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Glossary

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Abbe, Ernst
German mathematician and physicist who established the theoretical basis of image formation in the microscope.
Achromat Objective
Microscope objectives that reduce chromatic aberration by bringing the red and blue wavelengths to the same focal point by using combinations of flint and crown glass.
Airy Disk
The image of a point source of light which takes the form of a bright disc surrounded by a sequence of concentric dark and light rings.
Apochromat Objective
Microscope objectives that give negligible chromatic aberration by bringing the red, green and blue wavelengths to the same focal point by using glass combinations including fluorspar.
Autofluorescence
Fluorescence derived from naturally occurring biomolecules within a specimen.
Axial Resolution
Resolution (or resolving power) of a microscope objective measured in the axial (z) direction. This is sometimes referred to as the depth of field
Back Focal Plane
The focal plane of a lens which lies behind it when viewed in the direction of passage of light.
Barrier Filters
Filters designed to block excitation light but transmit emission from the fluorophore of interest.
Bit Depth
The number of different shades or brightness levels that can be represented in a pixel in an image. Common bit depths are 8 bit (256 brightness values), 10 bit (1024 levels), 12 bit (4096 levels), 14 bit (16384 levels), and 16 bit (65536 levels). Larger bit depths allow the capture of a larger signal dynamic range.
Chromatic
Coloured; pertaining to or consisting of colour; relating to wavelength.
Chromatic Aberration
Wavelength dependent artefacts of a lens in which light of different wavelengths is focussed at different points along the optical axis.
CMYK
The abbreviation for Cyan, Magenta, Yellow and Black (K). These coloured inks or dyes are used in the subtractive printing process.
Confocal Imaging
A technique in which a point source is imaged into a pinhole placed in front of the photodetector of a scanning optical microscope, resulting in strong optical sectioning which can be used to give three dimensional images. Resolving power is improved and stray light rejected.
Correction Collars
A mechanism provided on some objectives in order to adapt their correction for spherical aberration to compensate for deviations from correct optical pathlength in the medium or media between the objective and the object.
Coverslip
A rectangular or circular piece of thin glass used to cover a microscopical preparation. It is regarded as part of the objective and thus its thickness, refractive index and dispersion must be adapted to the demands of the objective.
Crown (glass)
An optical glass characterised by low refraction and low dispersion.
Dichroic Mirror
An interference filter designed to selectively reflect light of some wavelengths and transmit light of other wavelengths.
Dwell Time (see Pixel Dwell Time)
The time (usually expressed in microseconds) that is spent exciting the specimen by the laser and used to collect emitted photons.
Emission
Release of radiant energy.
Epifluorescence
Fluorescence resulting from epi-illumination, where the excitation light and emission light are on the same side of the specimen. A dichroic mirror is required for epi-illumination.
Excitation
The input of energy to matter leading to the emission of radiation.
Fluorescence
A form of photoluminescence which persists only for a short period of time (usually less than 100 nanoseconds) after the cessation of excitation.
Fluorite
Crystalline calcium fluoride. Because of its low dispersion this material is used as an additional lens material for the correction of chromatic aberration.
Frame Averaging
The collection of multiple frames (images) and displaying the average pixel value for each x,y pixel position in an image. Averaging is usually used to minimise the appearance of noise in confocal images. Frame averaging and line averaging usually produce identical results.
Gain
The voltage applied to the photomultiplier tube. Increasing the gain increases the final output signal from the detector. Gain is sometimes also referred to as high voltage (HV). High gain values usually produce more noise in images.
Greyscale Image
An image containing only brightness information. By definition, a pixel value of 0 (zero) is defined as pure black and the maximum value is defined as white. All values between these limits are shades of grey.
Immersion Oil
A synthetic immersion liquid specified as suitable for use in the space between the front of the immersion objective and the glass coverslip. This immersion liquid is considered in computing lens corrections and its refractive index and dispersion are critical.
Kohler Alignment
Kohler aligning the microscope components provides bright, even illumination throughout the field of view.
Laser
(Acronym for Light Amplification by Stimulated Emission of Radiation.) A source which emits coherent radiation of high spectral concentration and an extremely small solid angle (low divergence).
Laser Power
The power (usually expressed in mW or μW) of laser light used to excite a specimen. A value of 150μW per pixel is considered to be enough to fully saturate all fluorophores within the focal spot volume (voxel). On most confocal microscopes, the laser power is expressed in some arbitrary unit (often a numerical value or percentage of maximum), that varies with manufacturer and microscope configuration. Comparison between different microscopes is, therefore, not possible without direct measurement of laser power at the objective.
Lateral Resolution
Resolution in the plane perpendicular to the optical axis (ie the x,y plane).
Light Emitting Diode (LED)
A twin-element semiconductor that efficiently produces light. The materials and the relative proportions used in construction enable production of different colours of light.
Line Averaging
The collection of the same scan line multiple times and displaying the average pixel value for each x,y pixel position in that line of pixels before collecting the next scan line. Averaging is usually used to minimise the appearance of noise in confocal images. Line averaging and frame averaging usually produce identical results.
Look Up Table (LUT)
A table that converts the “real” pixel value into a colour value. A LUT is used on a display image only and does not change the underlying real pixel values. Special LUTs are frequently used to indicate pixels having minimum and maximum values, thereby indicating under- and over-saturation regions in an image.
Magnification
The process of enlarging or the degree by which the dimensions in an image are (or appear to be) enlarged with respect to the corresponding dimensions in the object.
Maximum Intensity
The highest pixel value from a series of pixels. A maximum intensity projection (MIP) is a way to display three dimensional data in a single two dimensional image, where, for each x,y position, the maximum pixel value (irrespective of which z slice that value occurred in) is used in the 2D projection image. MIPs are usually used on image stacks where the data is bright on a dark background (ie confocal or fluorescence images).
Minimum Intensity
The lowest pixel value from a series of pixels. A minimum intensity projection is a way to display three dimensional data in a single two dimensional image, where, for each x,y position, the minimum pixel value (irrespective of which z slice that value occurred in) is used in the 2D projection image. Minimum intensity projections are usually used on image stacks where the data is dark on a bright background (ie brightfield images).
Mounting Medium
The liquid or medium in which the specimen is placed for investigation in the microscope. It should be transparent, colourless, and of a specified refractive index and enclosed between the slide and the coverslip.
Noise
“Random” signal generated in the detection system (PMT and amplifiers) that is mixed with the real signal from the specimen. It is usually seen as a random pattern of speckles over the entire image. High gain values generate more noise in an image.
Numerical Aperture (NA)
Numerical aperture is a measure of the acceptance angle of an objective and can also be considered as the ability of the objective to gather light and resolve specimen detail at a fixed objective distance. It is defined by the expression: NA = n(sin(a)), where NA is numerical aperture, n is the refractive index of the medium between the objective front lens and the specimen, and a is the half angle aperture of the objective.
Objective
The first part of the imaging system. It forms the primary image of the object.
Offset
The dark current setting of a PMT detector. In effect, this control sets the black values in an image or display. It is essential that this control is set correctly, otherwise very weak signals may be suppressed (and lost) by too low an offset value.
Optical Section Thickness (OST)
The thickness of the optical section collected on a confocal microscope. It is mostly determined by the confocal pinhole diameter, objective numerical aperture and the wavelength of the light.
Oversampling
Setting the pixel or voxel dimensions smaller than the Nyquist sampling limit defined by the optical system (primarily determined by the numerical aperture of the objective). For an objective of NA=1.4, the pixel size (in the lateral x,y plane) should be no smaller than approximately 0.08μm.
Phase contrast
A form of interference in which contrast in the image is enhanced by altering the optical pathlength travelled by the diffracted light with respect to that travelled by the direct light. This is achieved by the action of a phase plate. Contrast is achieved by the conversion of phase differences in the light leaving the object to amplitude differences in the image.
Photobleaching
The irreversible destruction of a fluorescent molecule such that it no longer fluoresces.
PhotoMultiplier Tube (PMT)
A detector (commonly used in confocal microscopes) that contains a photosensitive surface to capture photons and convert and amplify them into an electronic signal.
Photon
Elementary quantity of radiant energy (quantum) whose value is equal to the product of Planck’s constant h and the frequency (hz) of the electromagnetic radiation.
Pinhole
An aperture which, in a confocal microscope, blocks out-of-focus light from reaching the detector.
Pixel
The smallest unit of information in a digital image. Thought to be a contraction of Picture + Element
Pixel Dwell Time
The time spent illuminating and collecting signal from a single pixel position.
Pixel Size
The actual (real world) dimensions of a pixel, usually quoted as (x dimension, y dimension).
Plan Objective
A specially corrected microscope objective that compensates for field curvature and produces sharp image detail from the centre to the edge of the field of view. Plan objectives are often referred to as “flat field” objectives.
Plane of Focus
A surface at right angles to the optical axis of a lens in which the image of an object lying at infinity is formed.
Point Spread Function (PSF)
The three dimensional pattern of light distribution from a subresolution point source.
Rayleigh

