Binning refers to the combination of charges from several neighbouring pixels into only one signal. This technology is used whenever the sensitivity of the sensor is not sufficient for the detection of weak signals or if very weak signals are to be detected.
This increased sensitivity causes however a smaller resolution: the 2 x 2 binning process combines 4 pixels into one single signal before the readout process has started; the resolution amounts to thus only a quarter of the actual resolution of the CCD sensor.
Blooming describes the crossing of charges into neighbouring pixels, if, by overexposure, more charge is produced in an individual pixel than can be stored up the discharge.
The CCD sensor is an electronic compartment containing a light-sensitive metallic surface. CCD stands for charge coupled device: Light in terms of photons reaching the sensor result in electrical charge, which can be measured (see photoelectric effect). The charge is proportional to the amount of light, which has reached the sensor. The CCD sensor consists of an array of single distinguished compartments, called pixels. The charge of any pixel can be measured independent from each other.
The CCD sensor has been developed in the Bell Laboratories of Willard Boyle and George E. Smith. First they were thought as data saving units. Later it was found, that CCDs react sensitive to light. Therefore it became obvious that CCDs are capable to become image sensors if a reasonable amount of pixels are arrayed.
Shortly after the first cameras with that kind of technology appeared. Willard Boyle and George E. Smith were honored with the noble price in physics in 2009, 40 years after they discovered the phenomenon.
Chemiluminescence describes a chemical process where it comes to the transition of electrons from an excited to an energetically lower condition as consequence of a chemical reaction. Energy is emitted in the form of light. The quantities of light emitted are usually very weak.
In contrast to fluorescence (excitation by an external source of light) the condition of excitement is reached by a chemical reaction when describing chemiluminescence.
The principle of chemilumineszenz was at first described by Heinrich Henning Brand in the year 1669.
CFR 21 Part 11
The Food and Drug Administration of the department Health and Human Services, USA, has passed rules, which regulate handling with electronic data and their use. Based on the rules of the current Good Manufacturing Practice, cGMP, written down as 21 CFR 210 and 211. They are binding for all organizations and institutions, which are part of the FDA or have report to this authority. Mainly these are institutions in the fields of pharmacy, food industry and control, and all further departments, that driectly deal with human health. Main topic of the rules of 21 CFR Part 11 is the aquiring of electronic signatures. They should allow ro recognize without any doubt, who, when and which data have been generated, used or altered.
Consequences: Strict, forgery-proof and clear rules to allow any system admission. This means, that even the system to make an admission has to be strictly controlled. All electronic data have to get a signature, which identifies the user. Any kind of additions and changes of electronic data has to generate a furher unique signature. It has to be ensured, that data can't be edited and manipulated from outside, if no unique signature is created.
VILBER has prepared some of the softwares to comply to the rules according to 21 CFR Part 11. It should ease the users to embed their system to their SOPs, that it can be certified and a report posiibility to the FDA is ensured.
Method for measuring the optical density. This can either occur by determination of the darkening (for example a film) or by direct measurement of light. It is possible to calculate the mass of a marked biomolecule under controlled experimental conditions in a CCD-Imager (Quantification).
The dynamic range is a measure for the maximum and the minimum intensity, which can be detected and represented in an image at the same time next to each other. The dynamic range is indicated in grey scale levels. The height of the dynamic range is especially essential if quantifications of the material are to be performed.
Popular chemiluminescent detection system for western blots based on luminol.
Chemical substance which is strongly fluorescing after intercalation into nucleic acids and which can therefore be used for detection of nucleic acid fragments on agarose gels. Ethidium bromide is considered carcinogenic. Alternatives to ethidium bromide are, for example, SYBR® Green, SYBR® Safe or SYBR® Gold.
The term fluorescence describes a process where energy is emitted in form of light through the transition of electrons from a level of high energy to a level of low energy. This light can be measured and quantified with suitable devices . Different to chemiluminescence the excitation to the level of high energy is reached by an external source of light when describing fluorescence.
The frame rate describes the fastest rate with which two following images can be read out and stored.
High Dynamic Range
The dynamic range or dynamic measurement range marks the maximum ratio of contrast in an image. A large dynamic measurement range is necessary to display weak and strong signals besides of each other in one image. A reduced dynamic measurement range causes the disappearance of the weak signals or the simultaneous overexposure of the strong signals. For quantification analyses a HDR regularly offers better data. However, the dynamic measurement range is often mixed up with grey scales!
Image Master Assistent
The Image Master Assistant helps the user to easily generate high-quality data for quantification without having to submit each individual image to an extensive and complicated image analysis. The Image Master Assistant is an integral component of every .CAPT-Software of the computer-assisted systems.
Multiplex Imaging means, that different fluorescently labeled detection antibodies are used in one single experiment, for example on one membrane. Selective detection of the single color channels, prevention, minimization or taking care of possible cross-talk require high demands on the technical platforms and experimental planning.
