Microscopy and Photography

Optimal Optics

Optimal image formation occurs when the image is in focus and the illumination is appropriately adjusted. First, the eyepieces should be adjusted for width (by sliding them back and forth) in order to form a single image. Second, each eye is closed in turn individually to adjust the focus of each eyepiece. Many people have subtle differences between their two eyes. Finally, the illumination is adjusted to ensure that the light is bright, evenly dispersed, glare free, and that good image contrast is achieved. This procedure is termed “Koehler illumination” after August Karl Johann Valentin Kohler (1866–1948) who first determined the optimal settings for his Ph.D. thesis in 1893.

Parts of the microscope used for Kohler illumination:

• Field diaphragm: This is an iris diaphragm at the base of the microscope that is adjusted by moving the surrounding circular ring. Closing the diaphragm limits the size of the illuminated field on the stage of the microscope.

• Substage condenser: This is located just below the stage. It can be moved up and down. It is centered using two screws.

• Aperture iris diaphragm: This iris diaphragm is located in the substage condenser. It is adjusted using a rotating ring on the front of the condenser.

Adjusting a microscope for Koehler illumination:

• 1.

  Open the aperture diaphragm and the field diaphragm completely. Using a 20× objective, focus on a slide on the stage.

• 2.

  Close the field diaphragm almost completely. Raise the condenser until the edges of the diaphragm are sharply focused (the condenser is usually at about its highest position).

• 3.

  Use the centering screws on the substage condenser to center the image of the field diaphragm. Slowly open the field diaphragm until it just disappears from view.

• 4.

  Remove one eyepiece objective and look into the tube. Open and close the aperture diaphragm until only 66–77% of the back lens is illuminated ( Fig. 7.1 ). This prevents unnecessary light from entering that creates glare.

  

The microscope is now optimally adjusted for the objective. Other objectives will require readjustment and in practical terms usually a compromise position is used that is adequate for all of the objectives. A reasonable solution is to center the field diaphragm for a 40× objective and maintain its opening for a 10× field. The substage condenser should be slightly below the level where dust is brought into focus using a 10× objective. As objectives are changed, only the aperture diaphragm need be changed to optimize contrast.

If the light intensity needs to be adjusted, it is accomplished by using the transformer, not by changing the position of the condenser or diaphragm.

Contrast can be increased by closing the aperture diaphragm below 60% or by lowering the substage condenser. However, resolution and sharpness are reduced. This maneuver may be useful if looking for refractile material (see section below).

Oil Immersion Microscopy

Oil immersion lenses (usually 100×) can achieve higher magnifications than dry lenses. Because the refractive index of oil is higher than that of air, light rays coming from the slide are bent to a greater degree and thus an oil objective can capture more of these light rays which results in greater resolution.

There are two major uses in surgical pathology for oil immersion magnification:

Hematologic specimens: Due to the generally small size of the cells and the importance of subtle nuclear and cytoplasmic features for characterization, higher magnifications are helpful.

Small microorganisms: Some organisms such as acid-fast bacilli or microsporidia are best seen at high magnification.

There is virtually no other use for oil immersion lenses. This has led to the old saying in pathology that “low power pathologists make high power diagnoses.” The use of oil should be avoided outside of the applications cited above due to the frequent contamination of the microscope and other objectives with oil if great care is not used.

Only immersion oil designed for microscopic use should be employed. Lint-free tissues or lens paper should always be available to wipe away any excess oil before it drips into the microscope or onto other surfaces.

Using an oil immersion lens:

• 1.

  Focus the microscope using the highest magnification available with a dry objective. It is helpful to identify and mark the area(s) of the slide to be examined under oil with ink to avoid the need to scan the slide after oil is applied.

• 2.

  Swing this objective away (in the direction to bring the oil objective into place) and place a small amount of oil on the slide.

• 3.

  Swing the oil objective into place, making sure that the space between the objective and the slide is filled with oil. The slide may now be observed.

