Theory of Confocal Microscopy
Glossary of Terms in Fluorescence and Confocal Microscopy
The complex nomenclature of fluorescence microscopy is often confusing to both beginning students and seasoned research microscopists alike. This resource is provided as a guide and reference tool for visitors who are exploring the large spectrum of specialized topics in fluorescence and laser scanning confocal microscopy.
Absorbance (Optical Density) - The quantity of light absorbed by a chemical or biological substance as measured in a spectrophotometer or similar device. Units of absorbance are equivalent to the logarithm of the reciprocal transmittance (the ratio of the transmitted light intensity to incident light intensity). Absorption bands typically cover a wide range (many tens or hundreds) of wavelengths and are commonly plotted as intensity, transmission, or optical density versus wavelength.
Acousto-Optic Tunable Filter (AOTF) - A filtering device that utilizes sound waves to modulate the wavelength or intensity of light emitted by a laser or non-coherent illumination source (primarily arc-discharge lamps). The filter consists of a specialized crystal, such as tellurium oxide or quartz, sandwiched between an acoustic transducer and absorber to induce standing sound waves with alternating domains of high and low refractive index. As either polarized or non-polarized light passes through the filter, the crystal acts as a diffraction grating to deflect the incident beam. Specific wavelengths are selected by altering the frequency of the sound waves delivered to the crystal, thus changing the period of the diffraction grating.
Acridine Orange - A nitrogen heterocyclic chromophore containing the acridine nucleus, which binds to DNA and RNA by intercalation between successive base pairs to produce a strong, but very broad emission band in the green to red wavelength region. Acridine orange is excited by blue-green laser light (488 nanometers) or by ultraviolet, violet, and blue interference filters coupled to an arc-discharge lamp (mercury or xenon). The fluorophore is an excellent candidate for displaying many cellular structures, but is not useful for multi-probe staining because of the broad emission spectrum.
Alexa Fluor Dyes - Synthetic fluorescent conjugate dyes, trademarked by Molecular Probes, that are useful for labeling antibodies, nucleic acids, and as general probes for proteins, cytoskeletal elements, lipids, and general neuroscience. The excitation and emission spectra for Alexa Fluor dyes span higher wavelength portions of the ultraviolet region and the entire visible light spectrum, even stretching into the near infrared frequencies. In general, Alexa Fluor dyes are noted for their high stability and resistance to photobleaching.
Attenuation (Blocking) Level - Reduction or suppression of a visible or ultraviolet light signal before it is detected in an optical system, such as a fluorescence microscope. The degree of attenuation is usually expressed in specific wavelengths or a wavelength range as a function of relative intensity or absolute optical density. Attenuation, also referred to as modulation or blocking of light intensity, can be accomplished with neutral density filters or an acousto-optic tunable filter (AOFT) in confocal microscopy.
Autofluorescence (Primary Fluorescence) - The generation of background fluorescence due to endogenous metabolites and organic or inorganic fluorescent compounds present in biological specimens and other materials. In some cases, autofluorescence is of high enough intensity to be confused with signals expected from fluorescently labeled molecules. Common sources of autofluorescence in biological cells and tissues are flavins, flavin proteins and nucleotides (FAD and FMN), B vitamins, reduced pyridine nucleotides (NADH and NADPH), fatty acids, cytochromes, porphyrins, lipofuchsin pigments, serotonin, and catecholamines. Autofluorescence in plant cells derives principally from chlorophyll (red fluorescence) and lignin (green fluorescence). In general, autofluorescence emission is greatest when live cells are examined with blue and ultraviolet excitation wavelengths.
Automated Fluorescence Image Cytometry (AFIC) - The application of fluorescent probes coupled with computerized microscopy systems (reflected light fluorescence microscope, low-light-level camera, and computer) to enable the high-speed automatic screening of stained specimens with reliable and accurate detection of almost all cells. Disease markers in clinical specimens can be monitored with this technique through the use of specific and sensitive fluorochromes attached to antibodies or nucleic acids. Selective excitation of the target fluorophore produces images with high signal-to-noise ratios that further increase the sensitivity of detection.
Average Transmission - In filter nomenclature, average transmission refers to the average level of light transmitted in a particular region (the useful transmission band) rather than over the entire spectrum. In a standard bandpass filter, the average transmission region spans the full width at half maximum (FWHM) of the transmission band. In longpass and shortpass filters, the term indicates the wavelength region transmitted past the cut-on and cut-off wavelengths, respectively.
Background - Detectable light (noise) that is not part of the emission signal from a fluorescent probe. Sources of background noise include crosstalk between excitation and emission filters, light leaking from pinhole artifacts in excitation and emission filters, bleeding of fluorochromes in mounting medium, non-specific fluorochrome staining, electronic noise in digital camera systems, and autofluorescence. Ideally, the background in fluorescence microscopy should be jet black to maximize the level of specimen contrast.
Bandpass Filter - A filter that transmits a defined region (or band) of wavelengths while attenuating light having wavelengths both shorter and longer than the passband. The central wavelength of the transmitted band is known as the center wavelength (commonly abbreviated CWL). The effective bandwidth is indicated by the full width at half maximum (FWHM) transmission, which is alternatively referred to as the half bandwidth (HBW). Bandpass filters are commonly employed as excitation, and less often, as barrier filters in fluorescence microscopy.
Barrier (Emission) Filter - An optical filter, usually designed as a longpass or with a well-defined bandpass region, which transmits fluorescence emission wavelengths collected from the specimen by the objective while blocking residual excitation light. Barrier filters are often fabricated using colored glass or with multiple interference cavities and are matched in sets with excitation filters and dichromatic mirrors to produce optimum results.
Beamsplitter - A common optical device used for separating an incident beam of light into two or more components that are subsequently projected in different directions. Beamsplitters are available in a variety of configurations to suit particular requirements. Trinocular observation tube assemblies for optical microscopes utilize prism beamsplitters to simultaneously direct the imaging beam to the eyepieces and a traditional or digital camera system. Polarizing beamsplitters are composed of a crystalline birefringent material to produce linearly polarized light. In fluorescence microscopy, dichromatic (often termed dichroic) mirrors act as beamsplitters to reflect excitation wavelengths back into the source while transmitting longer wavelength secondary fluorescence emission to the eyepieces or detector.
Bioluminescence - A biochemical oxidative process that results in the release of energy as emitted light. Firefly luminescence, which requires the enzyme luciferase to catalyze a reaction between the substrate luciferin and molecular oxygen (in the presence of adenosine triphosphate), is a commonly employed example of bioluminescence. The phenomenon occurs in a wide variety of marine organisms and insects.
Bleed-Through (Crossover) - Bleed-through or crossover occurs when unwanted wavelengths are transmitted through an optical filter designed to block them. The effect usually occurs when the design of a filter does not allow complete destructive interference of wavelengths excluded from the bandpass region. Bleed-through is also a problem when the incident light angle is highly oblique with respect to the filter surface, or when the excitation and emission spectra of fluorescent dyes overlap.