Near Infrared (NIR) is called the range of electromagnetical spectrum next to the visible range. Infrared (IR) comprises a spectral range of 780 nm to 3 µm.
Fluorescent based Optical Imaging with an NIR-excitation compared to excitation in the visible range (RGB) may exhibit some advantages in terms of autofluorescence.
Electromagnetic radiation can result in the activation of electrons when reaching a metal or a semiconductor surface. This allows a conversion of light in electrical energy. This effect was described as photoelectric effect, first in 1839 by the physician Alexandre Edmond Becquerel. After further studies by Heinrich Hertz (1886), Albert Einstein introduces in 1905 the light quantum, nowadays called photon, within the light quantum hypothesis. Albert Einstein was honored with the noble price in 1921. Annalen der Physik. 322, Nr. 6, 1905, S. 132–148 : Über einen die Erzeugung und Verwandlung des Lichtes betreffenden heuristischen Gesichtspunkt. Albert Einstein.
Qdot® fluorescent dyes
Qdot® fluorescent dyes are nano-crystalline structures which can easily be coupled to a multiplicity of biomolecules due to their special surface. Qdot® fluorescent dyes are characterized among other things by a very long photostability or by defined and relatively narrow excitation and emission spectra, which makes them for example very suited for multiplex applications.
Sterilisation by UV irradiation
The UV irradiation lamps are suited for contamination prevention in research laboratories, food producing industry or in the medical field.
They emit 254 nm UV light, which is well known to be very efficient in the destruction of bacteria, funghi, yeasts and viruses. The UV irradiation lamps are equipped with one, two or three UV tubes with power of 4 up to 40 watts each.
Super-Bright® describes the new generation of UV tables. The underlying technology was developed by Vilber Lourmat. It is based on a completely new filter plate, which is permeable for UV light only and which does not emit visible light any more. Super-BrightT® transilluminators offer many crucial advantages compared to standard transilluminators as for example very clear and homogeneous illumination. Super-Bright® transilluminators are particularly well suited as multi-application devices for many of the fluorescent dyes besides ethidium bromide (e.g. SYBR® Green, SYBR® Orange, SYBR® Gold, SYBR® Red, SYPRO® Ruby, Texas Red).
SYBR® Gold is a fluorescent dye for the labeling of single and double stranded RNA or DNA, which allows a very high sensitivity when detecting the nucleic acid (minimum 10x higher than ethidium bromide, depending of the application). Excitation and emission spectrum of SYBR® Gold. Source: invitrogen
SYBR® Green is an intercalating cyanid fluorescent dye with a very high specificity for double stranded DNA. After intercalation into the nucleic acid a complex of nucleic acid and fluorescent dye is formed which absorbs at a wavelength of ca. 498 nm and emits at a wavelengths of ca. 522 nm. As well, weaker maxima of absorbion are found at 370 nm and 300 nm. SYBR® Green is considered less carcinogen as for example ethidium bromide and is therefore more and more present on the market.
SYBR Safe™ is a fluorescent dye for the detection of nucleic acids, which does not show transforming effects in corresponding experiments with primary cells of hamster. SYBR Safe™ is thus considered an non-carcinogenic alternative to ethidium bromide.
SYPRO® Orange is a fluorescent dye from the SYPRO® group, closely related to SYPRO® Ruby or SYPRO® Red. However, the background signal is frequently a little bit increased compared to e.g. SYPRO® Red.
SYPRO® Red is a fluorescent dye for the labelling of proteins, closely related to SYPRO® Orange. The lowest detection limit for proteins is 1 - 2 ng of protein per band on a mini gel. SYPRO® Red therefore is more sensitive than a Coomassie or a silver stain.
SYPRO® Ruby is a highly sensitive fluorescent dye for the labelling of proteins.
SYPRO® Tangerine is a fluorescent dye for the labelling of proteins with the lowest detection limit of 4 - 8 ng of protein per band on a mini gel.
UV irradiation is an electromagnetic irradiation with a wavelength of approx. 1 nm - 380 nm. It therefore is located between the more short wave X-rays and the more long wave visible light. UV irradiation has been detected by Johann Wilhelm Ritter in the year 1802. UV irradiation usually is divided into UV-A (wavelengths of 320 - 400 nm), UV-B (280-320 nm) and UV-C (200-280 nm).
Western Blot characterizes an established protein chemical method to transfer electrophoretically separated proteins on a membrane (e.g. nylon, nitrocellulose, PVDF). The specific identification detection of the protein of interest on the membrane is done with antibodies which are selective for the protein of interest and in addition have a functional component.
In case of a detection by chemiluminescence a chemical substrate is enzymatically converted (AP or HRP); this chemical reaction emits light as a by-product.
Another option is to use antibody bound fluorophors which can be excited to emit light of a specific wavelength.
With the help of appropriate instruments (e.g. FUSION imagers) those proteins can be qualitatively characterized. The amount of light as an equivalent of the amount of protein can be measured, analysed and evaluated (densitometric quantification).