Glossary - Terms and Abreviations
Glossary

Terms and abbreviations

Microscopy & Imaging Glossary

  • A

    Aberration
    Geometric deviation of an image formed by an imaging grating from the ideal point image.

    Absorbance
    A.-The logarithm to the base 10 of the reciprocal of the transmittance,(T). A = log10 (1/T) = - log10 T

    Absorption Band
    A region of the absorption spectrum in which the absorbance passes through a maximum.

    ACE
    Automatic Component Extraction
    Statistical procedure for the detection of single dye spectra in a Lambda Stack.

    Accuracy
    Measurements of minimal changes and stability are directly dependent on each other and are mainly limited by the noise present in the electronics, as the stability of the light path is ensured in most spectrometers. As with all parameters, it is important how a value – here in the true sense of the word – is determined. For the data provided by the MMS, for example, an integration time of 10 ms is set and the standard deviation Dd is computed using 20 recordings. This supplies a measure of the accuracy Dl with which an intensity value can be determined.

    Dl = Inoise = Dd

    ADC
    Analog-to-Digital Converter

    Analytical Wavelength
    Any wavelength at which an absorbance measurement is made for the purpose of the determination of a constituent of a sample.

    AOM
    Acousto Optical Modulator

    AOTF
    Acousto Optical Tunable Filter
    Acoustically generated diffraction grating. The intensity of the laser excitation light can be tuned very quickly via AOTF.

    APD
    Avalanche Photo Diode
    Highly sensitive detector allowing single photons to be registered.

  • B

    Background
    Apparent absorbtion caused by anything other than the substance for which the analysis is being made.

    Baseline
    Any line drawn on an absorbtion spectrum to establish a reference point representing a function of the radiant power incident on a sample on a given wavelength.

    Beer’s Law 
    The absorbance of a homogeneous sample containing an absorbing substance is a directly proportional to the concentration of the absorbing substance.

    Blaze grating
    The facet or inclination angle of the longer profile edge (called glance angle or blaze angle) is usually determined by the wavelength l for which the diffraction efficiency of the first order should be a maximum in case the groove number G is given. Specially it is true sin = 0.5 l G.

    Blaze wavelength
    Monochromatic light is diffracted by gratings in numerous orders. The measure of the light intensity diffracted from a grating in a certain order is called its efficiency. For a number of reasons, gratings are not equally efficient for all wavelengths.

    The efficiency can be optimized by changing the form and depth of the grooves. Optimizing the efficiency via the groove form is known as “blazing”. The “blaze wavelength" is the wavelength for which the grating reaches its maximum efficiency.

     

  • C

    C-DIC
    High-contrast innovations Carl Zeiss provides an optical polarization technique that operates with circular polarized light (Circular DIC) in contrast to conventional DIC. Trendsetting in materials microscopy, circular polarization with DIC solves a familiar imaging problem. With the innovative C-DIC technique, sample structures that were visible only in a certain direction can now be seen in their entirety – regardless of their orientation and without rotating the sample stage. You simply adjust the knob on the DIC slider for complete and highcontrast imaging of all specimen structures. All information about the specimen is rendered visible, contrasts are sharper – and your workflow is substantially more efficient.

    CFP
    Cyan Fluorescent Protein
    A protein that can be excited by light and fluoresces in the blue spectral range. Developed from GFP through modification.

    Changing Arc Lamps
    Arc lamps in mercury and xenon burners work under high vacuum and high temperatures. These safety steps are highly recommended. Wear safety glasses. After switching the unit off do NOT switch power supply back on again before the burner has been permitted to cool down completely (typically 20 min.). Wear lint-free gloves or use lens tissue when handling the bare bulb. Let the burner cool completely before removing the bulb. Unplug the power supply.

    CIE
    The abbreviation for the French title of the International Commission on Illumination, Commission Internationale de l’Eclairage.

    Cleaning of Optical Components (Recommendations)
    Accessible optical surfaces (front lenses, eyepiece back lenses, condenser front lenses) should normally be cleaned using mild cleaners. Optics cleaning paper or a white linen cloth (both fluff-free) can be used. A wooden stick wrapped in medical-grade cotton wool is also suitable. Slight moistening with distilled water may also be of assistance in the cleaning procedure. Cleaning is always performed in circular movements, starting at the center and working toward the edge. Fluff or dust can be blown off with the bellow-like devices obtainable from camera stores Petroleum ether used for medical purposes should be used occasionally to remove stubborn dirt or oil. The benefit of this agent is that it evaporates easily and therefore does not enter gaps or joints, i.e. the lacquers are not damaged during the short reaction time of the agent. The dust cover supplied protects the microscope from dirt when it is not in use. In the event of severe contamination, please contact Zeiss Service personnel.