Caged Fluorophores - A caged fluorophore is usually covalently linked to the caging moiety (usually an organic structure), but can be photolytically released (uncaging) by a pulse of light. Typical fluorochromes are caged using nitrobenzyl derivatives, which lock the fluorescent species into a non-fluorescing tautomeric form. A variety of caged molecules have been synthesized and used experimentally.
Chromophore - A naturally occurring or synthetic pigment with characteristic optical absorption, usually containing a combination of alternating single and double bonds or a high degree of cyclic aromatic or heterocyclic conjugation. In optical microscopy, chromophores often refer to classical dyes, such as Eosin, Safranin, or Hematoxylin, utilized to stain tissues for brightfield observation. Fluorescent probes are also classified as chromophores because they exhibit strong absorption spectra in the ultraviolet, visible, and/or near infrared regions.
Coherent Anti-Stokes Raman Scattering (CARS) Microscopy - The major benefit of coherent anti-stokes Raman scattering microscopy is that it enables investigations of biomolecules without the addition of synthetic fluorescent labels or endogenous coupling to fluorescent proteins. Instead, the technique is based on the vibrational properties of the target molecule and does not require the species to be electronically excited by ultraviolet or visible light. In practice, fast (picosecond or lower) laser pulses in the near-infrared region from two sources are focused onto the specimen with a microscope objective and raster scanned in the lateral and axial planes. The pulses are separated in frequency by a selected molecular vibrational mode and generate a new beam, which has a wavelength shorter than the incident beams, at the objective focal point. The secondary beam produces a concentration profile of the target species and enables the construction of a three-dimensional image of the specimen.
Coherent Light - A light beam that is defined by the individual waves vibrating in the same phase, but not necessarily in the same plane. In order to maintain the same phase relationship over long distances, coherent light waves must be monochromatic (have the same wavelength). For example, laser light is highly coherent, almost monochromatic, and usually linearly polarized.
Cold Mirror - A specialized dichromatic interference filter that operates over a very wide temperature range to reflect the entire visible light spectrum while very efficiently transmitting infrared wavelengths. Similar to hot mirrors, cold mirrors can be designed for an incidence angle ranging between zero and 45 degrees, and are constructed with multi-layer dielectric coatings, in a manner similar to interference filters. Cold mirrors can be employed as dichromatic beamsplitters with laser systems to reflect visible light wavelengths while transmitting infrared.
Collector Lens - In widefield fluorescence microscopes, the collector lens is positioned between the arc-discharge lamp and the vertical illuminator. Modern lamphouses are equipped with an adjustment knob that enables translation of the collector lens to focus the lamp discharge plasma ball. The collector lens collects light over a wide area and directs the light to the rear of the vertical illuminator for transmission to the specimen. When a fluorescence microscope is being configured for proper Köhler illumination, the collector lens is used to focus a magnified real image of the arc plasma in the front aperture of the condenser (the objective).
Colored Glass Filters - Glass filters containing embedded colloids or dissolved metals designed to absorb specific wavelengths while freely transmitting others. In fluorescence microscopy, colored glass filters were once widely employed in excitation and barrier filter sets and as effective blockers of ultraviolet and infrared light.
Confocal Laser Scanning Microscopy (CLSM) - A popular mode of optical microscopy in which a focused laser beam is scanned laterally along the x and y axes of a specimen in a raster pattern. The emitted fluorescence (reflected light signal) is sensed by a photomultiplier tube and displayed in pixels on a computer monitor. The pixel display dimensions are determined by the sampling rate of the electronics and the dimensions of the raster. Signal photons that are emitted away from the focal plane are blocked by a pinhole aperture located in a plane confocal with the specimen. This technique enables the specimen to be optically sectioned along the z axis.
Composite View (Projection View) - A seemingly three-dimensional image created by adding together multiple optical sections acquired along the z-axis of a confocal or multiphoton fluorescence microscope (the technique may also be used with differential interference contrast optics). In conventional widefield fluorescence microscopy, images of three-dimensional specimens are often blurred due to emission occurring away from the immediate focal plane. The same images are often remarkably sharp when gathered with a confocal microscope.
Crosstalk - A term in standard filter nomenclature that describes the minimum attenuation (blocking) level over a specified range of two filters placed together in series (back-to-back). The degree of filter crosstalk should be seriously considered when matching excitation and barrier (emission) filters for fluorescence sets. Dichromatic mirrors are often included in crosstalk evaluation of fluorescence filter combinations. Reduction or elimination of crosstalk helps to provide images that are not complicated by fluorescence emission from fluorophores in multiply stained specimens whose spectra fall largely out of the filter set bandwidth.
Cyanine Dyes (Cyanine Fluorochromes) - Containing a centralized heterocyclic benzoxazole moiety, cyanine dyes are fluorescent probes first marketed by Amersham, Inc., which are known for their high degree of photostability, good water solubility, and relatively efficient quantum yield. Cyanine dyes find many applications as fluorescent labels for proteins, nucleic acids, and sub-cellular components. The fluorochromes can be fine-tuned on a molecular level to produce derivatives having a wide spectrum of excitation and emission wavelengths, and can be structurally altered to control solubility, non-specific interactions (reducing background noise), and target reactivity.
Deconvolution Fluorescence Microscopy - Deconvolution analysis is a technique that applies algorithms to a through-focus stack of images acquired along the optical (z) axis to enhance photon signals specific for a given image plane or multiple focal planes in an image stack. The microscope must be equipped with a stepper motor attached to the focus gearset to guarantee image acquisition at precisely defined intervals between focal planes in the specimen. In a typical application, deconvolution analysis is utilized to deblur and remove out-of-focus light from a particular focal plane of interest using fluorescence excitation and emission (although the technique is useful for other illumination modes as well). The most sophisticated applications apply deconvolution analysis to an entire image stack to produce projection views or three-dimensional models.
Descanning - The process of allowing light emitted by the specimen in confocal microscopy to be collected by the objective and retrace its path back through the scanning mirrors to the dichromatic mirror. When the descanning mechanism is properly aligned, the fluorescence image spot at the detector pinhole remains steady and does not wobble.
Dichromatic Beamsplitter (Dichroic Mirror) - An interference filter/mirror combination commonly used in fluorescence microscopy filter sets to produce a sharply defined transition between transmitted and reflected wavelengths. When inclined at a 45-degree angle with respect to the incident illumination and emission light beams, the dichromatic mirror reflects short excitation wavelengths through a 90-degree angle onto the specimen and transmits longer emitted fluorescence wavelengths straight through to the eyepieces and detector system. Dichromatic mirrors fabricated with interference thin films for fluorescence microscopy are capable of reflecting 90 percent or more of the excitation light while simultaneously transmitting approximately 90 percent of the emission band. The dichromatic mirror is usually the central element of three filters (excitation, barrier, and dichromatic mirror) contained within a fluorescence filter optical block.