• 4.

  After viewing, again swing the oil objective partially away from the slide.

• 5.

  Immediately wipe the objective with lens paper to prevent oil from dripping onto the microscope. If the oil is allowed to dry, it can be difficult to remove and efforts to do so can damage the lens.

• 6.

  Remove the slide and wipe off excess oil with lens paper. The slide may then be cleaned with a small amount of xylene. Peripheral blood smears are often viewed under oil without using a coverslip. The oil can be wiped directly off the slide.

Do not attempt to view a slide with a high power dry objective while there is oil on the slide! If it is necessary to scan a slide, place a 4× objective adjacent to the oil objective. One can safely alternate between these two objectives without contaminating the 4× objective with oil. It is very difficult to clean oil off an objective not designed to be used with oil (see the section on cleaning objectives below).

Optical Properties

The optical properties of microscopic objects reveal clues about their structure and identity ( Table 7.1 ).

Table 7.1

OPTICAL PROPERTIES OF COMMONLY SEEN NONCELLULAR MATERIAL

TYPE	POLARIZABLE	REFRACTILE	LOCATIONS USUALLY SEEN	APPEARANCE/STAINS	COMMENTS	HISTOLOGIC APPEARANCE
ENDOGENOUS Material
Amyloid	Yes	Yes/no	Bone marrow (multiple myeloma), medullary carcinoma of the thyroid, periarticular tissue in dialysis patients, many other sites	Acellular homogeneous pink material, sometimes with giant cells. Congo red +: orange/red without polarization and green, orange or yellow with polarization	Immunoperoxidase studies can be used to identify specific types of amyloid: Multiple myeloma—lambda and kappa chains Medullary carcinoma of the thyroid—calcitonin Dialysis related amyloidosis - ß 2 microglobulin EM can also be used to recognize amyloid
Bile	No	No	Liver—hepatocytes or intracanicular	Dark green–brown globules (intra or extracellular). PAS +	May be helpful for recognition of hepatocellular carcinoma
Bone and collagen	Yes	No	Joints and connective tissue	Normal bone—polarization shows regular osteoid seams (not seen in woven bone) Type I collagen is polarizable. Type III collagen (reticulin) is not polarizable	Bone and collagen overstained with Congo red will show green birefringence after polarization. A properly stained slide should not show background staining Trichrome stains and reticulin stains can be used to identify collagen Nodular sclerosing Hodgkin's disease is associated with polarizable collagen
Calcium oxalate	Yes	Yes	Apocrine cysts of the breast, benign thyroid follicles, giant cells in sarcoidosis	Flat rhomboid (sometimes needle shaped) colorless or pale yellow crystals. Can be difficult to see without polarization	Can be the source of mammographic calcifications in breast biopsies Also present in congenital hyperoxaluria
Calcium phosphate	No	No	Benign and malignant breast lesions, areas of chronic inflammation or necrosis, deposition on collagen (e.g. heart valves), pulmonary blue bodies	Purple granular material Calcium stain +	Most common source of mammographic calcifications
Calcium pyrophosphate dihydrate crystals	Yes	Yes	Large joints in periarticular tissues (uncommon in small joints of foot or hand)	Blue to purple short rhomboid crystals (but may be needle shaped)	“Pseudogout” or chondrocalcinosis The crystals are water soluble and require anaqueous processing for best demonstration. They can sometimes be demonstrated with polarization of an unstained tissue section
Charcot–Leyden crystals	No	Yes	Sites of eosinophil accumulation (e.g. chronic sinusitis, parasitic infections, asthma)	Bright-red, needle-like crystals	Formed from galectin-10 released when eosinophils undergo cytolysis
Corpora amylacea	No	No	Prostate, brain, lung	Extracellular laminated light pink spherical structures	Incidence increases with age