    Color (of an Object)
    The aspect of the appearance of an object dependent upon the spectral composition of the incident light, the spectral reflectance or transmittance of the object, and the spectral response of an observer.

    Concentration
    The quantity of a substance contained in a unit quantity of sample.

    Confocal Microscopy
    Unlike "normal" microscopy, only a spot of a minimum size in the specimen is illuminated in confocal microscopy. To enable an image to be formed from this, the specimen must be scanned. The image of the spot is directed through a pinhole stop in an intermediate image plane. As a result, only light from the focal plane can reach the detector (a photomultiplier). All other (out-of-focus) planes are blocked out. This results in an "optical section". The images are stored electronically and displayed on a monitor. A series of optical sections can be recorded by moving a motor a slight distance along the z-axis each time an image has been recorded, after which the next image is then also recorded. Such a z-series permits the electronic reconstruction of the three-dimensional structure using suitable computer programs. The procedure, which is restricted to incident-light techniques, has completely revolutionized fluorescence microscopy in biology in particular.
    The Confocal Scan Module is used to produce confocal images in the eyepiece, with the height of the reflecting specimen being color-coded in colors using a color-converting optical system. This enables even minute defects and contamination on wafers to be detected quickly and reliably.

    Coatings
    The coating used for the grating blank is dependent on the spectral range chosen. In the IR range, all coatings provided by Carl Zeiss feature reflectances of over 98%. Aluminium is mainly used for the shorter wavelength range λ = 600 nm. In special cases, a silver coating is used. Silver coatings are always overcoated with a protective layer of MgF2 or Si02. On request we also offer special coatings.

     

  • D

    Depth of field
    The depth of field (mm) is the area above and below the focal plane in the object which is still perceived as sharply imaged.

    Derivative Absorption
    A plot of rate of change of absorbance or of any function of absorbance with respect to wavelength or any function of wavelength, against wavelength or any function of wavelength.

    Determining the half-width
    The parabola fit also supplies qualitative data on the half-width. For this, Imax/2 must only be inserted in the parabola equation. There are only minor differences between the half-width of a parabola fit and that of a Gaussian fit (see below).
    The half-width which is displayed by a AS is also dependent on the position of a line relative to the individual pixels. Our specifications are valid for worst-case values. More adequate, but more complex, are fits to Gaussian or Lorentz curves which better correspond to the actual spectral distributions. These fits also have the advantage that the halfwidth calculated from them is not dependent on its position relative to the pixels.

    DlFWHM = 2[(b/2a)2 - (c - Imax)/a]1/2

    DIC
    Differential Interference Contrast (Normarski)
    Optical contrasting technique permitting the optical cutting of the specimen and the examination of height differences.

    Diffraction efficiency
    Fraction of the light diffracted in a certain order at a certain wavelength relative to the reflection of a comparison mirror or absolute to the incident light.

    Diffraction order
    In accordance with the grating equation l = g/m (sin a + sin b), where g is the grating constant, a is the angle of incidence; b is the diffraction angle and m is the order of the diffraction, the wavelengths m*l
    (m = 0; +/-1; +/-2) fall in the same direction b.

    Dispersion
    The term Dl / Pixel (= DlPixel) has nothing to do with spectral resolution; it is merely the linear dispersion of a diode array spectrometer. The pixel dispersion and the spectral resolution are related to each other via the width of the entrance slit and the imaging properties of the spectrometer. lf the entrance slit is imaged on approx. 3 pixels, the triple of the pixel dispersion approximately corresponds to DlRayleigh.
    Dl Rayleigh » 3 x DlPixel

    DSP
    Digital Signal Processor
    Controls all the processes of a laser scanning microscope.

    Dual Plate Guidance
    Ultrastable and Precise Focusing

    All Axioplan 2 Microscopes have the new designed Dual Plate Guidance (Patent) system. With this design we have significantly improved the short and long term stability of the focusing mechanism (long fluorescence photographs, or time lapse video recordings). Plates (1) and (3) are connected and move up and down on the four cross roller bearings of the fixed middle plate (2), providing stability in x, y and z. Conventional focusing mechanism use only a single fixed plate equipped with dual roller bearings. This new design was less susceptible to thermal and mechanical stress and instability.

