Definitions in Microscopy - for MDS Oral Pathology students

Collector lens. A focusable lens of the illuminator capable of collecting light over a wide area and directing it toward the specimen. In Koehler illumination, the collector lens is used to focus a magnified real image of the filament or arc of the bulb in the front aperture of the condenser.

Collimated beam. A beam in which rays proceed in the same direction and follow trajectories that are parallel to one another. Collimated light need not be monochromatic, polarized, or coherent.

Colored-glass filter. A slab of glass containing colloids or metals that absorb certain wavelengths while freely transmitting others. In microscopy, colored-glass filters are commonly employed in fluorescence filter sets and as effective blockers of UV and IR light.

Coma. An off-axis aberration of lenses, whereby rays from an off-axis point passing through the edge of the lens are focused closer to the optic axis than are rays that pass through the center of the lens, causing a point object to look like a comet with the tail extending toward the periphery of the field. Coma is the most prominent off-axis aberration. For lenses having the same focal length, coma is greater for lenses with wider apertures.

Compound light microscope. An optical instrument that forms a magnified image of an object through a two-step series of magnifications: The objective forms a magnified real image of the object, and the eyepiece forms a magnified virtual image of the real image made by the objective. This basic design forms the basis of all modern light microscopes.

Condenser annulus. In phase contrast and dark-field microscopy, a transparent annulus in an opaque black disk located in the front aperture of the condenser that serves as the source for illuminating the specimen.

Condenser lens. A lens assembly located near the specimen and specimen stage that collects light from the illuminator and focuses it on the specimen. Proper optical performance requires that the condenser be highly corrected to minimize chromatic and spherical aberration.

Confocal laser scanning microscope (CLSM). A mode of light microscopy whereby a focused laser beam scans the specimen in a raster and the emitted fluorescent light or reflected light signal, sensed by a photomultiplier tube, is displayed in pixels on a computer monitor. The dimensions of the pixel display depend on the sampling rate of the electronics and the dimensions of the raster. A variable pinhole aperture, located in a plane confocal with the specimen, rejects out-of-focus signals and allows for optical sectioning.

Conjugate focal planes. In light microscopy, two sets of field and aperture planes whose precise geometrical positioning in the microscope is assured by adjusting the focus of the objective, condenser, and lamp collector lenses as required for Koehler illumination. The two sets of focal planes are conjugate with each other but not with the focal planes belonging to the other set; as a consequence, looking from one focal plane along the optic axis simultaneously reveals the images of the other conjugate focal planes.

Constructive interference. In wave optics and image formation, the condition where the summation of the E vectors of the constituent waves results in an amplitude greater than that of the constituents. For interference to occur, a component of one wave must vibrate in the plane of the other.

Contrast. Optical contrast is the perceived difference in the brightness (intensity or irradiance) between an object and its surround, and is usually given as the ratio of the light intensity of an object Io to the light intensity of the object’s background Ib.

Contrast threshold. The minimal contrast required for visual detection. The contrast threshold is strongly dependent on the angular size, shape, and brightness of the specimen, the brightness of the viewing environment, the region of the retina used for detection, and other factors. For extended objects, the contrast threshold is usually given as 2–3% in bright light and 30–100% or even greater in dim light. 

Definitions in Microscopy - For MDS - Oral Pathology students

Azimuth angle. A term used to describe the orientation of an object in a plane such as the specimen plane of the microscope. On a graph with polar coordinates marked off in 360°, the angle subtended between a fixed reference (designated 0°) and a vector rotated about the origin.

Back aperture of the objective lens. An aperture plane of the light microscope located at or near the rear lens element of the objective lens and the site of formation of a diffraction image of the object. Optical devices such as phase plates, DIC prisms, and aperture masks used in forms of interference microscopy are located at or near this location.

Background subtraction. An operation in electronic imaging whereby an image of the featureless background near an object is subtracted from the image of the specimen to remove patterns of irregular illumination and other optical faults such as scratches and dust.

Beam splitter. An optical device for separating an incident beam of light into two or more beams. A prism beam splitter in the trinocular head of a microscope directs the imaging beam to the eyepieces and to the camera simultaneously. A polarizing beam splitter made of a crystalline birefringent material is used to produce linearly polarized light. A dichroic mirror beam splitter reflects excitation wavelengths while transmitting long-wavelength fluorescence emission.

Bertrand lens. A built-in telescope lens located behind the back aperture of the objective lens. When rotated into the optical path, the back aperture and diffraction plane are seen, and other planes that are conjugate to it, while looking in the oculars of the microscope.

Biaxial crystal. A class of birefringent crystals having two optic axes. Mica is an example of this class of crystals.

Bioluminescence. An oxidative process resulting in the release of energy as light emission— for example, firefly luminescence, which requires an enzyme, luciferase, to catalyze a reaction between the substrate luciferin and molecular oxygen in the presence of ATP. 181

Birefringence. The double refraction of light in transparent, molecularly ordered materials caused by the existence of orientation-dependent differences in refractive index. Also refers to the refractive index difference experienced by a transmitted ray through such a material. Incident beams of light on a birefringent specimen are split into O and E rays that can recombine after emergence from the object, giving linearly, elliptically, or circularly polarized light.

Brewster’s angle. The unique angle formed by an incident ray and the perpendicular to a reflective dielectric substance such as water or glass (the transmitting medium) at which the reflected ray is totally linearly polarized.