Gamna–Gandy nodules	No	Yes/no	Spleen, lymph nodes, thymus gland, thyroid, cardiac myxomas	Granulomas consisting of hemosiderin, calcium, foreign body giant cells, and ovoid or bamboo shaped structures	“Siderotic granulomas” found in sites of prior hemorrhage , Can mimic fungal mycelia or parasite eggs
Hamazaki–Wesenberg bodies	No	Yes	Areas of prior hemorrhage, lymph nodes (sinusoids)	Small round to ovoid brown bodies that may appear to be budding	Can mimic pigmented fungal forms or bacteria
Hemosiderin	No	Yes	Any area of hemorrhage Liver in hemochromatosis and hemosiderosis	Coarse granular brown intra and extracellular granules. Iron stain +	A complex of iron and ferritin Useful in distinguishing prior bleeding from intraoperative bleeding
Liesegang rings	No	No	Any area of old hemorrhage	Round extracellular concentric laminated or fibrillated concretions of precipitated proteins	May be mistaken for the Giant Kidney Worm or fungal organisms. , , Similar pattern to psammoma bodies
Lipids	No/yes	No	Polarizable lipids may be present in xanthomas, histiocytosis X, and other dermatopathologic entities, and fat necrosis secondary to pancreatitis Cholesterol crystals are often seen as empty clefts in areas of cell injury	Needle-shaped clear crystals of varying size, plate-like structures, or intracellular rounded structures. Oil red O +. Sudan black +	Must have special processing or frozen sections to avoid loss during processing Also present in metabolic storage diseases such as Gaucher, Neimann-Pick, and Wolman disease Polarized crystals in fat necrosis may be due to pancreatitis
Lipofuscin (oxidized lipid precursors)	No	No	Sites of atrophy or chronic injury	Fine yellow–brown granules to coarse granules resembling hemosiderin. PAS +	Ochrocytes are histiocytes containing lipofuscin
Malaria pigment (haemozoin, hematin)	Yes	No	Macrophages and erythrocytes	Brown to black granules inside macrophages	Present in malaria and schistosomal infections or severe hemolytic anemia Formalin pigment can resemble malaria pigment
Melanin	No	No	Normal melanocytes in basal epithelium, pigmented malignant melanomas	Fine brown or black granules but can sometimes resemble hemosiderin. Fontana Masson +
Michaelis–Gutmann body	No	No	Malakoplakia	Concentric targetoid bodies (“owl-eye”), intra and extracellular. Iron stain +, Von Kossa +, PAS +	These bodies form due to defective phagocytosis of bacteria—most patients have a chronic coliform infection. Forms masses that can mimic tumors
Prostatic crystalloids	No	Yes	Prostate carcinomas	Bright eosinophilic angulated crystals in lumens of adenocarcinomas	When present in the prostate, these crystals are always associated with cancer—if present on a core needle biopsy in the absence of cancer, additional levels should be examined Similar crystals are rarely seen in DCIS of the breast
Psammoma body	No	No	Papillary carcinoma of the thyroid, ovarian carcinoma, lactational-like changes in the breast, others	Laminated concentric rings of calcium phosphate	Very specific in the thyroid for papillary carcinoma Similar structure to Liesegang rings
Reinke crystals	No	Yes	Leydig cells of the testis, hilus cells of ovary, and their tumors	Rod-shaped eosinophilic crystals. Masson's trichrome + (magenta)	The crystals dissolve in formalin They are better preserved in frozen sections, touch imprints, and tissue fixed in alcohol
Schaumann bodies	No	No	Intracytoplasmic inclusions seen in the granulomas of sarcoid, tuberculosis, and chronic beryllium diseases	Concentric basophilic rings	These bodies are most commonly seen in sarcoidosis. Other types of inclusions in sarcoidosis are calcium oxalate crystals and asteroid bodies (spider-shaped inclusions). These inclusions are not specific for sarcoid
Spironolactone bodies	No	No	Adrenal adenomas associated with Conn syndrome	Concentric laminated eosinophilic inclusions (2–12 microns) in cytoplasm. PAS +	Found in tumors in patients with Conn syndrome, treated with spironolactone IHC can identify aldosterone in the bodies
Uric acid crystals (gout)	Yes	Yes	Periarticular tissues around joints, other areas of connective tissue	Long needle shaped crystals (sheaves of wheat) are characteristic but may be fractured and appear to be smaller crystals	Requires anaqueous processing for preservation In routine H&E sections only needle shaped holes may be seen where the crystals are dissolved. The crystals may be seen in routinely fixed tissue if the tissue is unstained
Iatrogenic Material
Barium sulfate	No/yes	Yes	In GI tract after radiologic examination, in peritoneum after perforation, within bone cement	Golden refractile granular material. May be intra- or extracellular	Rarely incites an inflammatory reaction
Bile acid sequestrants			In GI tract	Bright orange (but also black or red), rectangular shape (but may be rounded), smooth texture (fish scale texture uncommon); may have small dot-like inclusions. AFB dull yellow	These agents are usually used to treat diarrhea and do not cause bowel damage. Also see kayexalate and sevelamer as these agents are also seen in the GI tract
Cotton fibers	Yes	Yes	Around surgical sites	Hollow discoid fibers, present intra or extracellularly
Cornstarch	Yes	No	In surgical sites	3–20 micron spheres. Maltese cross appearance after polarization. PAS+, MSS+	Used to lubricate surgical gloves. However cornstarch should be avoided as it can incite a granulomatous response
Formalin pigment	No	Yes	Most commonly seen in bloody tissues	Brown or black finely granular extracellular deposits	Due to a reaction between formic acid and heme during fixation. Can be avoided by using buffered formalin Can be mistaken for malaria pigment
Gelfoam	No/yes	No/yes	Within vascular spaces of hemangiomas or other vascular lesions, sometimes used to mark breast core needle biopsy sites	Irregular fenestrated bluish or clear material	Numerous substances are used for embolization including polymer foams (such as gelfoam), poly (vinyl alcohol) particulates, gelatin products, microspheres, and drug- eluting beads. , , Autologous tissue including blood clots and tissue (subcutaneous tissue or muscle) have been used in the past. Metallic coils are also used
Gold	Yes	No	Skin, lymph nodes, organs of patients treated with gold for rheumatoid arthritis	Small intracellular black particles in histiocytes	Gold in intramammary LNs can mimic mammographic calcifications. This treatment is now uncommon. Black deposits in lymph nodes are more likely to be due to tattoo pigment
Graft material—Gortex or Dacron	Yes	Yes	Grafts	Numerous uniform round filaments with small black granules
India Ink (tattoo pigment)	No	No	Injected into the site of biopsied colonic polyps	Black granular pigment in stroma or within histiocytes	Used to document the site of a previously biopsied polyp on the serosal surface to aid in colonic resection. Indocyanine green and autologous blood are also used ,
Kayexalate (sodium polystyrene sulfonate)	No	Yes	GI tract	Rectangular purple to red crystals with a fish scale pattern. PAS + and AFB black	Used to treat patients with renal-failure as a potassium-binding agent. Can cause ulcers, perforation, necrosis, and pseudotumors (Gonzalez)
Melanosis coli	No	No	Lamina propria of the colon	Fine brown to black granules in macrophages. PAS +, silver stain +	Associated with anthracene-derived bowel cathartics. Can cause grossly pigmented colonic mucosa
Mercuric chloride	Yes/no	No	Tissues fixed in mercury containing fixatives	Dark brown granular extracellular deposits throughout the tissues	Should be removed by proper tissue processing
Metal	No	No	Tissue around prosthetic joints, rarely at surgical sites	Small black irregular angulated or needle shaped fragments that may be intra or extracellular
Minocycline	No	No	Thyroid, atheromatous plaques, substantia nigra	Black granular pigment	Found in patients treated with minocycline
Myospherulosis	No	No	Nasal cavity and paranasal sinuses and subcutaneous tissue	Sac-like structures with outer lipid surrounding endobodies (red blood cells)	Due to packing with a petroleum based ointment resulting in a benign mass formed by erythrocytes and exogeneous or endogenous lipids Can be mistaken for protothecosis or fungi, carcinoma, osteofibrosis, or metastasis