Dwell Time - A term in confocal microscopy that refers to the length of time that the scanned laser beam is allowed to remain in a unit of space corresponding to a single pixel in the image. Typically, dwell times range between 0.1 to 1 microsecond or longer, but setting the dwell to very long times increases the rate of photobleaching and decreases the viability of living cells.
Electronic State - The overall configuration of electrons in an atom or molecule, which in turn determines the distribution of negative charge (electrons) in the molecule and the molecular geometry. Any given molecule can exist in one of several electronic states (ground or multiple excited), depending upon the total electron energy and on the symmetry of the electron spins in the orbitals. At room temperature, the majority of molecules exist in the electronic state with the least energy (the ground state). When the molecule absorbs a photon, electrons are excited to higher energy states (excited states).
Emission Spectrum - The band (spectrum) of wavelengths emitted by an atom or molecule (fluorochrome) after it has been excited by a photon of light or energy from another radiation source. After the fluorochrome has emitted a photon, it returns to the ground-level energy state and is ready for another cycle of excitation and emission. Typically, the fluorescence emission spectrum, which is plotted as relative intensity as a function of wavelength, is positioned in a region of longer wavelengths than the stimulating excitation spectrum (in effect, the average wavelength of emission is longer than the average wavelength of excitation). In addition, the wavelength range and intensity profile of the fluorescence emission spectrum is generally independent of the excitation wavelength.
Epi-Illumination (Reflected Light) - The mode of illumination for fluorescence and reflected light microscopy. In a reflected or epi configuration, the illumination source (often termed a vertical illuminator) is placed on the same side of the specimen as the objective, which serves the dual role as both condenser and imaging lens system. The fluorescence microscope contains a dichromatic mirror that is strategically positioned within the light path to reflect excitation light from the lamp in the direction of the specimen and transmit emitted fluorescence to the eyepieces or detector system.
Excitation (Exciter) Filter - The first element from a matched filter set (or filter cube) in the optical train of a fluorescence microscope. The excitation filter, along with its partners in the set, is usually housed in the vertical (epi) illuminator and filters selected regions from a broadband light source (such as a mercury or xenon arc-discharge lamp) to produce the exciting band of wavelengths for fluorescence microscopy. Excitation filters are often produced as selective bandpass filters using interference filter technology, but they may also be composed of colored (dyed) glass.
Excitation Spectrum - The spectrum of wavelengths, usually spanning the ultraviolet and visible light spectrum, which are capable of exciting a fluorochrome (atom or molecule) to exhibit fluorescence. Excitation spectra are generated by scanning through the absorption spectrum of a fluorochrome or fluorophore while monitoring the emission at a single (peak) wavelength. In a manner similar to the absorption spectrum, the excitation is broadened due to vibrational and rotational relaxation (internal conversion) of excited molecules. Absorption and excitation spectra are distinct but often overlap, sometimes to the extent that they are nearly indistinguishable.
Filter Slope - The slope of an optical filter is an indication of the filter profile in the transition region between blocking and transmission. In general, the slope of a filter is defined by the wavelength at which the filter exhibits a specified blocking level and the rate of change in this region. Two filters can have the same cut-on or cut-off wavelengths, but still have dramatically different blocking levels and slopes. Very sharp slopes transmit a narrow bandwidth of wavelengths in the cut-off or cut-on region, while shallow slopes have large bandwidths.
Fluorescence - The process by which a suitable atom or molecule, which is transiently excited by absorption of external radiation at the proper energy level (usually ultraviolet or visible light), releases the absorbed energy as a photon having a wavelength longer than the absorbed energy. In fluorescence, an electron promoted into a singlet state of the excited orbital is paired (or the opposite spin) with respect to a second electron in the ground-state orbital. As a result, return of the electron to the ground state is spin-allowed and occurs rapidly with the accompanying emission of a photon. The fluorescence excitation and emission processes usually occur in less than a nanosecond.
Fluorescence Anisotropy - A term referring to the photoselective excitation of fluorophores due to the transient alignment of the absorption dipole moment with the electric vector of incoming photons. Similar to excitation, fluorescence emission by the excited fluorophore occurs in a plane oriented parallel to the emission dipole moment. The transition (dipole) moments for excitation and emission have a defined orientation with respect to the molecular axes of the dye molecule, and are separated from each other by an angle. During the excited state lifetime, rotation of the fluorophore will depolarize its emission with respect to the excitation vector, resulting in a mechanism with which to measure the rigidity (viscosity) of the environment containing the fluorophore. Fluorescence anisotropy is defined as the ratio of the difference between the emission intensity parallel to the polarized electric vector of the exciting light and the intensity perpendicular to the vector, divided by the total intensity.
Fluorescence Correlation Spectroscopy (FCS) - Used primarily with laser scanning confocal or multiphoton microscopy, fluorescence correlation spectroscopy is a technique designed to determine molecular dynamics in volumes containing only one or a few molecules, yielding information about chemical reaction rates, diffusion coefficients, molecular weights, flow rates, and aggregation. In FCS, a small volume (approximately one femtoliter) is irradiated with a focused laser beam to record spontaneous fluorescence intensity fluctuations arising from the number or quantum yield of the molecules occupying the volume as a function of time. Relatively small fluorophores diffuse rapidly through the illuminated volume to generate short, randomized bursts of intensity. In contrast, larger complexes (fluorophores bound to macromolecules) move more slowly and produce a longer, more sustained time-dependent fluorescence intensity pattern.
Fluorescence and DIC Combination Microscopy - Combining two of the most powerful contrast enhancing techniques in optical microscopy, fluorescence and differential interference contrast (DIC) can be coupled to image much of the fine detail in living cells while simultaneously observing the distribution of added fluorophores. Because DIC requires a complex arrangement of polarizers and birefringent beamsplitters (Nomarski or Wollaston prisms) using transmitted light, images of the target viewfield must be captured separately from those obtained with reflected light fluorescence. The resulting images are then combined (overlaid) to spatially map fluorescence intensity as a function of cellular architecture. In some cases, the fluorescence microscope dichromatic mirror can be employed to act as a polarizer (the analyzer) to enable simultaneous imaging in both modes.
Fluorescence Filter Set - Usually housed in cube-shaped optical blocks, fluorescence filter sets are composed of an excitation and barrier (emission) filter along with a dichromatic mirror. The filter block is positioned in the vertical illuminator, above the objective, so that incident illumination can be directed through the excitation filter and reflected from the surface of the dichromatic mirror onto the specimen. The fluorescence emitted by the specimen is gathered by the objective and transmitted through the dichromatic mirror to the eyepieces or detector system. Fluorescence filter sets are matched with respect to bandpass regions for the various filter combinations to minimize crosstalk between the filters and maximize recorded fluorescence intensity.