    Lambda = mean wavelength of white light = 0.00055 mm (green)
    NA = numerical aperture of the objective
    Mtotal = overall magnification in the microscope

     

    Dynamic range and intensity changes
    The dynamic response is defined as the ratio of the saturation value lsat and the noise Inoise » Dd and thus corresponds to the signal-to-noise ratio S/N. (The usable range is reduced by the dark current.) Dd does not only depend on the detector, but also on the digitization determining the smallest step width into which a measured signal can be decomposed.
    Dynamic range = S/N = Isat / Inoise
    The weakest link in the chain, of course, determines the signal to-noise ratio to be achieved. For instance, when using a 14 bit converter . this corresponds to 16,384 steps or increments and a noise of Dd = 1 count, a signal (full-scale display) can really be divided into 16,384 increments. Hence, the lowest measurable change is 1/16,384 of the saturation signal. At a noise of 4 counts an uncertainty of 4 counts also exists, i.e. a change of 4/16,384 of the saturation signal can only be definitively measured or the signal divided into 4,096 increments.
    It should be noted here that a wide dynamic range is only obtainable if the PDA (photodiode array) is near the saturation limit.
    The aim is always to reach high light intensity - here, the high sensitivity of the spectrometer modules is beneficial.

    Dynamic range = range ADC/ Dd

  • E

    Echelette grating
    Reflection grating with a triangular groove profile and la large grating constant, used with the Echelle spectrograph in particular.
    Grating with a sawtooth-shaped groove profile. The inclination angle of the longer profile edge is determined by wavelength ë in most cases, for which the diffraction efficiency of the first diffraction order should obtain the maximum level (grazing angle, blaze angle) if the groove number G is given.

    Educational Microscope
    A microscope for education, used at universities, medical schools and schools in general, must satisfy a number of specific requirements.

    Efficiency anomaly
    Minimum of the wavelength-dependent efficiency curve of a grating diffracting in several directions simultaneously but the light of one order doesn’t expand in the clearance but along the grating surface. Because of that it is missing in the energy balance.

    Efficiency
    The absolute efficiency is defined as the ratio of the diffracted to the incident intensity for a given wavelength. Relative efficiency, however, is defined as the ratio of the diffracted intensity in a specific diffraction order to the reflected intensity of a mirror with the same reflection coating as the grating.

    Spectral efficiency
    The variation of the relative efficiency with the wavelength and the efficiency maximum are mainly determined by the groove shape and, to a lesser degree, by the beam geometry. We distinguish between two domains: the scalar and the electromagnetic. In the scalar domain (λ </= 0,6 g), the relative spectral efficiency curve has a pronounced maximum which is typically over 80% with an echelette grating, 33% with a sinusoidal grating and 40% with a laminar grating.
    In the electromagnetic domain (λ </= 0,7 g), where the zero and the first orders mainly occur, the difference in efficiency of the different groove shapes vanishes with increasing groove depth (h >/= 0,3 g). Furthermore, the spectral efficiency curves differ for light polarized parallel and perpendicular to the grooves. In sinusoidal and laminar gratings, efficiency values of over 90% can also be attained, especially in the case of perpendicular polarization.

    Emission Fingerprinting
    Method available with the LSM 510 META for the recording, analysis and separation of emission signals in multi-fluorescence imaging; working also with widely overlapping emission spectra.

  • F

    FCS - Fluorescence Correlation Spectroscopy
    Statistical evaluation method where molecules are distinguished by their different diffusion speeds.

    Femtoliter
    10-15 Liter (i.e. one quadrillionth of a liter)

    Field of view number
    The field of view number designates the diameter of the visible intermediate image in millimeters. Therefore, the diaphragm limiting the intermediate image in the eyepiece is of decisive importance. Furthermore, the microscope must permit the bundle of rays to pass through the entire beam path. The Axioplan 2 imaging microscope has been designed for the field of view number 25, while the  Axioskop 2 plus,  Axioskop 40 and Axiovert 200 microscopes have been designed for 23 mm fields.  Axiovert 40 and  Axiostar plus are designed for 20 mm fields.  Plan APOCHROMAT,  Plan-NEOFLUAR and  Epiplan-NEOFLUAR objectives have been flattened for the 25 mm field of view,  A-PLAN and  ACHROPLAN objectives for the 23 mm field.
    Fields of view are decisive for the area of the object imaged. The object field therefore increases with the field of view number.

    Examples:

    • Plan-NEOFLUAR objective 40x and eyepiece 10x/25: object field = 0.625 mm 
    • ACHROPLAN objective 40x and eyepiece 10x/20: object field = 0.5 mm 
    • Plan-NEOFLUAR objective 1.25 and eyepiece 10/25x: object field = 20 mm 
    • Plan-NEOFLUAR objective 63x and eyepiece 10x/20 and Optovar 1.25x: object field = 0.254 mm 

    FLIP
    Fluorescence Loss in Photobleaching

    Fluorescence
    Phenomenon which occurs when high-energy radiation is absorbed by a molecule (fluorochrome) and when this molecule emits light of a longer wavelength than that absorbed (Stoke's shift). In microscopy, fluorescence is an important technique, since most  fluorochromes do not damage cells and can therefore be used in the microscopy of living specimens. Furthermore, they can be bound to antibodies and other specific molecules, permitting the exact localization, observation and measurement of changes. The phenomenon of autofluorescence also exists, where already fluorescent molecules are present in the material to be examined. 

