. The homogeneous black background was obtained by means of digital editing using Adobe Photoshop. (Courtesy Dr. C. O’Muireagain, Regional Veterinary Laboratory, Sligo, Ireland.)
APPENDIX Photographic Techniques in Veterinary Pathology
A
B
Figure 5 Outcomes of Koehler Illumination. A, Koehler illumination with optimal resolution and contrast. Brain, canine distemper, inclusion bodies. H&E stain. 40× planachromatic objective. B, Koehler illumination with diffraction and reduced resolution. Caseous exudate. The aperture diaphragm has been closed too far—to 50% cone. 40× planapochromatic objective. (A courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee. B courtesy Dr. P.W. Ladds, James Cook University, Australia.) Microscope eyepiece tube
A
Aperture iris diaphragm
B RFP
C 100% cone
80% cone
Figure 6 Koehler Illumination. The ocular (eyepiece) has been removed from the microscope eyepiece tube to reveal the rear focal plane (RFP) of the objective. A, RFP of the objective with the aperture (condenser) diaphragm fully open so that it is not visible and to reveal the full diameter of the objective’s aperture. B, RFP of the objective with the aperture diaphragm at 100% cone. The internal diameter of the image of the aperture diaphragm matches the internal diameter of the aperture of the objective. C, RFP of the objective with the aperture diaphragm at 80% cone. The internal diameter of the image of the aperture diaphragm is 80% of the aperture of the objective (gray). Yellow, eyepiece tube; gray, inner surface of eyepiece tube; green, aperture (condenser) iris diaphragm; white, rear focal plane. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee; and Dr. J.F. Zachary, College of Veterinary Medicine, University of Illinois.)
open the aperture diaphragm to 90% cone so the condenser’s circle of illumination is just visible inside the RFP. For well-stained histologic sections, use 80% to 90% cone diameter, but if the contrast of the stained section is low, as from inadequate density of the stain, it may be necessary to use 70% to 80% cone diameter. For very low-contrast specimens, it may be necessary to close the aperture diaphragm even more and risk diffraction if the specimen is to be visible. With histologic slides the secret is to have well-stained sections with good color and tonal (gray scale) contrasts. If the specimen is thick (7 to 9 µm in thickness), to have the image in focus it may be necessary to close the aperture diaphragm almost to the point of causing diffraction, particularly with high-magnification objectives, which have the shallowest depth of field (i.e., depth of the tissue in focus).
Evaluation of Photomicrographs 1. Focus. The image should be in focus across the entire monitor. If it is out of focus at the edges (“falloff”), this appearance could be caused by poorly corrected flat field (or plan objectives), or the aperture diaphragm has not been closed adequately to increase the depth of field. Before the use of digital cameras and focusing on the screen of the computer monitor, focusing was done on the aerial image of the microscope’s image, “floating” in
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front of the image of the graticule, when viewed through the focusing telescope of the camera. This method had significant problems. Corrections had to be made for the viewer’s eyesight and objectives with a large depth of field; the 4× and lower-power objectives were particularly difficult to focus. With these objectives it is necessary to focus on a plane in the section that gives the appearance of the whole field being in focus, and not on a single cell that may or may not be in a plane that would render those cells above and below it also in focus. However, focusing the image on the monitor’s screen is relatively reliable and avoids these problems. 2. Exposure. Exposure is evaluated by looking at the microscope’s clear background in the print or digital image. This background should have a faint density, usually gray unless there is a color cast. In a printed photomicrograph there should be just a faint density, slightly darker than that of the white of the blank page. 3. Color cast. The microscope’s clear background should be white and have no color casts. The usual color casts are yellow, orange, or blue arising from incorrect color temperature. Occasionally color casts are green from a chromatically uncorrected lens, usually in the lamp house. Color casts can easily be prevented in digital photomicrography by setting the white balance before each exposure, usually by selecting an area of faint density in the microscope’s clear background and pressing the “white button.” 4. Uneven illumination. Uneven illumination can be due to errors in setting up Koehler illumination but is also a common problem with low-power objectives, 4× and lower. To obtain even illumination with low-power objectives of less than 4×, special condensers and highly corrected objectives are frequently required, but an easier method is to use a software program to correct this problem in the image stored in the computer. 