Fluorescence in situ Hybridization (FISH) - Not directly related to aquatic life, the fluorescence FISH technique is based on hybridization between target sequences of chromosomal DNA with fluorescently labeled single-stranded complementary sequences (termed cDNA) to ascertain the location of specific genetic sequences. In a method referred to as direct FISH, fluorochromes are coupled directly to the complementary nucleic acid probe to enable the probe-target hybrid complexes to be visualized with a fluorescence microscope. Indirect FISH involves binding of a fluorescently labeled antibody to the complementary probe after hybridization in order to amplify the fluorescence signal and expand the number of fluorophores available for multiple labeling experiments.
Fluorescence Lifetime - The characteristic time that a molecule remains in an excited state prior to returning to the ground state. During the excited state lifetime, the fluorophore can undergo conformational changes as well as interacting with its local environment. The fluorescence lifetime is defined as the time in which the initial fluorescence intensity of a fluorophore decays to 1/e (approximately 37 percent) of the initial intensity. This quantity is the inverse of the rate constant of the fluorescence decay from the excited state to the ground state.
Fluorescence Lifetime Imaging Microscopy (FLIM) - A sophisticated technique that enables simultaneous recording of both the fluorescence lifetime and the spatial location of fluorophores throughout every location in the image. The methodology provides a mechanism to investigate environmental parameters, such as pH, ion concentration, solvent polarity, and oxygen tension in single living cells, presenting the data in a spatial and temporal array. FLIM measurements of the nanosecond excited state lifetime are independent of localized fluorophore concentration, photobleaching artifacts, and path length (specimen thickness), but are sensitive to excited state reactions such as resonance energy transfer.
Fluorescence Loss in Photobleaching (FLIP) - In a technique related to FRAP, a defined region of fluorescence within a living cell is subjected to repeated photobleaching by illumination with intense irradiation. Over a measured time period, this action will result in complete loss of fluorescence signal throughout the cell if all of the fluorophores are able to diffuse into the region that is being photobleached. By calculating the rate at which fluorescence is extinguished from the entire cell, the diffusional mobility of the target fluorophore can be determined.
Fluorescence Microscopy - A popular clinical and research technique in optical microscopy that relies on excitation of fluorescent molecules with a specific wavelength region to produce an image generated by the secondary fluorescence emission at longer wavelengths. Modern fluorescence microscopes are equipped with reflected light illuminators that incorporate neutral density filters and a specialized combination of interference filters to segregate incident illumination from the detected fluorescence emission.
Fluorescence and Phase Contrast Combination Microscopy - Traditional phase contrast optics can be coupled with fluorescence microscopy to observe the spatial distribution of fluorophores in living and fixed cells and map the emission intensity to specific structures and organelles. Because the phase contrast technique requires transmitted light illumination that is modified with a ring-shaped aperture (condenser annulus) and detected with a spatial filter (phase plate) positioned in the objective rear focal plane, viewfields must be captured independently from fluorescence in order to obtain the best contrast. The resulting images are then combined (overlaid) to spatially map fluorescence intensity as a function of cellular architecture. Some manufacturers offer low-density phase plates that enable simultaneous imaging of live cells in phase contrast and fluorescence.
Fluorescence Recovery After Photobleaching (FRAP) - Translational mobility (lateral diffusion coefficients) of fluorescently labeled macromolecules and small fluorophores can be determined by photobleaching recovery techniques. In FRAP, a very small, selected region (several micrometers in diameter) is subjected to intense illumination, usually with a laser, to produce complete photobleaching of fluorophores in the region. The result is a dramatic reduction or annihilation of fluorescence. After the photobleaching pulse, the rate and extent of fluorescence intensity recovery in the bleached region is monitored as a function of time to generate information about repopulation by fluorophores and the kinetics of recovery.
Fluorescence Resonance Energy Transfer (FRET) - An adaptation of the resonance energy transfer phenomenon to fluorescence microscopy in order to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, and nucleic acids in living cells. FRET microscopy is performed using either steady state or time-resolved techniques, but time-resolved FRET imaging has the advantage of more accurately mapping the donor-acceptor distance. A standard fluorescence microscope equipped with the proper excitation and emission filters and a sensitive video camera can be utilized to perform FRET imaging.
Fluorescence Speckle Microscopy (FSM) - A technique compatible with widefield and confocal microscopy that employs a very low concentration of fluorescent labeled subunits (for example, tubulin) to reduce fluorescence away from the focal plane, and thus improve visibility of labeled structures and their dynamics in thick regions of living cells. This is accomplished by labeling only a small fraction of the entire structure of interest. Fluorescence speckle microscopy has been very useful for defining the movement and polymerization kinetics of cytoskeletal elements, such as actin and microtubules, in live cells.
Fluorochrome - A natural or synthetic dye or molecule that is capable of exhibiting fluorescence. Fluorochromes (also termed fluorescent molecules, probes, or fluorescent dyes) are usually polynuclear heterocyclic molecules containing nitrogen, sulfur, and/or oxygen with delocalized electron systems and reactive moieties that enable the compounds to be attached to a biological species.
Fluorophore - The structural domain or specific region of a molecule that is capable of exhibiting fluorescence. Fluorophores are divided into two general classes, intrinsic and extrinsic. Intrinsic fluorophores are those that occur naturally in biological structures and other materials. Extrinsic fluorophores are added to a specimen that does not display the desired spectral (fluorescent) properties. In many cases, a fluorophore is composed of a smaller aromatic molecule (fluorochrome) attached through a chemical reaction or by absorption to a larger macromolecule. Typical examples include acridine orange intercalated between successive DNA base pairs, the fluorescein moiety in a conjugated protein or antibody, and the tetrapyrrole ring system in chlorophyll.
Franck-Condon Principle - A fundamental concept governing the fluorescence phenomenon, which is based on the fact that molecular nuclei are stationary during electronic transitions (represented by vertical lines in a Franck-Condon or Jablonski diagram). Electronic transitions from the ground state to a higher energy excited state occur in such a short time frame (measured in femtoseconds) that the nuclei do not have sufficient time to vibrate. As a result, the only electronic transitions from the ground state to the excited that can occur are those where the probabilities of the electron position in the ground and excited states maximally overlap.
Full Width at Half Maximum (FWHM) - The bandpass region of transmitted wavelengths by a glass or interference filter is described by a parameter known as the full width at half maximum (abbreviated FWHM). The cut-on and cut-off boundaries are defined as the lower and upper wavelengths yielding fifty percent of maximum transmittance by the filter, and the center wavelength (CWL) is the arithmetic mean of wavelengths in the bandpass region. For example a FWHM of 40 indicates that the transmitted bandwidth spans 40 nanometers and could have a CWL anywhere in the ultraviolet, visible, or infrared region. The FWHM is also referred to as the half bandwidth (HBW) in many textbooks.