    Fluorescence filter sets, consisting of exciter filter, beam splitter and barrier filter, are required, as well as a high-intensity lamp (usually super-pressure mercury or xenon lamps).The decisive factor for good visualization (high contrast of the fluorescent areas against a dark background), especially in the case of weakly dyed specimens, is the high numerical aperture of the  objectives used. Doubling of the objective aperture allows four times more fluorescent light to be detected.

    With the market introduction of three new products in October 2004 Carl Zeiss has set a special focus on  Fuorescence 

    Focal curves
    Plot of the focal distance of an imaging grating against the wavelength in case of an ideal point imaging in the direction of dispersion

    FRAP
    Fluorescence Recovery After Photobleaching

    FRET - Fluorescence Resonance Energy Transfer
    Transfer of the energy of a donor to an acceptor close to it which can then emit photons although it has not directly been excited by light.

  • G

    GFP - Green Fluorescent Protein
    A protein that can be excited by light and fluoresces in the green spectral range. It is widely used in cell biology.

    Grating equation
    sin a+ sin b= ml G
    light incidence angle, light diffraction angle, m diffraction order, light wavelength, G groove number.

    Groove frequency
    The shape of the grooves (lines) of a grating is described by the groove profile. The individual grooves repeat in a periodicity interval g, which is called the grating constant. The reciprocal value is the spatial frequency, also known as the line frequency N, which is usually stated in lines per mm(L/mm).

    Groove number
    Number G of lines of a grating with the dimension mm-1, reciprocal to the grating constant.

    Groove profile
    Cross section of the grating groove shape. It can be found a symmetric profile (sine, triangle, rectangle) or an asymmetric triangular one.

  • H

    The Harmonic Drive Gear Box
    Extremely high Reduction Ratio Capabilities Combined with very Accurate Motion

    All Axioplan 2 with motorized focus have the Harmonic Drive Gear Box&trade; system. From the figure you will see that the drive has essentially three parts. A full 360º rotation of the wave generator (1) results in displacement of two teeth on the flexspine (3) in relation to the circular spine (2). This outstanding transmission ration directly impacts the high resolution of the focusing drive. Rotating the focusing knob, rotates an encoder disc. The encoder measures the amount of rotation of the focusing knob by counting the number of lines printed on the encoder disc as they pass through the encoder. This information is sent to the focus motor controller. Together with the programmed focusing sensitivity this information is translated into a movement command for the DC focus motor. Voltage is applied to the focus motor and the motor starts to move. Attached to the shaft of the focus motor is another encoder, which feeds back information to the focus motor controller telling it the actual position of the motor. Once the focus motor reaches its destination, the focus motor controller switches off the voltage to the focus motor, and the movement stops.

    This makes it possible to have 40 different resolution levels in total which one can choose from to select the focusing sensitivity for a specific objective. Individual adjustment for an objective can be achieved by the user. Therefore smooth and precise movement of the focusing drive is guaranteed. The coarse focus is set to be five times of the fine focus which is programmed as a default but it can be also adjusted to the factors 1x to 40x.

    Specifications: 

    Resolution: 25 nm Minimum Step Size 
    Reproducibility: 100 nm 
    Drift: 0 
    Reversing Backlash: 300 nm 

    Holographic exposure
    In a photoresist layer deposited on a plane or curved substrate an interference figure is recorded formed by two point laser light sources. Plane gratings are exposed in case plane waves interfere, imaging ones in case plane or spherical/aspherical waves interfere. If the interfering bundles originate in the same hemisphere the groove profile is sinusoidal, if they originate in different hemispheres the profile is sawtooth-shaped with something rounded edges. After the exposure the photoresist have to be developed.

    Holographic grating
    The grooves of the master grating were generated by recording of an interference figure in a photoresist layer.

  • I

    ICS Optics 
    Objectives with astounding image quality are major components of the "pyramids" from Zeiss.

    Imaging grating
    The diffracting structure is placed on a substrate’s convex surface (convex grating with positive radius) or concave surface (concave grating with negative radius)

    Imaging properties
    Point resp. slit imaging by an imaging grating in amounting resulting in a minimum of astigmatism and coma.

    Infrared Spectrum
    Pertaining to the region of the electromagnetic spectrum from approximately 0.78 to 300µm.

    Ion etching
    A method to increase the blaze angle of holographic photoresist gratings by transfer of the grooves into the substrate material taking advantage of the different etching rates of resist and substrate. Because of that the spectral distribution of the grating efficiency is shifted to greater wavelengths. Ion etching is also a method to transform a sine profil in a rectangular one.

    lntensity resolution
    To measure intensity, the following properties which are dependent on each other are of interest.
    Relative:
    - smallest detectable change
    - signal stability
    - detection or dynamic range
    -  linearity
    Absolute:
    - lowest detectable amount of light or sensitivity.