5. Contrast. The factors involved in controlling contrast are as follows: • Quality and staining of the specimen and the specific stain chosen (e.g., trichrome vs. van Gieson). • Quality of the objective (e.g., apochromatic objectives have far higher contrast than achromatic objectives). • Correct positioning of the aperture diaphragm. With Koehler illumination, positioning of the aperture diaphragm controls resolution, contrast, and depth of field. Its correct positioning is particularly important with high dry and 100× oil immersion objectives. • Cover glass and mountant thickness, which together should be 0.17 mm, requiring a No. 1 coverslip. • Cleanliness of the surface of the coverslip. • Digital camera quality, including image correction software if provided. • Magnification of the objective: Lower-magnification objectives and high-power objectives (i.e., high dry and oil immersion) have lower contrast than 10× and 25× objectives of the same optical correction (e.g., all apochromatic objectives or all achromatic objectives). Troubleshooting of problems with photomicrography is described in the online Appendix. A convenient arrangement is for the operator to make a checklist that applies to the procedure for taking photomicrographs with a specific microscope and digital camera. Checklists may be boring but are essential for ensuring that the correct steps and sequence in Koehler illumination are followed. Unfortunately, Koehler illumination is not an intuitive process. Computer programs are available to correct out-of-focus areas from curvature of field, incorrect density of the microscope’s clear background, color casts, and contrast. More information on these topics is available at www.expertconsult.com.
Studio Lighting The optimal arrangement for the photography of isolated organs is a studio setup as depicted in Fig. 1, with a main light to the left to produce shadows and a fill light on the right to illuminate those shadow sufficiently to show detail in them without being so intense as to erase them. To achieve this, the fill light is 1.5 to 3 times the distance of the main light from the subject. A good default is 1.5 times the distance from the average specimen. To cast shadows downward, the axis of the main light should be approximately 30 degrees above the plane of the specimen and at the 10:30 o’clock position (315 degrees) on a clock face around the axis of the camera lens (see Fig. 1). This means that for an anatomically and correctly oriented specimen, the main source of light comes from the left and above. This seems natural to us because for millions of years man has been programmed to interpret images based on the assumption that there is one light source (the sun), and it comes from above. Lighting from below casts shadows upward and appears unnatural or eerie; it is called “spook” lighting and for that reason is used for movies of that name. To emphasize surface “hills and valleys” (i.e., texture) in a flat surface such as intestinal mucosa or skin, the main light is lowered to 15 to 25 degrees above the plane of the specimen to produce a “skimming light.” With a digital camera it is simple to try different angles of the main light and immediately review the images in the view finder.
Flash Photography If studio lighting is not available or if the specimen is too large to fit on the background or cannot be moved, then flash photography is the alternative. Point-and-shoot cameras produce photographs with excellent exposure and color balance, but the built-in flash unit produces axial lighting (i.e., light on the same axis as the axis of the camera lens). Therefore it casts no shadows, and it is shadows on the surface that are responsible for the depiction of three-dimensional shapes (modeling) of organs and the texture of their surface. The only answer is to move the axis of the light beam away from the axis of the camera lens. There are two main ways to do this. For small
specimens (a maximum dimension of approximately 9 inches), the flash unit can be mounted on a side bracket (Fig. 2), but for larger specimens the flash will have to be held by hand—either by the photographer or an assistant (Figs. 3 and 4). A black background from a glass-topped black box, painted blackboard, or black construction paper is used. These may become blotchy when wet by fluids, but this is easily corrected by Adobe Photoshop. The requirements for a photograph of a gross specimen include the following: • Appropriately dissected specimen • Correctly anatomically oriented specimen • Suitable framing so that anatomic landmarks are included and can be recognized and used for orientation by the viewer • Correctly focused with adequate depth of field • Lighting from above that renders surface modeling and texture • Interesting and aesthetically pleasing composition, if possible • Unobtrusive background—no stainless steel tables, tiled floors, floor drains, or colored backgrounds