Green Fluorescent Protein (GFP) - A naturally occurring protein fluorescent probe derived from the jellyfish Aequorea victoria, which is commonly employed to determine the location, concentration, interactions, and dynamics of a target protein in living cells and tissues. The excitation and emission spectra of enhanced GFP (a genetic derivative) have maxima at 489 nanometers and 508 nanometers, respectively. In order to incorporate the GFP (or any of its genetic derivatives) into a cell, the DNA sequence for the gene is ligated to the DNA encoding the protein of interest. After cultured cells have been transfected with the modified DNA, they are able to express chimeric fluorescent proteins for observation in the microscope.
Harmonic Generation Microscopy (HGM) - Harmonic generation occurs when an optical excitation event involving two or more photons at a particular frequency results in cooperative emission at multiple harmonics (primarily, the second and third) without absorption of the photons. Generation of the harmonic frequencies is essentially a non-linear scattering process yielding an emitted photon wavelength that is twice the frequency or half the wavelength (for second harmonic generation) of the incident illumination. In optical microscopy, transparent specimens that lack symmetry are ideal candidates for imaging with harmonic generation techniques. Unlike the situation with typical probes and traditional fluorescence microscopy illumination techniques, changing the excitation illumination wavelength produces a corresponding change in the emission wavelength. In addition, the emitted light is coherent and retains phase information about the specimen.
Hot Mirror - A specialized dichromatic interference filter often employed to protect optical systems by reflecting heat back into the light source. Hot mirrors can be designed to be inserted into the optical system at an incidence angle varying between zero and 45 degrees, and are useful in a variety of applications where heat build-up can damage components or adversely affect spectral characteristics of the illumination source. Wavelengths reflected by an infrared hot mirror range from about 750 to 1250 nanometers. By transmitting visible light wavelengths while reflecting infrared, hot mirrors can also serve as dichromatic beamsplitters for specialized applications in fluorescence microscopy.
Immunofluorescence Microscopy - A mode of fluorescence microscopy in which a targeted molecular species (protein, nucleic acid, membrane, etc.) in a specimen is labeled with a highly specific fluorescent antibody. After the labeled antibodies have been excited by a selected region of wavelengths, secondary fluorescence emission is gathered by the objective to form an image of the specimen. Antibodies are labeled either by coupling directly with a fluorochrome (fluorescent dye; termed direct immunofluorescence), or with a second fluorescent antibody that recognizes epitopes on the primary antibody (indirect immunofluorescence).
Intensifier Silicon-Intensifier Target (ISIT) Camera - A video camera tube designed for low-light level imaging applications, such as fluorescence microscopy of specimens having very low quantum yield. These video tubes are essentially a silicon-intensifier target (SIT) tube modified by the addition of an image intensifier coupled by fiber optics as a first stage of light amplification.
Interference Filter - A filter designed to transmit or reflect a specific region or band of wavelengths, commonly used in fluorescence microscopy to isolate excitation illumination from fluorescence emission. Interference filters are fabricated using alternating layers of several dielectric materials or successive layers of a dielectric material and a thin metal film. The spacings between dielectric layers (or between a dielectric and metal layer) are limited to either one-quarter or one-half of a wavelength to allow constructive interference and reinforce propagation of light through the filter at a specific wavelength. All other wavelengths produce destructive interference and are absorbed or reflected, but do not pass through the filter.
Intrinsic Lifetime - Defined as the lifetime of the excited state in the absence of any processes that compete for deactivation of excited state electrons, the intrinsic lifetime is measured as the inverse of the rate constant for the decay of fluorescence. In practice, the measured lifetime of a fluorophore is a combination of the intrinsic fluorescence lifetime and the non-fluorescent processes (quenching, non-radiative relaxation, etc.) that lead to relaxation of the excited state. The measured lifetime is always less than the intrinsic lifetime and is an indicator of the quantum yield.
Isosbestic Point - An isosbestic point is commonly observed when the excitation or emission spectra of a fluorochrome undergoing a chemical or physical reaction in equilibrium is recorded as a function of the concentration of each species. A curve of emission versus wavelength (or frequency) for such a mixture will often intersect at one or more points (wavelengths), termed the isosbestic points. The effect may also appear in the spectra for a set of two or more unrelated, non-interacting fluorochromes having the same total concentration. In a single chemical species, the isosbestic points will appear at all wavelengths in which the molar extinction coefficients are equal. As an example, the excitation spectrum of the fluorochrome fura-2 displays an isosbestic point at 362 nanometers when a dilute solution is titrated with calcium.
Jablonski Diagram - A graphical depiction of energy levels occupied by ground state and excited electrons in a fluorescent molecule. Singlet and triplet excited states are usually illustrated separately, but adjacent to one another. The Jablonski diagram identifies the relative energy positions of electronic states and vibrational energy levels as a series of horizontal lines. Electronic transitions between states are represented by straight vertical lines (excitation and emission), while internal conversion, vibrational relaxation, and quenching phenomena are indicated by wavy vertical lines. Intersystem crossing between singlet and triplet states is depicted by diagonal straight or wavy lines.
Liquid Crystal Tunable Filter (LCTF) - An electronically controlled device that enables imaging-quality filtration of light while providing a clear aperture for optical microscopy. These filters operate through a series of waveplates that are composed of a layer of birefringent material paired with a liquid crystal layer and sandwiched between two linear polarizers. The birefringence of the liquid crystal layer is fine-tuned by varying the voltage applied to transparent conductive coatings adjacent to the layer. Polarized light entering the filter undergoes a wavelength-dependent rotation by the waveplate, which is attenuated and converted into an amplitude variation by the analyzer (a second polarizer). LCTFs are designed to control filtering parameters by varying the birefringence character of the liquid crystal and employing multiple waveplates in series.
Longpass (LP) Filter - An optical interference or colored glass filter that attenuates shorter wavelengths and transmits (passes) longer wavelengths over the active range of the target spectrum (ultraviolet, visible, or infrared). Longpass filters, which can have a very sharp slope (referred to as edge filters), are described by the cut-on wavelength at 50 percent of peak transmission. In fluorescence microscopy, longpass filters are frequently utilized in dichromatic mirrors and barrier (emission) filters. Use of the older term of highpass to describe longpass filters is now discouraged because it more accurately refers to frequency rather than wavelength.
Luminescence - The emission of light from any substance (usually a molecule or atom) that occurs from an electronically excited state generated either by a physical (light absorption), mechanical, or chemical mechanism. Luminescence is formally divided into two categories, fluorescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway. The generation of luminescence can occur through excitation by ultraviolet or visible light photons (photoluminescence), an electron beam (cathodoluminescence), application of heat (thermoluminescence), chemical energy (chemiluminescence), a biochemical enzyme-driven reaction (bioluminescence), or by energy from a mechanical action (triboluminescence), such as friction.