  • L

    Lambda Stack
    Image stack with information in x, y and lambda dimensions, combinable with z and / or time series; for determination of spectral signatures at any location of a specimen.

    Laminar grating  
    The grating groove profile is rectangular.

    Linear Unmixing
    Mathematical method for the spectral deconvolution of multiple emission signals.

    Linear Dispersion   
    The derivative , dx/dl,where x is the distance along the spectrum, in the plane of the exit slit, and l ist the wavelength.

    Linearity  
    The previous remarks will be completely accurate only if the detector and the post-detector electronics provide ideal linearity, i.e. if the dependence of the measured charge on the irradiated intensity is exactly linear. For quantification, the admissible deviation must be specified. Fortunately, the behavior of modern semiconductor detectors is almost perfectly linear within a wide range. Before saturation (the extreme case of non-linearity) is reached, however, the increase of the current (carrier of the intensity information) supplied is no longer linear to the number of photons striking the photosensitive material. For this reason, the range of linearity is smaller than the dynamic range.

  • M

    Magnification and Lateral Magnification
    The total magnification of the microscope is calculated from the magnifying power of the objective multiplied by the magnification of the eyepiece and, where applicable, multiplied by intermediate magnifications.

    Mechanically ruled grating
    The grooves of the master grating were cutted or pressed in a ductile material by a ruling diamond.

    Metatracking
    Scanning mode available with the LSM 510 META, similar to Multitracking, yet with additional fast switching between detection settings.

    Monochromator  
    A device or instrument that with an appropriate energy source may be used to provide a continous calibrated series of electromagnetic energy bands of determinable wavelength or frequency range.

  • N

    NLO
    Non-Linear Optics (multiphoton imaging)

    Numerical Aperture   
    Abbreviation: NA

    This is the value designating the sine of half of the aperture angle of the objective. It only applies if there is air between the objective and the specimen. To put it more precisely: the refractive index of the immersion medium must also be taken into account. The formula therefore is: 

    NA = n x sin (* x alpha)

    n = refractive index of the medium between the objective front lens and the specimen or cover glass 

  • O

    Objective Classes
    Description of Classes of Objectives
    Learn more

    Objective With Correction Ring   
    Why cover glass correction for an oil objective? Oil objectives should function equally well, regardless of whether they are used with or without a cover glass because the cover glasses and the oil should display an almost identical refractive index (homogeneous immersion). Unfortunately, however, theory and practice are not always compatible. Manufacturers often provide cover glasses with a refractive index other than the 1.525 ± 0.0015, and a thickness other than the 0.17 (+0/-0.02) mm required especially for high-aperture objectives. Deviations of only 0.01 mm in the cover glass thickness can result in unsatisfactory image quality. The refractive index of the immersion medium must also be kept precisely to ensure a perfect image. The Zeiss immersion oil with the refractive index 1.518 is therefore ideal. It is particularly important to compensate for these deviations in the case of high-aperture dry objectives. All objectives with numerical apertures from 0.85 upwards therefore feature a correction ring. 


    Objectives for Research Microscopy

    Objectives for Routine and Research Microscopy

    Objectives for Routine Microscopy

    Opacity  
    The degree of obstruction to the transmission of visible light. (D 16)a.

    Optical interface  
    Interfaces must be mechanically and optically defined. A useful mechanical interface for optical systems is the SMA connector as used in the modules. Together with the well-defined light guidance factor of a fiber bundle, this results in a unique interface.

  • P

    Photometer  
    A device so designed that it furnishes the ratio, or a function of the ratio, of the radiant power of two electromagnetic beams. These two beams may be separated in time, space, or both.

    Photometric Linearity  
    The ability of a photometric system to yield a linear relationship between the radiant power incident on ist detector and some measurable quantity provided by the system.

    Plane grating  
    The grating has a plane substrate and straight and equidistant grooves.

    Push & Click Filter Cubes
    With so many options to choose from, flexibility is the key. Filter sets can be quickly removed or added to the turret using the newly designed, interchangeable filter cubes. No more screws, no problems with alignment: the mechanism ensures the filters are held securely in place and are correctly positioned. All in a matter of seconds.

  • R

    RealROI Scan
    Scanning mode in which freely definable specimen areas are excited and imaged; guarantees maximum specimen preservation thanks to exact blanking of laser lines outside the selected specimen areas. 

    Reflectance   
    The ratio of reflected to incident radiation. (A practical definition requires that basic term be modified by adjectives to indicate the spectral and geometric weighting of the incident and reflected radiation).

    Reflection grating
    The grating is used in reflection which the incident light is getting a directional reversal. Reflective coatings are preferred aluminium and gold.

