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Basic information about absorbance microplate readers
An absorbance microplate reader, (longform: absorbance microplate reader; shortform: absorbance reader), is a piece of equipment capable of detecting and quantifying the light photons absorbed/transmitted by a liquid sample present in a microplate, when exposed to light at a specific wavelength.
As opposed to fluorescence and luminescence, absorbance detection is an absolute measurement quantified in Optical Densities (ODs). OD is defined as the logarithmic ratio between the intensity of light hitting a sample and the intensity of the light transmitted through the sample. Alternatively, transmission, the portion of light that passes the sample, can also be used.
Absorbance microplate readers, also called photometer plate readers, are used for different applications in various fields, such as academic life science research, drug discovery and screening, clinical laboratories, synthetic biology and food or water quality monitoring.
Compared to a standard spectrophotometer that measures one sample at a time in a cuvette, an absorbance microplate reader can measure the same application with higher throughput. It can measure a full 96 well microplate in seconds, or 384 or 1536 well plates in a few minutes.
What is an absorbance microplate reader?
An absorbance microplate reader is quite a complex instrument, optically speaking. Because of the nature of the signal, these microplate readers require an excitation light source, optics for the selection of the incoming wavelengths, a detector, and a reference channel.
The sample is illuminated by light with a specific intensity. The total light intensity the sample is exposed to is assessed by a reference channel measured ahead of the sample. The light detector is located on the opposite side of the microplate relative to the light source. It measures how much of the total light intensity is transmitted through the sample and at which wavelengths. Light that does not pass through to the detector is absorbed by the sample.
The measured amount of transmitted light can be typically used to calculate the concentration of a molecule of interest, since the amount of light absorbed by a sample relates to its concentration (Beer-Lambert law). Alternatively, a standard curve in parallel to samples of unknown concentrations can be used. This way nucleic acids, proteins and specific molecules via ELISA can be quantified.
Absorbance microplate readers are available as stand alone, dedicated readers or, as part of multi-mode microplate readers, when combined to fluorescence intensity and/or luminescence detection.
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What to consider when choosing an absorbance microplate reader?
If you are on the market for a new absorbance microplate reader, there are a few factors to take into account, as the performance of the instrument can significantly affect the quality of your research data.
A wavelength selection capability is mandatory for an absorbance microplate reader and is dependent on the type of detectors that are used.
Two different types of detectors are commonly used: PhotoMultiplier Tubes (PMTs) or Charge Coupled Device (CCD) spectrometers.
The PMT-based microplate reader requires a wavelength selection tool between light source and sample. Optical filters or monochromators select the desired wavelength before the light is sent to the sample. This way, only the light at a desired wavelength is transmitted through the sample, reaches the PMT, and is quantified.
To scan spectra or a range of wavelengths, the PMT-based microplate reader requires a monochromator. This selects the desired wavelengths and measures them sequentially upon mechanical movement. For measurements of multiple discrete wavelengths, multiple single measurements with different filters or a monochromator scan are required.
The CCD-spectrometer based microplate reader does not require a wavelength selection tool. The spectrometer separates the different wavelengths present in the light transmitted through the sample as these are directed onto the CCD detector. This captures the intensity of all wavelengths at once. This way, spectra, single, or multiple discrete wavelengths can be acquired in a short amount of time.
If your application needs spectral acquisition, CCD spectrometers are significantly faster than monochromators, as mechanical spectral scanning is not required.
BMG LABTECH instruments are equipped with a UV/vis spectrometer for absorbance measurements. This captures a full range spectrum from 220 to 1000 nm in less than a second/well.
Compatible microplate formats
Commonly in life science applications, basic absorbance assays like ELISA are measured in 96 well microplates. However, if you have multiple samples and/or wish to save precious reagents or time, 384 and 1536 well formats can also be used. Just make sure that your absorbance microplate reader of choice is also capable of reading the microplate format you plan on using.
Shaking and incubation
When looking for an absorbance microplate reader, you should consider the nature of your research and of the assay you want to run. Generally, shaking and incubation are two important features. Incubation is critically important when running enzymatic assays at stable temperatures, e.g. 37° C. Multiple shaking options allow a proper mixing of the samples. For applications like cell growth assays in bacteria or yeast (OD600), shaking is crucial for a good oxygenation and consequent fast cell replication.
Compatibility with cuvettes
Traditionally, absorbance measurements were performed in cuvettes with a normalized path length of 1 cm. In microplates, the path length changes dependent on volume and plate format. If aqueous solutions are measured, the microplate measurement can be normalized by using the water-peak path length correction. If despite this option, for your research you still occasionally require measuring samples in cuvettes, make sure that the microplate reader of choice has either an integrated cuvette port like the SPECTROstar Nano, or plate adapters for measuring cuvettes horizontally like the LVis Plate.
Software for data reduction
Generally, the software that runs the microplate reader is a very underestimated part of the package. For some reader, the software only controls the measurement process and provides a raw data output. In other cases, it can perform complex data analysis.
A comprehensive, easy-to-use and flexible software can make life easier, simplify detection and data analysis so that you can get the most out of your instrument.
The most useful calculation features include automatic blank subtraction, automatic calculation of sample concentrations based on standard curves for ELISA assays or on the Beer-Lambert law, 260/280 ratios, path length corrections, and enzymatic constants such as Km and Vmax.
On the BMG LABTECH software package, the most common assays have predefined quick-run assay-specific protocols and dedicated data reduction templates. Through these dedicated solutions, data acquisition and analysis are performed with a single mouse click, enabling researchers to carry out experiments quickly and easily.
Our absorbance microplate readers
Absorbance detection can be performed on the PHERAstar FSX, CLARIOstar Plus, FLUOstar, POLARstar and SPECTROstar Omega, and SPECTROstar Nano plate readers.
All our absorbance microplate readers are equipped with a high-intensity xenon flash lamp and CCD spectrometer.
BMG LABTECH was the first company to employ a UV/vis spectrometer instead of filters or monochromators for absorbance detection. Upon illumination of the sample by a high-power xenon flash lamp, BMG LABTECH´s ultra-fast UV/vis spectrometer collects the entire spectrum instead of a specific wavelength. It captures all wavelengths from 220 to 1000 nm at a resolution of 1 – 10 nm in less than 1 second per well.
Alternatively, filter-based absorbance detection (PMT-based) is available on the FLUOstar Omega.
Which assays can be measured by an absorbance microplate reader?
Absorbance is one of the most popular detection modes for life science applications. Accordingly, there is a multitude of kits and reagents available on the market for the most different application and assay types that an absorbance microplate reader can cover both as single- or multi-mode instrument. Several colorimetric assays have been adapted or directly developed for use in a microplate reader.
One of the most common applications is DNA/RNA quantification at 260 nm. It is also possible to quantify a specific protein in solution, typically by enzyme-linked immunosorbent assay (ELISA). Several colorimetric methods are available to measure the whole protein content of a sample. In our blog article “Protein measurement: find a suitable method” you will find detailed descriptions of Bradford, BCA and other methods and tips to help you choose.
Another application absorbance microplate reader can measure is cell growth in bacteria and yeast by measuring the “optical density at 600 nm” (OD600) or cell viability by colorimetric assays.
Examples of measurements with BMG LABTECH absorbance microplate readers:
The following are examples of absorbance measurements taken on BMG LABTECH microplate readers: