There are three basic methods of obtaining useful optical data from microplate-based samples. The simplest method is absorbance measurement. Where greater sensitivity is required, fluorescence measurements are preferred. Sensitivity can be ten times greater than simple absorbance measurements. The third method involves luminescence, a naturally occurring phenomenon exhibited by certain animal and plant species which can emit light.
Microplate readers are designed to read from either the top or the bottom of a microplate. Top reading instruments rely on measuring reflected light above the wells. A good solid bright white plate is best for these absorbance measurements and a solid black plate for fluorescence readings. Bottom reading units illuminate the sample from above and then use detectors placed below the plate to measure the absorption or fluorescence/luminescence emission. This necessitates the use of clear-bottomed plates. The requirement is to transmit the light wavelengths of interest. Visible wavelength range (900-350nm) measurements require only clear plastic bases, whilst readings between 220nm and 350nm will require a UV-transparent material. This can be Quartz sheet or a modern polymer such as Cyclo-Olefin Co-Polymer (COP/COC).
Optical glass sheet is used where visible range detection is combined with confocal optics or whole plate imaging which requires a very clear uniformly-flat base.
Simple 96-well microplates for ELISA type assays are made from solid clear polystyrene with no additives. These are adequate for clinical and diagnostic tests, ELISA assays and any colour endpoint determination with relatively high absorbance. Typically they are available with flat well bottoms, giving high surface area, round well bottoms for good mixing or V-wells for high liquid recovery.
Crosstalk can also be an issue in bottom-reading absorbance and fluorescence measurements. To address this applications challenge, Porvair Sciences has developed the Krystal 2000 zero-crosstalk plates in which individual clear wells are moulded into either a white or black matrix. The black or white base material also projects down below the clear well bottom to further reduce the possibility of crosstalk.
By carefully selecting the correct plate type for the assay, it is possible to significantly improve results. By following the simple guidelines set out here, those tasked with assay development can ensure that their final assay has the best possible chance of success.
The range of clear or opaque solid bottom assay plates is specifically designed for absorbance, fluorescence, luminescence and scintillation applications. The design uses standard ‘chimney’ wells to overcome optical crosstalk and contamination. These plates are designed to give optimum results from any instrument which reads from the top of the plate. The acclaimed composite black/white plate has been shown to be ten times more sensitive than a standard white plate for chemi-luminesence assays.
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The Krystal series clear bottom plates in 24-, 96- and 384-well configuration are now recognised as industry-leading tools for Pharma drug discovery and screening. Our standard clear polystyrene bottom plates have bases just 0.4mm thick and are moulded from highly polished tools to reduce flow lines, artefacts and scratches, guaranteeing good results with all types of bottom-reading instruments. For the more modern demanding confocal readers, the Glass Bottom plates have been shown by leading research institutes to be unsurpassed for flatness, linearity and optical clarity. Our sparkling pure borosilicate glass is just 175μm thick with a flatness tolerance across the plate of +/- 15μm ensuring superb crisp images every time. We supply our Krystal clear plates treated for cell culture with a low pressure gas plasma that produces an even surface treatment right across the plate. It is particularly suited to cell lines which do not grow easily on plastics.
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Manufactured for whole-plate CCD imaging and laser detection applications, Krystal glass bottom plates consist of a polystyrene upper part and a 175μm thick clear borosilicate glass sheet fixed to the base with a proprietary adhesive. This process results in consistent flatness of the base and gives improved light transmission whilst maintaining a flat optical plane for growing cells. The nominal cut-off wavelength of 335nm allows most fluorescence assays to be excited or read through the glass bottom. All plates are supplied lidded.
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Recently, scientists have begun using assay chemistries which require excitation or detection wavelengths in the far UV region, below 350nm. To enable our customers to develop assays in this area, Porvair Sciences has introduced a very high specification range of COP-bottomed, UV-transparent microplates. Precision engineered and assembled, these plates are available in 96- and 384-well black matrix only for the most sensitive UV range fluorescence assays using whole-plate imaging or confocal microscopy.
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Filtration plates are used in their simplest form to remove particulate matter from liquid. Either the particulate matter or the filtrate is needed for further study. Porvair Sciences has a range of filter plates to suit most filtration applications. The company has optimised filter plates for applications including cell harvesting, DNA separations, binding studies, Plasmid isolation, general filtration and sample clean up. Porvair has a full range of 48-, 96- and 384-well microplates with a choice of glass fibre, nylon, PVDF or polyethylene filtration materials and well volumes ranging from 350μl to 5ml.
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PCR plates are made from polypropylene for extra rigidity. These plates are compatible with the majority of 96- and 384-well block PCR and sequencing instruments, including FAST sequencers. Our expanded range includes traditional no-skirt, half-skirt and fully skirted plates with standard 350μl wells and in addition 200μl Low Profile versions of these. The latest ‘elevated’ or ‘chimney’ well plates are also catered for with several new products. To ensure full compatibility with robotic systems, these new DNase/RNase- and pyrogen-free thermal cycler plates feature high rigidity to minimise distortion before and after thermal cycling.
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Bacti-growth plates are specially packaged plates to allow the growth of bacteria, yeast, mammalian or insect cell lines. Using only the highest-grade raw materials, our plates are tested to ensure no moulding or polymer contaminants leach out into the samples. This guarantees reproducibility and maintains customer confidence. Each Bactigrowth plate consists of a deep well plate and a tight fitting lid, packed sterile in bags.
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