    Replication  
    Profile-true multiplication method to mass-produce diffraction gratings. The grating structure is replicated in epoxy or UV-cured adhesive. Usually the replicated gratings are duplicates of a higher generation (copies of copies) but their efficiency comes closest to that of the master gratings.

    Resolution
    The resolving power of the microscope is determined by the ability to make points or lines which are closely adjacent in an object distinguishable in an image. The distance between these distinguishable points or lines is designated as do. This distance can be calculated using the following formula:  

            1.22 x Lambda 
    do = -------------------------------------- 
            NAObjecive + NACondensor 

    Lambda = Mean wavelength of white light, e.g. 550 nm (green)

    NA = numerical aperture   

    Resolution capability  
    Minimum spacing of 2 wavelengths separable by Resolving power a grating, proportional to the grating area and inversely proportional to the wavelength.

    Resolving Power

    The resolving power R of a grating is a measure of its ability to separate adjacent spectral lines of average wavelength λ. It is usually expressed as the dimensionless quantity.

    R= λ / Δλ
    Here Δλ is the limit of resolution, the difference in wavelength between two lines of equal intensity that can be distinguished.
    Often the Rayleigh criterion is used to determine Δλ – that is, the intensity maxima of two neighboring wavelengths are resolvable if the intensity maximum of one wavelength coincides with the intensity minimum of the other wavelength.
    The theoretical resolving power of a planar diffraction grating is given as

    R=mN
    where m is the diffraction order and N is the total number of grooves illuminated on the surface of the grating.

    For negative orders (m < 0), the absolute value of R is considered.
    R is not dependent explicitly on the spectral order or the number of grooves; these parameters are contained within the ruled width and the angles of incidence and diffraction. Since

    | sin a + sin b| <2
    the maximum attainable resolving power is

    R max = 2W/ λ
    The degree to which the theoretical resolving power is attained depends not only on the angles a and b, but also on the optical quality of the grating surface, the uniformity of the groove spacing, the quality of the associated optics in the system, and the width of the slits (or detector elements). Any departure of the diffracted wavefront greater than λ/10 from a plane (for a plane grating) or from a sphere (for a spherical grating) will result in a loss of resolving power due to aberrations at the image plane.

     

    Spectral resolution
    Due to the fixed position of the pixels with respect to the wavelength of the incident light, the resolution provided by AS differs from that provided by monochromators/spectrometers with moving components: resolution defined as the “separation of two adjacent lines” is dependent on the relative position of these lines with respect to the pixels: lf two adjacent lines are imaged on the pixels in such a way that the minimum falls on the central pixel (I2) and the maxima on the adjacent pixels (I1, I3) the lines can be separated if the intensity displayed is I2 < 0.81 x I1 (I3), Dl is then exactly two pixels (2 x DlPixel). In this case, it is sufficient to evaluate a total of 3 pixels; the locations of the maxima correspond almost exactly to the central wavelengths of the pixels displayed.

    ROI Bleaching
    Defined photobleaching of several freely defined specimen areas, e.g. for FRAP or Uncaging experiments.

    rROI
    real Region of Interest

  • S

    Scattered light  
    The specification of scattered light data is only useful in connection with the measuring instructions. Scattered light data for the spectrometer modules are determined using three different light sources to measure the different spectral components of scattered light: a deuterium lamp for UV, a xenon lamp for VIS and a halogen lamp for VIS-NIR. The level of scattered light is defined as the ratio of the respective measurement using Schott GG495 and KG3 filters to the maximum useful signal and is therefore specified for the short wavelength range. This reveals that the main components of scattered light in the spectrometer modules come from the NIR range. These spectral components are easy to filter out as they are far away from the spectral range of interest. The scattered light value for the PGS NIR is reduced to 0.1 % (measured at 1450 nm, halogen lamp, Schott RG 850 filter and 10 mm water absorption). Scattered light influences the dynamic range as the full range is no longer available. However, changes in the radiation used only affect the dynamic range in proportion to the scattered light present: for example, a change pf 10 % in the radiation used causes a change of 10–4 if the scattered light component is 0.1 %. lf the radiation causing the scattered light is not used, the amount of scattered light can be further reduced by filtering this radiation. A blocking of 103 results in a change of 10–7 in the case described. Thus, the measurement of minimal changes is only impaired to a very limited extent, as noise is the bigger problem in most cases. In addition, if the signal causing the scattered light is known, the scattered light component can be eliminated by computation.