Microchannel Plate - A plate, consisting of a compact array of capillary tubes with metallized walls, which is designed for amplifying the photoelectron signal in Gen II (and higher generation) image intensifiers. Photoelectrons from the intensifier target are accelerated onto the plate and undergo multiple collision and electron amplification events with the tube wall, resulting in a large electron cascade and amplification of the original photon signal. The microchannel plate is useful for detecting very faint emission signals in fluorescence microscopy.
Mirror Image Rule - The fluorescence emission spectrum is usually a mirror image of the excitation spectrum when plotted as a function of relative intensity versus wavenumber (as opposed to wavelength). This phenomenon occurs because the probability of an excited electron returning to a particular ground state vibrational energy level is related to the degree of similarity between the vibrational and rotational energy states in the ground state versus those present in the excited state. In effect, the most likely return pathway for the excitation transition of an electron from the zeroth vibrational ground state to the second vibrational level in the first excited state is from the zeroth vibrational level in the excited state to the second vibrational level in the ground state (excitation from S(0)=0 to S(1)=2, followed by emission from S(1)=0 to S(0)=2).
Molar Extinction Coefficient - Widely employed in the fields of spectroscopy, microscopy, and fluorescence, molar extinction coefficients are usually denoted by the Greek symbol epsilon (e) and are useful in converting units of absorbance into units of molar concentration for a variety of chemical substances. The extinction coefficient is determined by measuring the absorbance at a reference wavelength (characteristic of the absorbing molecule) for a 1 molar (M) concentration (one mole per liter) of the target chemical in a cuvette having a 1-centimeter path length. The reference wavelength is usually the wavelength of maximum absorption in the ultraviolet or visible light spectrum. Extinction coefficients are a direct measure of the ability of a molecule to absorb light.
Monochromatic - A light beam that is composed of a single wavelength. The concept of monochromatic light is easy to visualize but the phenomenon, owing to the Heisenberg uncertainty principle, does not actually exist in nature. In practice, monochromatic light is not truly limited to a single wavelength, but a narrow band consisting of two or more wavelengths. Even the monochromatic emission from a laser, an excited atomic source, or a narrow bandpass interference filter has a measurable bandwidth.
Multiple Fluorescence Filter Set - Designed for simultaneous viewing, photomicrography, or digital imaging of multiple fluorescence signals, this filter set contains specialized interference filters tuned to pass two or more wavelength regions. The transmission profile of each filter in the set contains multiple peaks and troughs for the transmission and reflection of specific excitation and emission wavebands similar to a conventional single probe filter combination. Registration errors and image shifts that occur when multiple probes are sequentially imaged with separate filter combinations are eliminated with the multiple probe filter set. However, bandwidth constraints, coupled to the inability to reject certain wavelengths and the limited steepness of transmission profiles, reduces the performance of these filter sets compared to individual filters for specific fluorochromes.
Near-Field Scanning Optical Microscopy (NSOM) - Near-field imaging occurs when a sub-micron optical probe is positioned a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe. The near-field is defined as the region above a surface with dimensions less than a single wavelength of the light incident on the surface. Within the near-field region, evanescent light is not diffraction limited and nanometer spatial resolution is possible. This phenomenon enables non-diffraction limited imaging and spectroscopy of a sample that is not possible with conventional optical imaging techniques.
Neutral Density (ND) Filter - Extensively utilized for a variety of applications in optical microscopy, neutral density filters are neutral gray in color (resembling smoked glass) and are designed to reduce transmitted light intensity evenly across either a small number of wavelengths or the entire wavelength spectrum without altering the spectral profile of illumination. Neutral density filters are ideal for controlling the intensity of illumination in fluorescence microscopy because the high intensity arc lamps commonly employed as light sources cannot be regulated with an adjustable power supply to control voltage.
Nipkow Disk - A thin opaque disk used in confocal microscopy to produce a real image that can be inspected visually or recorded on a high-resolution digital camera system. In contrast, conventional single-spot scanning instruments produce images that are reconstructed from signals generated by a photomultiplier and are displayed on a computer monitor. The Nipkow disk contains thousands of minute pinholes, which when rotated at high speed, provide parallel scanning of the specimen with miniature diffraction-limited spots from each pinhole. Fluorescence emission from the specimen is refocused at the same pinhole in the disk to provide the same function in rejecting out-of-focus light as the single pinhole in a conventional confocal microscope.
Optical (Molecular) Highlighters - A unique group of fluorescent proteins that can be spectrally altered by irradiation at specific wavelengths (usually with a laser). Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or are capable of being optically converted from one fluorescence emission bandwidth to another (photoconversion) enable the direct and controlled highlighting of distinct molecular pools within a cell.
Optical Tweezers (Laser Trapping) - Optical tweezers, alternatively termed a single beam laser trap employs the radiation pressure from a photon stream emitted by an infrared laser to capture or trap and relocate microscopic objects, such as a protein-coated bead. This technique has been applied to measure the forces generated by motor protein movement and the strength of cellular adhesions. Although laser tweezers or most commonly used in widefield fluorescence microscopy, they have also been incorporated into confocal and multiphoton imaging systems.
Peltier Thermoelectric Cooling - A common feature on digital cameras designed for fluorescence microscopy, Peltier cooling systems consist of a bimetallic strip that becomes hot on one surface and cold on the other during application of a current. These devices can be employed to quickly and efficiently cool a charge-coupled device (CCD) between 50 and 60 degrees below ambient temperature in a compact space. Cooled CCD camera systems can integrate charge in the photodiodes for much longer periods than conventional uncooled devices without significantly increasing noise levels.
Phosphorescence - The emission of light (photons) from an excited triplet state where the electron in the excited orbital has the same spin orientation as its partner in the ground state. Because a triplet state transition to the ground state is forbidden by the rules of quantum mechanics, the phosphorescence emission rate is much slower (measured in milliseconds to seconds) than that observed for fluorescence emission. In general, phosphorescence is not usually observed in solution, which includes most chemical and biochemical environments, at room temperature.
Photoactivation - Genetically encoded fluorescent proteins that generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength, are termed photoactivatable. Among the proteins that demonstrate photoactivation, a green fluorescent protein variant, termed PA-GFP, has been the most widely studied.
Photobleaching (Fading) - The permanent loss of fluorescence by a molecule due to photon-induced chemical damage or covalent modification. In general, photobleaching occurs when the fluorochrome undergoes intersystem crossing from an excited singlet to a triplet state. Molecules in the triplet state are easily modified by complex chemical reactions with adjacent molecules or oxygen. Reactions with the latter species will permanently destroy the fluorochrome and produce singlet oxygen free radicals that can chemically modify other biomolecules. After a fluorochrome has been photobleached, it usually does not recover. The rate of photobleaching can be dramatically reduced by lowering the excitation light flux or by limiting the oxygen concentration.