Photomicrography Automatic exposure determination, automatic color balancing, and on-the-monitor focusing have made digital photomicrography easier, but some steps to obtain optimal photomicrographs (Fig. 5) are still the responsibility of the operator. Koehler illumination is particularly important and has two steps that need to be carried out after the microscope has been focused on the specimen: (1) focusing the image of the field diaphragm in the microscope’s field of view (easier to do than describe) and (2) setting the position of the aperture diaphragm. The aperture diaphragm controls three important features of the image: (1) resolution, defined as the ability of the lens to separate adjacent points in the image; (2) contrast, the differences between the light and dark tones (gray scale) of colors; and (3) depth of field (i.e., the thickness of the specimen in focus). Maximum resolution occurs when the aperture diaphragm is open so its circle of light in the rear focal plane (RFP) of the objective (visible only when the ocular has been removed) is the same diameter as the circle of light of the objective (Fig. 6). This arrangement is called 100% cone and is an expression of the relative diameters of the illuminated circles from the objective and condenser as viewed in the RFP. Closing the aperture diaphragm increases contrast (similar to “lowering” the condenser), but if the aperture diaphragm is closed too far, diffraction occurs and resolution is markedly reduced (see Fig. 5, B). Thus the choice is a balance between resolution and contrast. There are some rules of thumb for obtaining maximum resolution and good contrast. With thin specimens, such as blood smears and thin tissue sections (3 to 4 µm in thickness),
1319
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APPENDIX Photographic Techniques in Veterinary Pathology
45°
30°
10:30 12 9
3 6
Figure 1 Studio Lighting Arrangement. Note that the axis of main light (left) is 30 degrees above the plane of the specimen to produce shadows that will show the topography of the surface. Also, the main light is at the 10:30 o’clock position to ensure that the light comes from the top left. The fill light on the right is at the 3 o’clock position, but further away (1.5 to 3 times) from the specimen than the main light, to ensure that the shadows cast by the main light are not obliterated. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.)
Figure 2 Camera, Bracket, and Flash Unit. The bracket holds the flash unit approximately 6 inches to the side of the camera with the result that its light produces shadows and thus modeling for small specimens less than 9 inches in length. At greater distances the flash is so close to the axis of the camera lens that it does not produce shadows large enough to depict modeling. Note also that the axis of the camera lens is directed onto the specimen from approximately the 10:30 o’clock position. The handle is extremely handy when holding the camera in an autopsy laboratory. (Courtesy Mr. P.D. Snow, College of Veterinary Medicine, University of Tennessee.)
Figure 3 Flash, Off the Camera. To obtain modeling for specimens whose dimensions are greater than approximately 9 inches, the main light has to be moved further to the left than is possible using a camera bracket. Thus the flash unit is held by hand at the 10:30 o’clock position and approximately 30 degrees above the specimen plane by the photographer, but for larger specimens an assistant is necessary. The background in this case is a glasscovered black box. (Courtesy Dr. C. O’Muireagain, Regional Veterinary Laboratory, Sligo, Ireland.)
Figure 4 Thoracic Viscera, Lungs, Badger, Tuberculosis. The flash unit was held by hand off the camera at the 10:30 o’clock position, as is evident from the shadows cast down by the heart. Note the excellent rendition of modeling of the lung lobes, tuberculosis tubercles in the top left lobe (right cranial) and heart. The homogeneous black background was obtained by means of digital editing using Adobe Photoshop. (Courtesy Dr. C. O’Muireagain, Regional Veterinary Laboratory, Sligo, Ireland.)
APPENDIX Photographic Techniques in Veterinary Pathology
A
B
Figure 5 Outcomes of Koehler Illumination. A, Koehler illumination with optimal resolution and contrast. Brain, canine distemper, inclusion bodies. H&E stain. 40× planachromatic objective. B, Koehler illumination with diffraction and reduced resolution. Caseous exudate. The aperture diaphragm has been closed too far—to 50% cone. 40× planapochromatic objective. (A courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee. B courtesy Dr. P.W. Ladds, James Cook University, Australia.) Microscope eyepiece tube
A
Aperture iris diaphragm
B RFP
C 100% cone
80% cone
Figure 6 Koehler Illumination. The ocular (eyepiece) has been removed from the microscope eyepiece tube to reveal the rear focal plane (RFP) of the objective. A, RFP of the objective with the aperture (condenser) diaphragm fully open so that it is not visible and to reveal the full diameter of the objective’s aperture. B, RFP of the objective with the aperture diaphragm at 100% cone. The internal diameter of the image of the aperture diaphragm matches the internal diameter of the aperture of the objective. C, RFP of the objective with the aperture diaphragm at 80% cone. The internal diameter of the image of the aperture diaphragm is 80% of the aperture of the objective (gray). Yellow, eyepiece tube; gray, inner surface of eyepiece tube; green, aperture (condenser) iris diaphragm; white, rear focal plane. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee; and Dr. J.F. Zachary, College of Veterinary Medicine, University of Illinois.)