    Sensitivity  
    The smallest detectable change is a relative specification. Much more difficult to specify is the lowest detectable amount of light or: how many photons are needed for the detection electronics to record a change. The difficulties result from determining the light intensity of a light source and the coupling efficiency. Furthermore, these parameters are wavelength-dependent. There is, on the one hand, a direct dependence, as all components feature wavelength dependent efficiencies – including the coupling – in device; on the other, there is a dependence, as the bandwidth is of decisive importance for sensitivity measurements. The simplest case is a light source with a very narrow band, as displayed by most of the lasers. lf the bandwidth of the light source used is markedly smaller than the bandwidth of the spectrometer used, the situation is clear. The MMS value of more than 1013 counts / Ws has been measured with a red HeNe laser.

    Sinusoidal grating  
    The grating groove profile is sinusoidal.

    Spectral Band Width   
    The wavelength or frequency interval of the radiation leaving the exit slit of a mono-chromator between limits set at a radiant power level half way between the continuous background and the peak of an emission line or an absorption band of negligible intrinsic width.

    Spectral resolution  
    The following three terms are often used to describe "spectral" resolution:
    1. Line width, mostly full width at half maximum – DlFWHM
    2. Sub-pixel-resolution (also termed "software resolution")
    3. Mean spectral pixel pitch – DlPixel
    lt is the actual application which provides a useful definition in this respect. There are mainly three different purposes for which a spectrometer is used (these can also occur in combination, of course):
    1. Separation of two or more lines within a spectrum – analysis of compositions
    2. Determining the line shape mostly determining the width of a line or band (FWHM or 1/e2-width)
    3. Measurement of a line with respect to its peak wavelength and intensity at the maximum.

    Spline Scan
    Scanning along a freehand-defined line for recording fast (physiological) processes, e.g. along neurons.

    Spectral resolution (Diode Array Spectrometer)  
    Due to the fixed position of the pixels with respect to the wavelength of the incident light, the resolution provided by AS differs from that provided by monochromators/spectrometers with moving components: resolution defined as the “separation of two adjacent lines” is dependent on the relative position of these lines with respect to the pixels:
    lf two adjacent lines are imaged on the pixels in such a way that the minimum falls on the central pixel (I2) and the maxima on the adjacent pixels (I1, I3) the lines can be separated if the intensity displayed is I2 < 0.81 x I1 (I3), Dl is then exactly two pixels (2 x DlPixel). In this case, it is sufficient to evaluate a total of 3 pixels; the locations of the maxima correspond almost exactly to the central wavelengths of the pixels displayed. lf the maximum of a line is imaged on the separating line between two pixels (I1,I2) however, a total of 4 pixels is required to be able to detect a clear reduction in the pixel intensities. Both pixels record about the same intensity, with the result that a reduction to 81 % is not displayed until in the next pixel (I3) Here, the actual maxima are separated by fewer than 3 pixels; the AS displays a spectral spacing of 3 x DlPixel as a diode array can only detect discrete values using the step width of the pixel dispersion. A total of 4 pixels are needed for processing.

    Spectral resolving power  
    According to DIN, the Rayleigh criterion is relevant to the separation of spectral lines. The criterion indicates how wide the spectral distance between two lines DlRayleigh must be to allow their recognition as separate lines. Here, the spectral width of the individual lines DlLine, (see above) must be markedly smaller than their spacing. This is the only significant definition of spectral resolving power.
    2 lines with Imax,1 = Imax,2 are separated if Dl decrease ³19 %

    Spectrograph  
    An instrument with one slit that uses photography to obtain a record of a special range simulaneously.

    Spectrometer  
    An instrument with an entrance slit and one or more exit slits, with which measurements are made eigther by scanning the spectral range point by point or by simultaneous measuements at several spectral positions.

    Spectrophotometer  
    A spectrometer with associated equipment so designed that it furnishes the ratio or a function of the ratio of the radiant power of two beams as a function of spectral position.

    Spot Scan
    Scanning mode in which the signal intensity at a confocal point can be tracked with extremely high temporal resolution

    Step Scan
    Fast overview scan in which intermediate lines are added by interpolation.

    Sub-pixel resolution or the parabola fit  
    To determine the peak wavelength lmax (and/or peak intensity Im) the spectral line to be measured must be imaged on at least 3 pixels (see below). Three pairs of values (intensity per pixel I 1,2,3 and the related central wavelength of the pixel l 1,2,3 allow relatively easy fitting of the line to a parabola. The equation for the parabola then gives the peak of the curve including the data for the peak wavelength and peak intensity. The accuracy of this method largely depends on the absolute accuracy of the central wavelength. In a diode array spectrometer, this wavelength can be determined, in principle, to almost any accuracy required. lf necessary, each pixel can be individually calibrated. However, this will only make sense if the module features the necessary stability. Otherwise, the wavelength specification will only remain alid until the next shock or temperature change. lf the imaging performance (and the dispersion) of a AS has been chosen such that fewer than 3 pixels are illuminated, no extrema can be determined, resulting in a parodox: an apparently ideal situation – a line is very narrow at the output – leads to considerably increased inaccuracy. lf, for example, a line is only imaged on a single pixel, the spectral inaccuracy is DlPixel in this case.
    Parabola equation
    I (l) = a x l 2 + b x l +c
    Coefficients
    a = (I3 + I1 - 2 I2) / 2 Dl2
    b = (I3 - I1) / 2 Dl- 2a x l2
    c = I2 - a x l2 - b x l2
    Maximum at l max = -b / 2a

    Student Microscope
    A student microscope, used at universities, medical schools and schools in general, must satisfy a number of specific requirements.