Photomultiplier Tube (PMT) - An electrical device designed to collect and amplify photon signals. Incoming photons strike a target in the face of the photomultiplier to liberate free electrons, which are accelerated onto a dynode that in turn liberates an amplified stream of electrons. Several dynodes are arranged in series to produce a tremendous degree of amplification from each original photon and then transmit the signal to a processing circuit. Unlike area-array detectors such as charge-coupled devices (CCDs), photomultipliers do not form an image.
Phototoxicity - Damage to a living specimen following excessive exposure to high intensity illumination. The level of phototoxicity generally increases with decreasing wavelength, but the potentially numerous mechanisms behind the phenomenon are poorly understood. Most living cells display an enhanced level of phototoxicity when simultaneously exposed to synthetic fluorophores that target specific sub-cellular locations, such as the nucleus, mitochondria, Golgi apparatus, or endoplasmic reticulum.
Pinhole - In confocal microscopy, variable diaphragm pinhole apertures placed near the light source and detector enable the microscope to produce thin optical sections of focal planes in the specimen. In filter terminology, a pinhole refers to a small break in the coating (primarily arising in the thin-films of interference filters) produced by dust particles or debris on the substrate during application. Pinhole size is measured against a standard under specific conditions using a high-intensity illumination.
Polychromatic Mirror (Polychroic Beamsplitter) - A specialized mirror or beamsplitter that is designed to transmit multiple bandpass regions of fluorescence emission from the specimen, while reflecting other defined wavelength regions that correspond to the excitation bands. Polychromatic mirrors are a critical member of multi-band fluorescence filter combinations tailored to eliminate registration shifts when imaging several probes in a single specimen. The most complex polychromatic mirrors can reflect three or more excitation bands and transmit at least three emission bands.
Quantum Efficiency - A term commonly utilized by the digital image sensor industry, quantum efficiency is a measure of the effectiveness of an image sensor (such as a charge-coupled device, CCD) to generate electronic charge from incident photons. Quantitatively, quantum efficiency refers to the number of electrons generated by a photodiode per incident photon during a reference time period. Quantum efficiency varys as a function of the incident wavelength due to a dependence of the substrate absorption coefficient on incoming electromagnetic radiation frequency. In practice, interference effects due to the thin films of silicon dioxide, silicon nitride, and polysilicon on the surface of an image sensor will also result in a wavelength dependence for quantum efficiency. Most manufacturers present imager quantum efficiency as a plot versus wavelength for comparison purposes.
Quantum Yield - A quantitative measure of fluorescence emission efficiency, the quantum yield of a fluorochrome or fluorophore is expressed as the ratio of the number of photons emitted to the number of photons absorbed. In other words, the quantum yield represents the probability that a given excited fluorochrome will produce an emitted (fluorescence) photon. Quantum yields typically range between a value of 0 and 1, and fluorescent molecules commonly employed as probes in microscopy have quantum yields ranging from very low (0.05 or less) to almost unity. In general, a high quantum yield is desirable in most imaging applications. The quantum yield of a given fluorophore varies, sometimes to large extremes, with environmental factors, such as pH, concentration, and solvent polarity.
Quenching - Reduction in fluorescence emission by a fluorochrome due to environmental conditions such as ionic strength, pH, solvent effects, or a locally high concentration of fluorochromes that reduces the efficiency of emission. Quenching is manifested by non-radiative relaxation of excited state electrons to the ground state. Excited fluorophores can be quenched by resonance energy transfer when they are in close proximity to acceptor molecules to which the excited state energy can be transferred.
Red Fluorescent Protein (RFP) - A fluorescent protein derived from a sea anemone belonging to the genus Discosoma, which is used as a fluorescent probe to determine the location, concentration, interactions, and dynamics of a target protein in living cells and tissues. In order to incorporate the RFP into a cell, the DNA sequence for the gene is ligated to the DNA encoding the protein of interest. After cultured cells have been transfected with the modified DNA, they are able to express chimeric fluorescent proteins for observation in the microscope.
Resonance Energy Transfer (RET) - A process by which a fluorophore donor in an excited state may transfer its excitation energy to a neighboring (within 10 nanometers) acceptor chromophore non-radiatively through dipole-dipole interactions. Spectral overlap between the donor emission and acceptor absorption bands is required for the transfer of energy by this mechanism. The energy transfer manifests itself by both quenching of donor fluorescence in the presence of the acceptor and increased (sensitized) emission of the acceptor fluorescence.
Scratches and Digs - Imperfections found on optical surfaces that are measured in terms of military specification standards. Scratches are surface imperfections with a length far exceeding the width, while digs refer to particles and small bubbles that are imbedded internally or present on the exposed surface coating of a filter. The thickness of a scratch is measured and specified in microns and the diameter of digs, pits, and bubbles is recorded in 10-micrometer units. For example, a 80/50 scratch/dig filter specification establishes limits of 80 micrometers for scratches and 500 micrometers for digs. The complete specification also limits the number of allowable blemishes over the entire surface area.
Shortpass (SP) Filter - An optical interference or colored glass filter that attenuates longer wavelengths and transmits (passes) shorter wavelengths over the active range of the target spectrum (usually the ultraviolet and visible region). Shortpass filters, which can have a very sharp slope (referred to as edge filters), are described by the cut-off wavelength at 50 percent of peak transmission. In fluorescence microscopy, shortpass filters are frequently employed in dichromatic mirrors and excitation filters. Use of the older term of lowpass to describe shortpass filters is now discouraged because it more accurately refers to frequency rather than wavelength.
Signal-to-Noise Ratio (S/N) - The standard ratio of the optical signal from a specimen to the noise (optical) of the surrounding background. Noise is defined as the square root of the sum of the variances of contributing noise components. In situations where noise is photon limited and background noise may be approximated by the square root of the background signal, signal-to-noise is defined as the number of standard deviations that distinguish the specimen signal from the mean signal of the background. In fluorescence microscopy, the signal-to-noise ratio can be compromised by excessive background signal due to poor staining techniques or very low signal from the probe of interest.
Silicon-Intensifier Target (SIT) Camera - A specialized video camera designed to operate under the low-light conditions that often prevail in fluorescence microscopy. The SIT camera tube contains a photocathode that accelerates photoelectrons onto a silicon diode target plate to dramatically amplify the signal. A scanning electron beam subsequently neutralizes the target while generating a beam current containing the signal.
Singlet State - For any many-electron system (in atoms and molecules), when the electron spins are paired, the electronic configuration is referred to as a singlet state. The spin quantum number (S) of an atom or molecule is the absolute value of the sum of electronic spins within the system. The multiplicity of such a system is defined as the quantity 2S + 1, and for a two-electron system in the singlet state with antiparallel (paired) spins, the multiplicity is 1 and the spin quantum number is zero.
