Microplate readers and spectrophotometers leverage a photomultiplier tube (PMT) to detect fluorescence and luminescence signals.
Photons are emitted at specific wavelengths during these processes. The PMT initially converts photons into electrons, then amplifies the signal, allowing it to be detected and expressed in relative fluorescence/luminescence units (RFU, RLU).
This article focuses on implementing PMT with xenon flash-lamp-based microplate readers, highlighting Molecular Devices’ unique patented AutoPMT™ data normalization functionality.
Understanding PMT
The PMT counts incoming photons that hit the photocathode, converting them into electrons. The electrons are initially deflected, hitting the primary dynode before being amplified over a series of subsequent dynodes (Figure 1).
The total number of generated electrons increases with the gain or voltage applied to the PMT. This number is proportional to the number of incoming photons.
Figure 1. Diagram of a photomultiplier tube (PMT). PMTs convert photons into electrons and amplify the signal. Image Credit: Molecular Devices UK Ltd
Material and Methods
- SpectraMax M5e Multi-Mode Microplate Reader
- 10 μM fluorescein in 1x phosphate-buffered saline (PBS)
- 96-well plate
An initial concentration of 10 μM fluorescein in 1x PBS was used to prepare the standard curve, yielding a final concentration of 0.1 nM fluorescein. This approach was designed to highlight the different aspects of PMT gain and data normalization.
Samples were plated in triplicate, with PBS included to serve as a blank.
The plate was read a total of four times using either the high, medium, low, or auto PMT. This was performed at an excitation wavelength of 485 nm and an emission wavelength of 525 nm, implementing a cutoff filter at 515 nm.
Endpoint measurements were all performed on the SpectraMax M5e Microplate Reader, with RFUs collated and displayed using the SoftMax® Pro 7.1 software.
PMT Gain Adjustment
The PMT gain should be adjusted based on the signal generated by the well to ensure optimal sensitivity and dynamic range for the assay.
Low sample concentrations emit few photons, meaning a higher PMT gain or voltage should be used. High sample concentrations emit more photons, however, meaning they require a lower PMT gain or voltage.
The PMT gain is set manually or automatically to a fixed value on most plate readers and spectrophotometers, and this value is used to read the entire plate.
The SoftMax Pro data analysis software allows a selection of pre-defined PMT gain options, depending on the plate reader model. These include automatic, high, medium, low, and a manual option (Figure 2).
Selecting the Manual PMT Gain option allows a PMT gain value to be set between 200 and 1000 volts for the SpectraMax M Series and the Gemini Microplate Readers, or between 500 and 1000 volts for the SpectraMax i Series Multi-Mode Microplate Readers (i3x, iD3, and iD5).
The high, medium, or low PMT gain options each offer a nominal dynamic range of three decades. This allows the user to select a voltage setting high enough to maximize sensitivity but low enough to avoid PMT saturation (Figure 3).
The ideal PMT gain is the highest value that avoids PMT saturation while offering the highest signal samples. The result is shown as ‘#SAT’ in the SoftMax Pro and the exported data if the PMT cannot detect the signal due to saturation. Reading lower-concentration samples with a low PMT setting results in reduced sensitivity at low signal intensities (Figure 3).
The automatic PMT gain option should be selected in instances where the range of fluorescence intensities within a single microplate is more than three to four orders of magnitude. This option uses the patented AutoPMT™ feature, unique to Molecular Devices M series, Flex, i3x, iDx, and Gemini microplate readers.
This feature allows a wide range of fluorescence signal intensities to be read within the same plate in a single read. The AutoPMT™ feature automatically determines the optimal gain for each well.
A pre-read of the microplate is completed at high PMT. The reads are then performed at high PMT if no wells saturate; if any wells do saturate, they will be read at medium PMT. If any wells still saturate at medium PMT, they are read with low PMT.
This means that a dynamic range of six or higher can be automatically acquired in a single plate read using AutoPMT™ mode (Figure 3).
Figure 2. Adjustable PMT Settings in SoftMax Pro. Image Credit: Molecular Devices UK Ltd
Figure 3. Standard curve with pre-defined PMT settings compared to AutoPMT™. A. High PMT gain shows a quick saturation of the PMT at a concentration of 33 nm. B. Medium PMT gain shows saturation at a concentration of 333 nM; PMT Low can read the full concentration range but loses sensitivity at low concentration. D. Automatic PMT allows maximal dynamic range with high sensitivity at all concentrations. Image Credit: Molecular Devices UK Ltd
Data Normalization
If the same samples are read with different PMT gains, the resulting unnormalized RFU values will differ for the same sample, leading to different standard curves (Figure 4A).
Samples read with a higher PMT gain will report higher RFU values than the same samples read at a lower PMT gain. Depending on the concentration range used on the plate, differences in RFU values pose challenges when comparing data from the same assay.
It is important to normalize RFU to ensure it becomes independent of the voltage.
Molecular Devices’ range of microplate readers employs a PMT calibration coefficient determined by measuring a fluorescent sample of known intensity to normalize the signal. This sample is fixed within the instrument.
The SoftMax Pro software offers full, automatic data normalization (Figure 4B).
No additional data adjustment is required after the measurements. This rapid, automatic normalization saves time and is easy to use because it is completed without the need for multiple measurements in different PMT settings.
The collected data is simultaneously normalized to achieve full linearity over the maximum dynamic range.
A key advantage of this normalization is evident when using kinetic assays, where signal intensities change over time, from day to day, or under different assay conditions.
The PMT settings must be fixed for kinetic reads, and it is advisable to begin with a medium PMT. There is an option to adjust this to low PMT if some wells saturate, or to high PMT if a low signal is generated by all wells.
Kinetic curves and final results can be compared across experiments with different PMT gains because the collected data is normalized simultaneously.
Figure 4. Standard curve read at three different PMT gains (325 V, 425 V, and 525 V) without data normalization (A. Un-normalized RFU) and with data normalization (B. Normalized RFU). C. Graph showing the data normalization for each PMT voltage when the Automatic PMT option is used. Image Credit: Molecular Devices UK Ltd
Conclusion
It is well known that results vary slightly from instrument to instrument and day to day due to factors such as the sensitivity of the PMT, the lamp, and the monochromator’s efficiency.
Microplate readers with AutoPMT™ functionality compensate for all these factors, leveraging a combination of instrument design, calibration, and full RFU normalization to acquire comparable high-quality fluorescence data and optimum reproducibility over time.
About Molecular Devices UK Ltd
Molecular Devices is one of the world’s leading providers of high-performance life science technology. We make advanced scientific discovery possible for academia, pharma, and biotech customers with platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. From cancer to COVID-19, we've contributed to scientific breakthroughs described in over 230,000 peer-reviewed publications.
Over 160,000 of our innovative solutions are incorporated into laboratories worldwide, enabling scientists to improve productivity and effectiveness – ultimately accelerating research and the development of new therapeutics. Molecular Devices is headquartered in Silicon Valley, Calif., with best-in-class teams around the globe. Over 1,000 associates are guided by our diverse leadership team and female president who prioritize a culture of collaboration, engagement, diversity, and inclusion.
To learn more about how Molecular Devices helps fast-track scientific discovery, visit www.moleculardevices.com.
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