Optimization systems in a scanning fluorometer
First Claim
1. A method of obtaining a wide dynamic range in a scanning fluorometer, the method comprising the steps of:
- multiplexing an output from a photomultiplier tube detector in said scanning fluorometer through a plurality of gain amplifiers, each of said plurality of gain amplifiers providing a different level of gain, wherein said plurality of gain amplifiers includes a high gain amplifier providing the highest gain of said plurality of gain amplifiers;
determining a maximum photomultiplier tube detector supply voltage, wherein said maximum photomultiplier tube detector supply voltage provides an output signal from said high gain amplifier near a maximum value for a low reference; and
determining at least one additional photomultiplier tube detector supply voltage, wherein said at least one additional photomultiplier tube detector supply voltage is lower than said maximum photomultiplier tube detector supply voltage.
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Abstract
A method and apparatus for determining the fluorescence, luminescence, or absorption of a sample is provided. The sample may either be contained within a cuvette or within one or more sample wells within a multi-assay plate. A combination of a broadband source, monochromator, and a series of optical filters are used to tune the excitation wavelength to a predetermined value within a relatively wide wavelength band. A similar configuration is used to tune the detection wavelength. In one aspect, multiple optical filters are coupled to the excitation source subassembly, thus allowing the system to be quickly converted from one optical configuration to another. In another aspect, the excitation light and the detected sample emissions pass to and from an optical head assembly via a pair of optical fibers. In another aspect, an optical scanning head assembly is used that includes mirrored optics for coupling the excitation source to the sample and the emitted light to the detector. In another aspect, time tags are recorded for samples contained within a multi-assay plate to monitor compositional time dependent properties or to ensure the accurate comparison of individual samples.
56 Citations
29 Claims
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1. A method of obtaining a wide dynamic range in a scanning fluorometer, the method comprising the steps of:
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multiplexing an output from a photomultiplier tube detector in said scanning fluorometer through a plurality of gain amplifiers, each of said plurality of gain amplifiers providing a different level of gain, wherein said plurality of gain amplifiers includes a high gain amplifier providing the highest gain of said plurality of gain amplifiers;
determining a maximum photomultiplier tube detector supply voltage, wherein said maximum photomultiplier tube detector supply voltage provides an output signal from said high gain amplifier near a maximum value for a low reference; and
determining at least one additional photomultiplier tube detector supply voltage, wherein said at least one additional photomultiplier tube detector supply voltage is lower than said maximum photomultiplier tube detector supply voltage. - View Dependent Claims (2, 3, 4, 5, 6)
introducing said low reference into a measurement zone of said scanning fluorometer;
irradiating said low reference with light from an excitation source; and
monitoring said photomultiplier tube detector output for said low reference.
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5. The method of claim 4, wherein said low reference is a polystyrene sample.
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6. The method of claim 1, wherein said plurality of gain amplifiers is comprised of four gain amplifiers.
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7. A method of scanning a multi-well assay plate with a scanning fluorometer, the method comprising the steps of:
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multiplexing an output from a photomultiplier tube detector in said scanning fluorometer through a plurality of gain amplifiers, each of said plurality of gain amplifiers providing a different level of gain, wherein said plurality of gain amplifiers includes a high gain amplifier providing the highest gain of said plurality of gain amplifiers;
determining a maximum photomultiplier tube detector supply voltage, wherein said maximum photomultiplier tube detector supply voltage provides an output signal from said high gain amplifier near a maximum value for a low reference;
determining at least one additional photomultiplier tube detector supply voltage, wherein said at least one additional photomultiplier tube detector supply voltage is lower than said maximum photomultiplier tube detector supply voltage, and wherein a first output range from said plurality of gain amplifiers when said photomultiplier tube detector supply voltage is set at said maximum supply voltage overlaps with a second output range from said plurality of gain amplifiers when said photomultiplier tube detector supply voltage is set at said at least one additional supply voltage;
scanning a plurality of wells of said multi-well assay plate with said scanning fluorometer a first time, wherein each scanned well of said multi-well assay plate is irradiated with light from an excitation source, wherein said photomultiplier tube detector supply voltage is set at said maximum photomultiplier tube detector supply voltage during said first scan;
monitoring said photomultiplier tube detector output for each scanned well of said multi-well assay plate;
optimizing said photomultiplier tube detector output with said plurality of gain amplifiers;
tagging wells of said plurality of wells of said multi-well assay plate when said photomultiplier tube detector output is outside of an acceptable range for all of said plurality of gain amplifiers;
at least scanning said tagged wells of said plurality of wells with said scanning fluorometer a second time, wherein each of said at tagged wells of said multi-well assay plate is irradiated with light from said excitation source, wherein said photomultiplier tube detector supply voltage is set at said at least one additional photomultiplier tube detector supply voltage during said second scan;
monitoring said photomultiplier tube detector output for at least said tagged wells of said plurality of wells; and
optimizing said photomultiplier tube detector output with said plurality of gain amplifiers for at least said tagged wells of said plurality of wells. - View Dependent Claims (8, 9, 10, 11, 12)
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13. A method of scanning a multi-well assay plate with a scanning fluorometer, the method comprising the steps of:
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selecting a photomultiplier tube detector supply voltage;
multiplexing an output from said photomultiplier tube detector in said scanning fluorometer through a plurality of gain amplifiers, each of said plurality of gain amplifiers providing a different level of gain;
scanning a plurality of wells of said multi-well assay plate with said scanning fluorometer, wherein each scanned well of said multi-well assay plate is irradiated with light from an excitation source;
monitoring said photomultiplier tube detector output for each scanned well of said multi-well assay plate;
optimizing said photomultiplier tube detector output with said plurality of gain amplifiers; and
processing said optimized photomultiplier tube detector output.
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14. A method of scanning a multi-well assay plate with a scanning fluorometer, the method comprising the steps of:
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multiplexing an output from a photomultiplier tube detector in said scanning fluorometer through a plurality of gain amplifiers, each of said plurality of gain amplifiers providing a different level of gain, wherein said plurality of gain amplifiers includes a high gain amplifier providing the highest gain of said plurality of gain amplifiers;
determining a maximum photomultiplier tube detector supply voltage, wherein said maximum photomultiplier tube detector supply voltage provides an output signal from said high gain amplifier near a maximum value for a low reference;
determining at least one additional photomultiplier tube detector supply voltage, wherein said at least one additional photomultiplier tube detector supply voltage is lower than said maximum photomultiplier tube detector supply voltage, and wherein a first output range from said plurality of gain amplifiers when said photomultiplier tube detector supply voltage is set at said maximum supply voltage overlaps with a second output range from said plurality of gain amplifiers when said photomultiplier tube detector supply voltage is set at said at least one additional supply voltage;
scanning a plurality of wells of said multi-well assay plate with said scanning fluorometer a first time, wherein each scanned well of said multi-well assay plate is irradiated with a single flash of light from an excitation source, wherein said photomultiplier tube detector supply voltage is set at said at least one additional photomultiplier tube detector supply voltage during said first scan;
monitoring said photomultiplier tube detector output for each scanned well of said multi-well assay plate;
determining a preferred photomultiplier tube detector supply voltage for each scanned well of said multi-well assay plate;
scanning said plurality of wells of said multi-well assay plate with said scanning fluorometer a second time, wherein each scanned well of said multi-well assay plate is irradiated with a predetermined number of flashes flash of light from said excitation source, wherein said photomultiplier tube detector supply voltage is set at said preferred photomultiplier tube detector supply voltage during said second scan; and
monitoring said photomultiplier tube detector output for said plurality of wells for said second scan. - View Dependent Claims (15)
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16. A wide dynamic range scanning fluorometer, comprising:
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a sample testing region;
a source of excitation light coupled to said sample testing region;
a photomultiplier tube detection system coupled to said sample testing region;
a scanning mechanism, said scanning mechanism moving said excitation light source and said photomultiplier tube detection system relative to said sample testing region along at least one axis;
a plurality of gain amplifiers coupled to said photomultiplier tube detection system, wherein each of said plurality of gain amplifiers provides a different level of gain, said plurality of gain amplifiers providing a range of gain;
a photomultiplier tube power supply coupled to said photomultiplier tube, said power supply providing at least a first voltage and a second voltage to said photomultiplier tube, wherein a first gain range from said plurality of gain amplifiers when said power supply provides said first voltage overlaps a second gain range from said plurality of gain amplifiers when said power supply provides said second voltage; and
a multiplexer coupled to said plurality of gain amplifiers, said multiplexer determining which of said plurality of gain amplifiers is coupled to a data processing system. - View Dependent Claims (17, 18, 19, 20, 21)
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22. A method of optimizing a fluorometer prior to performing a sample scan, the method comprising the steps of:
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determining an expected emission peak;
selecting a first emission system wavelength, wherein said first emission system wavelength is higher than said expected emission peak;
scanning an excitation source from a first excitation source wavelength to a second excitation source wavelength, wherein both said first excitation source wavelength and said second excitation source wavelength are less than said first emission system wavelength;
determining a peak excitation wavelength between said first excitation source wavelength and second excitation source wavelength;
fixing said excitation source at a third excitation source wavelength, said third excitation source wavelength between said first excitation source wavelength and said second excitation source wavelength;
scanning an emission system from a second emission system wavelength to a third emission system wavelength;
determining a peak emission wavelength between said second emission system wavelength and said third emission system wavelength;
selecting a preferred emission filter;
fixing said preferred emission filter; and
fixing said emission system at a fourth emission system wavelength. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29)
sequentially inserting a plurality of emission filters into said fluorometer;
measuring a signal-to-background ratio for each emission filter of said plurality of emission filters; and
determining said preferred emission filter from said plurality of emission filters, wherein said preferred emission filter has an optimal signal-to-background ratio.
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29. The method of claim 22, wherein said fourth emission system wavelength is approximately 10 nanometers greater than a cutoff associated with said preferred emission filter.
Specification