The radius of the first dark concentric ring of the Airy disk, expressed by the equation r = 0.61λ/NA.

Refractive index
The ratio of the speed of light in a vacuum to that in a second medium of greater density.
Refractive Index Mismatch
This occurs where the specimen is mounted in such a way that the optical properties (usually refractive index or coverslip thickness) of the mounted sample are not in compliance with the design requirements of the objective. This can result in severe spherical and/or chromatic aberration in the image and is one of the most common problems in confocal microscopy.
Resolution
The minimum distance separating two points such that these points can be seen as separate objects. Sufficient specimen contrast is also required to separate the two points.
RGB
The abbreviation for Red, Green and Blue. It is often used in the context of an RGB image (colour image).
Saturation
The state at which the pixel intensity reaches the maximum recordable value. Regions of saturated pixels contain no “detail”.
Scan Speed
The speed at which an x,y raster scan (a frame) is performed by the laser on the specimen. It is usually quoted in frames per second (fps).
Sequential Image Collection
The collection of a multichannel image where each channel image is collected with just one laser exciting the specimen at a time. This imaging collection mode is slower but usually effectively minimises spectral bleed-through between channels.
Simultaneous Image Collection
The collection of a multichannel image where all lasers are exciting the specimen at the same time. This imaging mode is fast but spectral bleed-through between channels can often be significant.
Spherical Aberration
A defect of a lens where rays of light intersect at different positions along the optical axis.
Undersampling
Setting the pixel or voxel dimensions larger than the Nyquist sampling limit defined by the optical system (primarily determined by the numerical aperture of the objective). For the optimum resolution when using an objective of NA=1.4, the pixel size (in the lateral x,y plane) should be no smaller than approximately 0.08μm.
Voxel
The three dimensional equivalent of a pixel. Thought to be derived from the term Volume Element.
Water Immersion
An optical system where water is the immersion medium between the objective lens and the coverslip.
Wavelength
The distance on a periodic wave between two successive points at which the phase is the same. It is represented by the symbol (λ) and is usually expressed in nanometres and commonly refers to the colour of light.
Widefield Microscopy
Conventional optical microscopy.
Working distance
The distance from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus.
Z Step
The dimension along the optical axis between successive frames in a z stack, usually given in μm.
Zoom
The additional magnification provided in a confocal microscope by altering the angle through which the galvanometer mirrors rotate. It is an optical magnification and the maximum zoom is usually limited by the numerical aperture of the objective.