Spectral Imaging - An advanced fluorescence technique that utilizes hardware (usually incorporated into a confocal detector unit) to separate the emitted light from multiple fluorophores into separate spectral components. Linear unmixing is the accompanying computational process, related to deconvolution, which uses the spectra from each fluorophore as though it were a point-spread function of fixed location to separate or unmix the component signals. The technique is a powerful analytical tool that can be used to discriminate distinct fluorophores with highly overlapping spectral profiles.
Steady-State Fluorescence - Encompassing all imaging and measurements performed with constant illumination and observation, steady-state fluorescence is the most common type of fluorescence experiment. The specimen is illuminated with a continuous beam of filtered light, and the fluorescence intensity or emission spectrum is recorded with a detector. A majority of the specimens observed in fluorescence microscopy reach a steady state immediately upon being exposed to excitation illumination.
Stimulated Emission Depletion Microscopy (STED) - Exhibiting spatial resolution well beyond the diffraction limit, stimulated emission depletion microscopy is an emerging technique that uses opposing objective lenses to achieve an axial resolution lower than 50 nanometers. The technique relies on inhibiting fluorescence of excited molecules at the periphery of a laser scanning focal spot using synchronized laser pulses for excitation of fluorophores and spatially coordinated circular STED pulses to deplete emission. Resulting fluorescence is inhibited at the periphery of the spot, but not in the center, thus dramatically reducing the fluorescence spot size with a commitment increase in resolution.
Stokes Shift - The difference in energy or wavelength between the photons involved in fluorescence excitation and emission is commonly referred to as the Stokes shift in honor of George G. Stokes, who first observed the phenomenon in 1852 while studying at Cambridge University. In practice, the Stokes shift is determined by the wavelength difference between excitation and emission maxima in the respective spectra of a fluorescent molecule. This value varies widely, depending on the electronic configuration of the fluorochrome, but can range from just a few to several hundred nanometers. For example, the Stokes shift for fluorescein is approximately 20 nanometers, while that for the porphyrins is over 200 nanometers. The Stokes shift is of great utility in fluorescence microscopy because it enables the isolation of excitation and emission wavelengths using interference filters.
Surface Flatness (Filters) - A term widely applied in optics, surface flatness is a measure of the surface deviation from a perfect plane in an optical element. In fluorescence filters designed for optical microscopy, surface flatness is measured in fractions or multiples of a visible light wavelength corresponding to green (550 nanometers), red-orange (630 nanometers), or using the center wavelength of a specific bandpass filter. When a plane wavefront is reflected from the surface of a mirror or filter, the actual wavefront distortion is twice the value of the surface flatness. In dichromatic mirrors, the surface flatness is determined by the wavefront distortion of light reflected from the front (reflecting) surface.
Thin-Film Interference Coating - The main component of interference filters, these coatings are fabricated by applying alternating layers of several dielectric materials or successive layers of a dielectric material and a thin metal film. The spacings between dielectric layers (or between a dielectric and metal layer) are limited to either one-quarter or one-half of a wavelength to allow constructive interference and reinforce propagation of light through the filter at a specific wavelength. All other wavelengths produce destructive interference and are absorbed or reflected, but do not pass through the filter. Each layer of the interference coating is colorless, but reflections created at the multiple interfaces between layers combine through interference to selectively reflect some wavelengths and transmit others, rendering the filter with an apparent color.
Time-Resolved Fluorescence - Employed for measuring intensity or anisotropic decays, time-resolved fluorescence measurements rely on exposing the specimen to a short pulse of light, where the pulse width is typically shorter than the decay time of the fluorophore. The intensity decay of fluorescence emission is usually recorded with a high-speed detection system that permits the intensity or anisotropy to be measured on a nanosecond time scale.
Total Internal Reflection Fluorescence Microscopy (TIRFM) - A technique designed to probe the surface of fluorescently-labeled living cells with an evanescent wave generated by a light beam traveling between two media of differing refractive indices. In practice, an incident laser beam is reflected at a critical angle (total internal reflection) when it encounters the interface between a microscope glass slide and the aqueous medium containing the cells. Fluorophores within a few nanometers of the surface are excited by the evanescent wave, while those farther away are unaffected. The technique is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology.
Transmitted Wavefront Distortion (TWD) - The degree of distortion introduced into a plane wavefront when it is transmitted through an optical element. Transmitted wavefront distortion is measured in fractions or multiples of a wavelength (usually 550 or 630 nanometers). Distortion of the transmitted wavefront arises from surface flatness variations along the outer surfaces of the optical element, as well as from internal discontinuities and inhomogeneity of refractive index.
Triplet State - For any many-electron system (in atoms and molecules), when the electron spins are unpaired, the electronic configuration is referred to as a triplet state. Also, for equal values of the other quantum numbers, states of higher multiplicity are states of lower energy. Therefore, an excited triplet state has lower energy than the corresponding singlet state. The spin quantum number (S) of an atom or molecule is the absolute value of the sum of electronic spins within the system. The multiplicity of such a system is defined as the quantity 2S + 1, and for a two-electron system in the triplet state with parallel (unpaired) spins, the spin quantum number is 1 and the multiplicity is 3.
Two Photon (Multiphoton) Microscopy - A derivative technique of laser scanning confocal microscopy where fluorochrome excitation is based on an infrared or long wavelength visible light laser beam whose energy density is adjusted to allow frequency doubling or tripling at the point of beam focus in the specimen. Fluorophores in the specimen are simultaneously excited by two or three photons to produce excited state transitions that are equivalent to single-photon fluorescence. For example, two and three photon excitation at 900 nanometers is equivalent to excitation by higher energy photons of 450 and 300 nanometers, respectively. Multiphoton microscopy enables deep penetration into thick tissues and eliminates the need for a pinhole aperture because fluorescence emission is restricted to a single focal plane.
Volume Rendering - A technique using algorithms to create a computer visualization of three-dimensional images from arbitrary angles without any intermediate conversion of the data set to represent surface geometry. In laser scanning confocal microscopy, volume rendering is usually conducted on optical section stacks gathered from fluorescent specimens to produce a semi-realistic model of the specimen in three dimensions.
Wedge (Parallelism) - A measurement in arc-minutes or arc-seconds of the variation from parallel between the outer surfaces of a flat optical element (usually a filter or window). Significant wedge angles can introduce an angular deviation to a wavefront passing through the element, leading to image shifts and registration errors when the optic is located in the imaging path. For a typical filter element or window, the level of angular deviation is approximately one-half the wedge angle. Wedge artifacts in internal coating surfaces can produce ghost images as a result of off-axis internal reflections.
Zoom - In laser scanning confocal microscopy, the zoom magnification control allows a considerable level of flexibility in varying the raster area scanned on the specimen, and therefore provides continuous control of pixel size in the lateral (x-y) specimen plane. The zoom control assists in optimizing image information content by enabling operation of the microscope in conformance with the Nyquist/Shannon sampling criteria of more than two pixels per smallest optically resolvable image point. The zoom control modulates the degree of scan mirror deflection by regulating the galvanometer current levels.