Polyethylene	Yes	Yes	Tissue around prosthetic joints	Large fragments, filaments, shards, or small intracellular fragments. Often with a giant cell reaction. Oil red O +
Polymethylmethacrylate (bone cement)	No	No	Tissue around prosthetic joints	Round to oval holes surrounded by a giant cell reaction	Dissolves in xylene. Barium sulfate may be present within the bone cement
Sevelamer			GI tract	Rectangular crystals with a fish scale texture, usually pink center and yellow edges (also brown, red, purple). AFB magenta	This is used as a phosphate-lowering medication in patients with renal failure. Can cause mucosal injury
Silicone	No	Yes	In tissue around implants, rarely in draining lymph nodes	Silicone may be removed during processing and appear as empty holes with residual refractile material around the edge	Intracellular silicone appears like multiple vacuoles in histiocytes that can be mistaken for lipoblasts Other organic oils can have the same appearance
Surgical gut sutures	Yes	No	Prior biopsy sites (often seen in breast)	Ovoid deeply eosinophilic monofilament often surrounded by a chronic inflammatory response and giant cells	Comprised of connective tissue (predominantly collagen) from cows or sheep. Nuclei may be spindle shaped (due to sterilization by cautery) and the suture may develop jagged edges during resorption. Gut sutures may be mistaken for metaplastic bone
Other sutures	Yes	Yes	Surgical sites	May be monofilament or polyfilament. Often colorless	Absorbable sutures may be surrounded by chronic inflammation
Talc	Yes	Yes	Pleura after talc pleurodesis, abdominal sites after perineal talc use, granulomas in intravenous drug abusers	Irregular clear to yellow crystalline material	Talc can be present in pelvic region lymph nodes, cervix, uterine corpus, fallopian tubes, and ovaries of women using perineal talc and has been associated with ovarian carcinoma ,
Tattoo pigment	No	No	Dermis of skin at site of tattoo or in draining lymph nodes	Black (or other colors) granules, may be in histiocytes	Tattoo pigment in axillary lymph nodes can mimic mammographic calcifications. , , It can also mimic melanin in patients with melanoma. During surgery, the darker nodes can mimic sentinel nodes marked with blue dye
Thorotrast (thorium dioxide)	No	No	Liver, spleen, and lymph nodes	Coarse light brown or gray granules in histiocytes or stroma—similar to the appearance of hemosiderin	This radiocontrast agent was used in the 1930s–1940s until it was realized it was associated with cirrhosis, hepatocellular carcinoma, bile duct carcinoma, and angiosarcomas of the liver and spleen (Takekawa). It has a half-life of 400 years. Slides of tissue with thorotrast are radioactive, which can be demonstrated by autoradiography
Environmental Material
Anthracotic pigment (carbon)	No	No	Lymph nodes of the respiratory tract	Black granular deposits in macrophages	The appearance is very similar to tattoo pigment and gold. However, these substances are seen in peripheral lymph nodes
Asbestos fibers	No/yes	No	Lung	Thin fibers encrusted with beaded protein and iron (ferruginous bodies)	Specific identification requires spectroscopic analysis. Similar bodies can be seen with aluminum silicate, fiberglass, or lung elastin Quantification and identification of fibers can be performed by energy dispersive X-ray analysis
Insects (flies and ticks)	Yes	Yes	Skin and subcutaneous tissue	Variable
Silica	Yes	No	Lymph nodes of the respiratory tract, silicotic nodules	Minute polarizable material in histiocytes and fibrotic nodules	May be seen in workers exposed to silica Lung disease is often complicated by superimposed infections
Plant material	Yes	Yes	Colonic rupture, lung (if aspirated)	Cell walls are readily identifiable by polarization	Can be useful to document colonic rupture

