Definitions in Microscopy

Diffraction grating. A transparent or reflective substrate containing an array of parallel lines having the form of alternating grooves and ridges with spacings close to the wavelength of light. Light that is reflected by or transmitted through such a grating becomes strongly diffracted. Depending on the geometry of illumination and wavelength, a grating can generate color spectra and patterns of diffraction spots.

Diffraction plane. One of the aperture planes of the light microscope containing the focused diffraction image of the object. Under conditions of Koehler illumination, the diffraction plane is located in or near the back focal plane of the objective lens.

Distortion. An aberration of lenses, where the magnification factor describing an image varies continuously between the central and peripheral portions of the image. Depending on whether the magnification is greater at the center or at the periphery, the distortion can be of the barrel or the pincushion type, respectively.

Double refraction. In polarization optics, the splitting of light into distinct O and E rays in a birefringent material. When a birefringent crystal of calcite is placed on a page  of printed words, the effects of double refraction are clearly observed as an overlapping, double image of the text.

Emission filter. In fluorescence microscopy, the final element in a fluorescence filter cube, which transmits fluorescence emission wavelengths while blocking residual excitation wavelengths. Commonly called a barrier filter. Emission filters are colored glass or interference filters and have the transmission properties of a bandpass or long-pass filter.

Emission spectrum. In fluorescence, the spectrum of wavelengths emitted by an atom or molecule after excitation by a light or other radiation source. Typically, the emission spectrum of a dye covers a spectrum of wavelengths longer than the corresponding excitation spectrum.

Epi-illumination. A common method of illumination in fluorescence microscopy, where the illuminator is placed on the same side of the specimen as the objective lens, and the objective performs a dual role as both a condenser and an objective. A dichroic mirror is placed in the light path to reflect excitatory light from the lamp toward the specimen and transmit emitted fluorescent wavelengths to the eye or camera.

Excitation filter. In fluorescence microscopy, the first element in a fluorescence filter cube and the filter that produces the exciting band of wavelengths from a broadband light source such as a mercury or xenon arc lamp. Commonly the excitation filter is a high-quality bandpass interference filter.

Excitation spectrum. In fluorescence, the spectrum of wavelengths capable of exciting an atom or a molecule to exhibit fluorescence. Typically the excitation spectrum covers a range of wavelengths shorter than the corresponding  fluorescence emission spectrum.

Eyepiece or ocular. The second magnifying lens of the microscope used to focus a real magnified image on the retina of the real intermediate image produced by the objective. The added magnification provided by the eyepiece increases the angular magnification of the virtual image perceived by the eye. The typical range of eyepiece magnifications is 5–25.

Eyepiece telescope. See Bertrand lens.

Field diaphragm. A variable diaphragm located in or near the aperture plane of the light source that is used to reduce the amount of stray light in the object image. Since the edge of the diaphragm is conjugate with the object plane under conditions of Koehler illumination, the field diaphragm is used as an aid in centering and focusing the condenser lens.

Field planes. That set of conjugate focal planes representing the field diaphragm, the object, the real intermediate image, and the retina.

Flat-field correction. In image processing, the procedure used to obtain a photometrically accurate image from a raw image. A so-called dark frame containing bias and thermal counts is subtracted from the raw image and from a “flat” or “background” image. The dark-subtracted raw image is then divided by the dark-subtracted flatfield image to produce the corrected image. With operation, all optical faults are removed. The photometric relation of pixel values to photoelectron count is also lost during division, although the relative amplitudes of pixel values within an image are retained. See also Dark frame and Flat-field frame.

Fluorescence. The process by which a suitable molecule, transiently excited by absorption of external radiation (including light) of the proper energy, releases the energy as a longer-wavelength photon. This process usually takes less than a nanosecond.

Fluorescence microscopy. A mode of light microscopy whereby the wavelengths of fluo-rescence emission from an excited fluorescent specimen are used to form an image.

Fluorite or semiapochromat lens. Objective lenses made of fluorite or Ca2F, a highly transparent material of low color dispersion. The excellent color correction afforded by simple fluorite elements accounts for their alternative designation as semiapochromats.  The maximum numerical aperture is usually limited at 1.3.

Fluorochrome. A dye or molecule capable of exhibiting fluorescence.

Fluorophore. The specific region or structural domain of a molecule capable of exhibiting fluorescence. Examples include the fluorescein moiety in a fluoresceinconjugated protein and the tetrapyrrole ring in chlorophyll.

Focal length. The distance along the optic axis between the principal plane of a lens and its focal plane. For a simple converging (positive) lens illuminated by an infinitely distant point source of light, the image of the point lies precisely one focal length away from the principal plane.

Focal ratio or f-number. The ratio of the focal length of a lens to the diameter of its aperture.

Fovea. A 0.2–0.3 mm diameter spot in the center of the macula on the retina that lies on the optic axis of the eye and contains a high concentration of cone cell photoreceptors for color vision and visual acuity in bright light conditions.

Frame averaging or Kalman averaging. In electronic imaging, the method of averaging a number of raw image frames to reduce noise and improve the signal-to-noise ratio. The signal-to-noise ratio varies as the square root of the number of frames averaged.

Halo. In phase contrast microscopy, characteristic contrast patterns of light or dark gradients flanking the edges of objects in a phase contrast image. Halos are caused by the phase contrast optical design that requires that the image of the condenser annulus and objective phase plate annulus have slightly different dimensions in the back focal plane of the objective.

Huygens’ principle. A geometrical method used to show the successive locations occupied by an advancing wavefront. An initial source or wavefront is treated as a point source or a collection of point sources of light, each of which emits a spherical wave known as a Huygens’wavelet. The surface of an imaginary envelope encompassing an entire group of wavelet profiles describes the location of the wavefront at a later time, t. Huygens’ principle is commonly used to describe the distribution of light energy in multiple interacting wavefronts as occurs during diffraction and interference.