Bright-field microscopy. A mode of optics employing the basic components of objective and condenser lenses for the examination of amplitude objects such as stained histological specimens.

Brightness. A qualitative expression for the intensity of light.

Chromatic aberration. An aberration of lenses, whereby light waves of different wavelength are brought to focus at different locations along the optic axis. In the typical case of a simple thin lens, the focal length is shorter for blue wavelengths than it is for red ones. 5

Coherent light. A beam of light defined by waves vibrating in the same phase, although not necessarily in the same plane of vibration. To maintain the same phase over long distances, coherent waves must be monochromatic (have the same wavelength). Laser light is monochromatic, linearly polarized, and highly coherent. 

Definitions in Microscopy - For MDS - Oral Pathology students

Aberrations of a lens: Faults in lens design that cause optical performance to deviate from that of an ideal lens. Usually attributed to materials composing the lens and the spherical curvatures of lens surfaces. Lens aberrations include chromatic and spherical aberration, astigmatism, coma, distortion, and field curvature.

Absorbance or optical density:  The amount of absorption of light by a substance as measured in a spectrophotometer and given as the log of the reciprocal of the transmittance, where transmittance is the ratio of the transmitted light intensity to incident light intensity.

Achromat: A lens corrected for chromatic aberration at two wavelengths (red and blue) and for spherical aberration (green).

Airy disk: The central diffraction spot in the focused image of a point source of light. Diffraction at the front aperture of the lens disturbs the incident wavefront, causing the diffraction pattern. The Airy disk diameter is determined by the wavelength of light and the angular diameter of the lens as seen from the image plane.

Amplitude object. Objects that absorb light as opposed to those that shift the phase of light (phase objects) as the basis for image formation.

Amplitude of an electromagnetic wave. The magnitude of the electric field vector of an electromagnetic wave. Amplitude is distinguished from intensity (irradiance), a measure of the amount of light energy or photon flux, which in visual perception is proportional to amplitude squared.

Anisotropic. In describing the optical properties of an object or propagation medium, having dissimilar properties in different directions.

Annulus. In phase contrast microscopy, the transparent ring at the front aperture of the condenser that provides illumination of the specimen.

Aperture angle. The angle subtended by the edges of a lens as seen from a point in the specimen plane or in the image plane. Aperture angle is included in the expression for numerical aperture (NA/  n sin), where n is the refractive index and  is onehalf of the full aperture angle. See also Numerical aperture (NA).

Aperture plane. In a microscope adjusted for Koehler illumination, the set of conjugate focal planes located at the light source, the front aperture of the condenser, the back aperture of the objective lens, and the iris of the eye. Adjustable aperture diaphragms at these locations are used to limit stray light and determine the numerical aperture, and hence the spatial resolution, of the instrument.

Apochromat A lens especially designed to correct for chromatic aberration at three or four wavelengths (red, green, blue, UV) and at two or more wavelengths (green, red) for spherical aberration. The high degree of color correction makes these lenses suitable for fluorescence microscopy and stained histological specimens in bright-field microscopy.

Astigmatism. An off-axis aberration of lenses whereby rays from an off-axis object passing through the horizontal and vertical diameters of a lens are focused as a short streak at two different focal planes. The streaks appear as ellipses drawn out in horizontal and vertical directions at either side of best focus, where the point image is a small disk. Off-axis astigmatism increases with increasing displacement of the object from the optic axis. Astigmatism is also caused by asymmetric lens curvature due to mistakes in manufacture or improper mounting of a lens in its barrel. 

Stage Viva in Staining for MDS - Oral Pathology students

1.      Thickness of section

2.      Serial Section

3.      Artifacts in sections

4.      Types of Hematoxylin and eosin

5.      Principle of Hematoxylin

6.      Oxidation (Natural, Chemical) Ripening of Hematoxylin

7.      Fixative – Types, principles, components

8.      Bluing , Reverse bluing

9.      Mordants , common mordants

10.  Charge of Hematoxylin

11.  Classification of Hematoxylin

12.  Advantage and disadvantage of various types of Hematoxylin

13.  Esoin belongs to _________ Class of dyes

14.  End point determination of decalcification

15.  Classify decalcification agents

16.  Medavars law

17.  Properties of ideal fixatives

18.  Ideal fixation timing

19.  Common agents for fixations

20.  pH, Temperature of fixation

21.  penetration of fixants, methelyne glycol and carbonyl form

22.  Why alcohol and xylene are used before paraffin embedding

23.  Melting point of paraffin wax

24.  Miscibility of a solution 

25.  Idea behind fixation

26.  Cross linking of proteins, denaturation of proteins

27.  Antigen retrival – methods and principle

28.  Types of microtomes

29.  Principle behind each type of microtomes

30.  Blades – types, mechanical

31.  Horning and strapping

32.  Formalin pigments – identification & removal

33.  Post fixation treatments

34.  Tissue cassette dimensions, material,  

35.  What is

Dehydration

Clearing

Embedding media

Section adhesive – common ones, composition

Antiroll plate

Quenching

Thawing

Special stains

Fluorescent staining

IHC

36.  Conditions for a water bath, hot plate for section cutting – temperature

37.  Frozen sections, cryostats, frozen section staining

38.  Enzyme histochemistry

39.  Mountants – curing time, type and specific advantage of each

40.  Alternatives for paraffin wax

41.  Fixatives for EM studies