  • T

    Tile Scan
    Produces an overview image consisting of a number of tiled partial images using the motorized XY stage; for recording larger objects with improved resolution.

    Transmission grating  
    The grating is used in transmission.

    Transmittance  
    The ratio of radiant power transmitted by the sample to the radiant power incident on the sample.

  • U

    Ultraviolet   
    Pertaining to the region of the electromagnetic spectrum from approximately 10 to 380 nm. The term ultraviolet without further qualification usually refers to the region from 200 to 380 nm.

    Useful Magnification
    This is obtained when the magnification is between 500 and 1000 times the numerical aperture of the objective. Since the eye has a limited resolving power, the magnification should be selected so that image details can still be resolved by the eye. If the overall magnification is below this range, details can no longer be recognized by the eye. If the overall magnification is above this range, this is called empty magnification. The objective is no longer able to resolve the structures. The image therefore seems out of focus.Examples:

    Plan-NEOFLUAR objective 40x/0.75 + eyepiece 10x = 400x 375x ... 750x OK 

    ACHROPLAN objective 100x/1.25 + eyepiece 16x = 1600x 625x ... 1250x not OK 

    Plan-APOCHROMAT objective 20x/0.75 + photo eyepiece 10x + camera factor 0.25 + additional magnification 10x = 500x 375x ... 750x OK .  

  • V

    VAREL-Contrast
    A contrast technique, in which phase contrast and inclined unilateral illumination are mixed, has been developed for the examination of living cells in culture vessels. An additional stop shaped like a ring sector is used on the illumination side, permitting unilateral inclined illumination. Unilateral darkfield, VAREL contrast superimposed on phase contrast and inclined brightfield are set by shifting the stop in the radial direction from the outside to the inside. This is a very low-price method of imaging cells in culture vessels. The image below shovvs pseudo-relief. Even vessels vvith a curved bottom allow a useful image to be produced. Phase contrast alone sometimes fails in such cases because a curved chamber base acts like a lens and impairs the superimposition of phase rings. The method is successfully used for the examination of living objects (micromanipulation)

    A slider contains two of the mentioned sectors to allow illumination to be performed from the left or right, as required. This makes it possible to contrast cells even in the "holes" of microtiter plates in the vicinity of the "hole" edges.

    Visible  
    Pertaining to radiant energy in the electromagnetic spectral range visible to the normal human eye (approximately 380 to 780 nm.

  • W

    Wavelength accuracy  
    To determine the spectral position l – with a specific accuracy Dl± – of a single line, a spectrometer with at least this absolute wavelength accuracy Dl± is required. This parameter is dependent on the accuracy of the positions of the readout elements (pixels or slit/ detector) or the stability of these positions characterized by repeatability. Contrary to this, the absolute wavelength accuracy only depends indirectly on the dispersive and focal properties of the spectrometer and is not “resolution” in the classic sense. The stability (or repeatability) of a spectral sensor is dependent on the mechanical stability of the module and the temperaturerelated wavelength drift. The former is completely uncritical in the spectrometer modules, and the drift can be more or less neglected.

    Wavelength   
    The distance measurred along the line of propagation between two points that are in phase on adjacent waves.

    Wavenumber   
    The number of Waves per unitlength.

    Width of spectral lines  
    To enable the measurement of the width of a spectral line DlLinie, the expansion of this line by the spectrometer must be smaller than the spectral width of the line itself. To ensure this, it is important to know the expansion DlFWHM produced by the spectrometer. This property is related to the Rayleigh criterion.
    DlFWHM = l 2(Imax/2) - l1(Imax/2)
    DlFWHM »0.8 x DlRayleigh

    Working Distance   
    The distance between the front lens of the objective and the surface over the cover slip is called working distance (or free working distance), when the focus is adjusted to the uppermost specimen details right below the cover glass. By making use of reflected light optics the focus has to be at the specimen surface. The higher the numerical aperture , the lower is the working distance. To reach the specimen with manipulators or capillaries one has to take long distance (LD-) objectives. For applications in reflected light for example under high temperature conditions (heating stage) one selects LD-Epiplan objectives.

  • Y

    YFP - Yellow Fluorescent Protein
    A protein that can be excited by light and fluoresces in the yellow spectral range. Developed from GFP through modification.

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