Picking the perfect plate

With a wealth of microplates on the market, it’s hard to know which one to chose. Steven Knight provides tips on how to pick the best plate for your application

Today most biological assays are conducted in the universally-accepted microplate format. Since their introduction 30 years ago, “microtitre” plates have grown in popularity as they provide a convenient, standardised and high density format for handling large numbers of samples. In Life Science research, the microplate has become ubiquitous, yet there are many different types and styles of microplate all designed to optimise certain parameters. This might be cell adhesion, chemical resistance or the spectroscopic measurement technique in use. Choosing the correct microplate for your application can mean the difference between indifferent and great results.

There are three basic methods of obtaining useful optical data from microplate-based samples. The simplest method is absorbance measurement. Following the Beer-Lambert law, concentration of a given compound in solution is directly proportional to the quanta of light absorbed at a given wavelength and at a constant optical path-length. Thus a beam of monochromatic light can be directed into a microplate well and the transmitted or absorbed light can be measured. A simple calculation gives the relative concentration in each well. The majority of laboratory microplate readers use this method.

Where greater sensitivity is required, fluorescence measurements are preferred. In this case an excitation beam of a given wavelength is directed into the well. The fluorophore within the compound of interest absorbs photons from the incident light and re-emits photons of lower energy, which consequently appear at a longer wavelength. In a fluorimeter, simple cut-off filters can be used to remove reflected excitation wavelengths from the detected light, leaving only the emission wavelength. Sensitivity can be ten times greater than simple absorbance measurements. Greater sensitivity can be achieved by reducing the band-width of both excitation and emission wavelengths using monochromators. Such spectrofluorimeters can offer another order of magnitude increase in sensitivity.

The third method involves luminescence. Bioluminescence is a naturally occurring phenomenon exhibited by certain animal and plant species which can emit light through one of two common mechanisms, the Aequorin and Luciferin pathways.  Firefly beetles (Lampyridae), the marine copepod Metridia Longa and the Sea Pansy (Renilla Reniformis) all contain enzymes in the class luciferase which catalyse the oxidation of Luciferin substrate in the presence of ATP resulting in the emission of light. In luminescent jellyfish of the family Aequorea, the substrate Aequorin is excited in the presence of coelenterazine and Ca2+ ions. In returning to the ground state, blue light is emitted. These are biological adaptations of a process which can also be seen as purely chemically-driven reactions, in which case it is referred to as chemiluminescence.  Detection is normally in a luminometer or a multi-label reader, which is a multiple purpose spectrometer capable of reading in absorbance, fluorescence or luminescence modes.

Microplate readers are designed to read from either the top or the bottom of a microplate. Bottom reading instruments tend to be more complex and expensive, but give better results for sensitive assays, relying as they do, on transmitted rather than reflected light.

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 with monochromatic light 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. These may have plastic, glass, quartz or some other clear polymer base. The requirement is to transmit the light wavelengths of interest.

Visible wavelength range (900-335nm) measurements require only clear plastic bases, whilst readings between 220nm and 335nm 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 end-point determination with relatively high absorbance. They can be sourced from most laboratory plastics manufacturers worldwide and their simple design allows cheapness of manufacture and robustness in use. 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.

However, for more demanding applications, simple ELISA-type plates will not do. Microplates for use in assay development and high throughput screening are usually manufactured from ultra-pure polystyrene. To this is added an optical brightener, such as titanium dioxide, to increase the reflectivity of the plate surface for white plates. In addition, the steel tools used to mould these plates are highly polished to give a very smooth bright surface inside the wells. These attributes ensure that emitted light within the wells is reflected back up towards the top of the micro well. This is where the sensitive optical detector of the measuring instrument is positioned during the reading cycle.

For fluorescence measurements, “quenching” the natural auto-fluorescence of the polystyrene substrate is achieved by adding carbon black to the mix. This is very effective in fluorescence assays and gives much better results than a white plate in these cases. Again, all the internal surfaces are polished to increase reflectivity.

Difficulties can arise when using luciferase assays in screening where a high dynamic range is observed across the plate. In such cases, adjacent wells may exhibit very strong or very weak signals and this can lead to optical cross-talk between the wells and consequently to erroneous results. A certain amount of visible light can “leak” through the white plastic walls of the plate and is erroneously detected in the adjacent cells as an additional, albeit low, signal. The error is worst when reading a low level signal adjacent to a far brighter well. Although plate designs using separated “chimney” wells can help to reduce this, they cannot eliminate it. In a 96- “chimney well” plate each well has individually formed well walls which are separated from each other and held in a “web” of struts across the plate.

To overcome this problem of light-piping by the white plastic, Porvair Sciences developed a unique patented 96-well Black & White plate. A black polystyrene plate matrix with individual white cells moulded into it at the time of production.

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.

With the proliferation of cell-based assays in recent years, a need has arisen for plasticware on which adherent cell lines can grow efficiently. Using Polystyrene plasticware and applying a surface treatment to the plastic is found to increase the adhesion of cells. This is known as tissue culture treatment. The T/C treatment can be effected in different ways, but the most common are a high-voltage arc discharge into the wells of the plate and incubation in a gas plasma T/C chamber. The ionised gas disrupts the polymer surface and lays down functional groups which better enable the cells to adhere to the plastic. Varying the gas mixture can lead to surface treatments optimised for different cells, proteins or for other biomolecules to adhere.

In the case of proteins, plates are said to be low-, medium- or high- protein binding according to the treatment they have undergone. Some of these will have physical modifications to the plate surface as described above, whilst others may involve the laying down of a coating over the plastic. Popular coatings include Streptavadin (for binding of Biotinylated compounds), collagen and poly-d-lysine. Plates coated with biomolecules may have a restricted shelf life.

There are many different manufacturers of assay microplates and between them they offer a bewildering variety of plate types. It is therefore not surprising that many scientists are unsure about the type of plate that will give them the best results.  By carefully selecting the correct plate for the assay, it is possible to significantly improve results and when comparing fluorescence measurements in black plates with the same assay in white plates, results are improved by as much as an order of magnitude.

By following the simple guidelines set out here and ensuring that only quality plates from reputable manufacturers are in use, those tasked with assay development can ensure that their final assay has the best possible chance of success.

Choosing the correct assay plate - a summery of the choices available for assay plate selection