open the aperture diaphragm to 90% cone so the condenser’s circle of illumination is just visible inside the RFP. For well-stained histologic sections, use 80% to 90% cone diameter, but if the contrast of the stained section is low, as from inadequate density of the stain, it may be necessary to use 70% to 80% cone diameter. For very low-contrast specimens, it may be necessary to close the aperture diaphragm even more and risk diffraction if the specimen is to be visible. With histologic slides the secret is to have well-stained sections with good color and tonal (gray scale) contrasts. If the specimen is thick (7 to 9 µm in thickness), to have the image in focus it may be necessary to close the aperture diaphragm almost to the point of causing diffraction, particularly with high-magnification objectives, which have the shallowest depth of field (i.e., depth of the tissue in focus).
Evaluation of Photomicrographs 1. Focus. The image should be in focus across the entire monitor. If it is out of focus at the edges (“falloff”), this appearance could be caused by poorly corrected flat field (or plan objectives), or the aperture diaphragm has not been closed adequately to increase the depth of field. Before the use of digital cameras and focusing on the screen of the computer monitor, focusing was done on the aerial image of the microscope’s image, “floating” in
1321
front of the image of the graticule, when viewed through the focusing telescope of the camera. This method had significant problems. Corrections had to be made for the viewer’s eyesight and objectives with a large depth of field; the 4× and lower-power objectives were particularly difficult to focus. With these objectives it is necessary to focus on a plane in the section that gives the appearance of the whole field being in focus, and not on a single cell that may or may not be in a plane that would render those cells above and below it also in focus. However, focusing the image on the monitor’s screen is relatively reliable and avoids these problems. 2. Exposure. Exposure is evaluated by looking at the microscope’s clear background in the print or digital image. This background should have a faint density, usually gray unless there is a color cast. In a printed photomicrograph there should be just a faint density, slightly darker than that of the white of the blank page. 3. Color cast. The microscope’s clear background should be white and have no color casts. The usual color casts are yellow, orange, or blue arising from incorrect color temperature. Occasionally color casts are green from a chromatically uncorrected lens, usually in the lamp house. Color casts can easily be prevented in digital photomicrography by setting the white balance before each exposure, usually by selecting an area of faint density in the microscope’s clear background and pressing the “white button.” 4. Uneven illumination. Uneven illumination can be due to errors in setting up Koehler illumination but is also a common problem with low-power objectives, 4× and lower. To obtain even illumination with low-power objectives of less than 4×, special condensers and highly corrected objectives are frequently required, but an easier method is to use a software program to correct this problem in the image stored in the computer. 5. Contrast. The factors involved in controlling contrast are as follows: • Quality and staining of the specimen and the specific stain chosen (e.g., trichrome vs. van Gieson). • Quality of the objective (e.g., apochromatic objectives have far higher contrast than achromatic objectives). • Correct positioning of the aperture diaphragm. With Koehler illumination, positioning of the aperture diaphragm controls resolution, contrast, and depth of field. Its correct positioning is particularly important with high dry and 100× oil immersion objectives. • Cover glass and mountant thickness, which together should be 0.17 mm, requiring a No. 1 coverslip. • Cleanliness of the surface of the coverslip. • Digital camera quality, including image correction software if provided. • Magnification of the objective: Lower-magnification objectives and high-power objectives (i.e., high dry and oil immersion) have lower contrast than 10× and 25× objectives of the same optical correction (e.g., all apochromatic objectives or all achromatic objectives). Troubleshooting of problems with photomicrography is described in the online Appendix. A convenient arrangement is for the operator to make a checklist that applies to the procedure for taking photomicrographs with a specific microscope and digital camera. Checklists may be boring but are essential for ensuring that the correct steps and sequence in Koehler illumination are followed. Unfortunately, Koehler illumination is not an intuitive process. Computer programs are available to correct out-of-focus areas from curvature of field, incorrect density of the microscope’s clear background, color casts, and contrast. More information on these topics is available at www.expertconsult.com.