The table lists the most common findings for these materials. However, materials can be altered by in vivo responses, fixation conditions, and staining. There are several general resources for the evaluation of foreign material. ^,

“Refractile” objects: These objects have a refractive index different than that of normal tissue. Refractility can be highlighted by increasing the contrast (i.e., by lowering the condenser or closing the aperture diaphragm). Refractile objects look brighter and shinier than tissues. This material is usually foreign (e.g., suture material) but can be endogenous such as calcium oxalate crystals. “Doubly refractile” is sometimes used to describe objects that are polarizable.

“Polarizable” objects: Polarized light is light oriented in one specific plane and is produced by using two crossed polarizing filters. Most tissues are isotropic and do not change the quality of light passing through them. They appear dark under polarized light, as very little light passes through the filters in any given plane.

“Polarizable,” “birefringent,” and “anisotropic” are terms used to describe substances that change the direction and speed of the light passing through them. Polarized light passing through such an object is deviated in a particular plane or planes and can pass through a second polarizing filter at an angle to the first. A “polarizable” object appears bright in comparison to the surrounding dark nonpolarizable tissue. Some substances can reflect light at two different wavelengths (e.g., the “dichroic birefringence” of amyloid). Many of these polarizable substances have regular repeating structures (e.g., crystalline) and may be biologic (e.g., amyloid or collagen) or synthetic (e.g., polyethylene).

How to use polarizing filters:

Some microscopes have built-in high-quality polarizing filters. Polarizing material may also be purchased as sheets that can be cut into squares. Polarizers and polarizing material in sheets are available from Edmund Optics ( www.edmundoptics.com or 1-800-363-1992) as well as from other businesses. These lower-cost polarizers can be used to detect foreign material but may not detect other types of substances. Tissues (especially with suspected amyloid) should be observed using the higher-quality built-in polarizers before it is determined that polarizable material is not present.

Definitions:

• Polarizer: The polarizing disk below the condenser.

• Analyzer: The polarizing disk above the specimen (laid on top of the slide or built into microscope above the objective).

When the polarizer and the analyzer are at 90° to each other, no light can pass through and the field is dark. As the angle between the filters is changed by rotating the polarizer, substances that preferentially reflect light in a specific direction (i.e., polarizable materials) allow light to pass through the analyzer and appear bright.

The determination of “positive” and “negative” birefringence requires using a compensating first-order red filter under polarized light and can be used to distinguish uric acid crystals from CPPD crystals. However, this determination is best performed on crystals in solution and cannot be reliably performed on fixed tissue.

Substances may be neither refractile or polarizable (most tissues and cells), refractile but not polarizable (e.g., hemosiderin), polarizable but not (or poorly) refractile (e.g., amyloid), or both (e.g., suture material). Amyloid can be identified by the apple-green color, as well as other colors, seen under polarization. ^,

Measuring With the Microscope

The microscope can be used to precisely measure objects and distances in microns. Measurement can be important for grading and staging cancers. For example measurements are necessary for the following:

• Depth of invasion of malignant melanomas and cervical carcinomas

• Fuhrman nuclear grading

• Grading breast carcinomas and sarcomas (the size of the microscopic field size must be known in order to score mitoses)

• Classification of the size of lymph node metastases for AJCC N category (isolated tumor cells vs. micrometastasis vs. macrometastasis)

Approximate measurements of very small objects can be made by comparison to cells with relatively constant sizes:

• a lobe of a neutrophil nucleus2 µm

• nucleus of a small lymphocyte5–6 µm

• a red blood cell7 µm

• a histiocyte nucleus10 µm

However, more commonly larger objects or distances need to be measured. Four different methods can be used depending on the need for accuracy, the size of the object to be measured, and the equipment available ( Table 7.2 ).