WO2024144798A9 - Hybrid fluorescence-absorbance measurement system and method for sub-microliter and sub-nanoliter volume measurement - Google Patents

Abstract

A hybrid system and method for calibrating a liquid dispensing apparatus combining fluorescence and absorbance information. At least one fluorophore and at least one chromophore are inserted into a sample solution that is dispensed. The fluorescence and absorbance measurements are combined to take advantage of the positive attributes of each. The invention includes a method for establishing a traceability of the fluorescence measurements through a range of volumes by correlating the fluorescence measurements with stable absorbance measurements. A diluent may be added to wells of a microtiter plate before or after adding the sample solution. The diluent does not contain the fluorophore and may include chromophore that is not in the sample solution to establish the stable absorbance measurements. The hybrid system and method may be used to carry out daily traceability information by determining instrument drift with absorbance measurements and accounting for that instrument drift in the fluorescence measurements.

Description

HYBRID FLUORESCENCE-ABSORBANCE MEASUREMENT SYSTEM AND METHOD

FOR SUB-MICROLITER AND SUB-NANOLITER VOLUME MEASUREMENT

BACKGROUND OF THE INVENTION

1. Field of the Invention.

[0001] The present invention relates to systems and methods for calibrating liquid handling devices. More particularly, the present invention relates to systems and methods of volume measurement in a wide range of volumes. Still more particularly, the present invention relates to systems and methods of accurate volume measurements in the nanoliter and picoliter volume range using a combination of fluorescence and absorbance analysis techniques.

2. Description of the Prior Art.

[0002] Accurate measurement of sub-microliter and sub-nanoliter volumes is of increasing interest for biopharma, clinical diagnostic, and other life sciences laboratories. Liquid delivery devices that dispense individual droplets in the picoliter and nanoliter volume range have been developed over the past 20 years and are becoming common tools. With their increased use comes the requirement for optimizing their performance, which is only achieved by measuring the volume dispensed by these liquid delivery devices. Hence, what is needed is a method and system for accurately measuring volumes in the sub-microliter (nanoliter) and sub-nanoliter (picoliter) volume ranges.

[0003] Artel, Inc. of Westbrook, Maine, has for years provided systems and methods to calibrate liquid handling devices with great accuracy at lower and lower volumes. Dual-dye ratiometric absorbance methods implemented in both the Artel PCS® and Artel MVS® systems provide measurements with low uncertainties down to about 30-100 nanoliters (nL). At volumes lower than this, the dye concentration being measured becomes sufficiently low that the uncertainties associated with the measurement become large. Thus, using absorbance spectroscopy alone does not provide a good measurement platform for measuring such low volumes.

[0004] Fluorescence spectroscopy has been used for some time to attain low volume measurements. One advantage of using fluorescence is the inherent sensitivity of this method, which results in a much lower limit of detection than can be measured using absorbance spectroscopy (i.e., much lower concentrations of dye can be measured using fluorescence spectroscopy). Another advantage is that fluorescence spectroscopy and fluorescent dyes can provide measurable signals over a very large dynamic range, much larger than absorbance dyes are capable of producing, which results in the need for fewer test solutions (i.e., a standard absorbance dye can cover a dilution range of approximately 1 :5 to 1 : 10, but a fluorescent dye can cover a dilution range of 1 :300 to 1 :7,000 using one detector gain setting, and a 1 : 1,000,000 dilution range or higher when using different detector gain settings. In principle this means that one fluorescent dye solution of a given concentration would be needed to measure the same range of dilutions as several absorbance dyes of increasing concentration) . However, the disadvantage of fluorescence is the well-known instability of the fluorescent signal from dye solutions, and the instability of fluorescence readers. What is needed is a system and method that combines the advantages of fluorescence and absorbance spectroscopies to achieve reliably accurate volume measurements at sub-microliter and sub-nanoliter volumes. In particular, what is needed is a system and method that combines the advantages of fluorescence and absorbance spectroscopies to attain the limit of detection and large dynamic range of fluorescent dyes with the traceability and stability of absorbance dyes.

SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide a system and method that combines the advantages of fluorescence and absorbance spectroscopies to achieve reliably accurate volume measurements at sub-microliter and sub-nanoliter volumes. The present invention combines fluorescence and absorbance spectroscopies to create a method and a system that can attain the limit of detection and large dynamic range of fluorescent dyes with the traceability and stability of absorbance dyes. That is, the present invention is a hybrid absorbance-fluorescence system and a related method for accurate, reliable volume measurements at sub-microliter and sub- nanoliter volumes.

[0006] Two embodiments of the system and method of the invention are described. In a first embodiment, the method provides the average dispense volume calculated from a summation of multiple dispenses and an estimate of the coefficient of variation of the individual dispenses. In a second embodiment, the method provides quantitation of individual dispense volumes and the coefficient of variation for those dispenses. The method described in the second embodiment uses an overlap of the absorbance and fluorescence response ranges to calibrate the fluorescence response using the highly accurate and traceable absorbance result. Both embodiments rely upon a system comprising sample solutions that contain a mixture of fluorescence and absorbance dyes. Target volumes of these sample solutions are dispensed into wells of a microtiter plate by a liquid delivery device under test. A diluent or baseline solution is also dispensed into the plate wells with the sample either before or after sample addition, and the solutions are mixed, creating a dilution of the sample solution. Absorbance and fluorescence measurements are collected for each filled well, and these measurements are used to determine the volume of sample solution dispensed by the device under test.

[0007] The present invention includes a method for calibrating a liquid dispensing apparatus by measuring a volume of a sample solution through a selectable range of volumes dispensed by the liquid dispensing apparatus into one or more wells of a microtiter plate. The method includes the steps of dispensing the sample solution into each of the one or more of the wells of the microtiter plate, wherein the sample solution includes one or more chromophores and one or more fluorophores, measuring with a spectrometer fluorescence of the sample solution in the one or more wells to obtain one or more fluorescence measurements, measuring with the spectrometer absorbance of the sample solution to obtain one or more absorbance measurements, and calculating a volume dispense size of the dispensed sample solution for each of the one or more wells using the one or more fluorescence measurements and the one or more absorbance measurements. The calculating may be done using only the one or more absorbance measurements or only the one or more fluorescence measurements. An empirically determined fluorescence response constant based on excitation intensity, fluorophore concentration, fluorophore quantum yield, the environment, detector sensitivity, and optical configuration of the plate reader, can be used to provide calibration curves if the fluorescence measurements are not sufficiently linear but are reproducible and correlate with the absorbance measurements for the solution, which curves can be used to calculate the volume dispense size. The one or more chromophores are selected to have absorbance characteristics substantially distinct from excitation and emission characteristics of the one or more fluorophores.

[0008] The sample solution in each of the one or more wells may be excited with the spectrometer at a first wavelength, the absorbance measurements may be carried out at a second wavelength, and the fluorescence measurements may be carried out at a third wavelength. The volume dispense size calculation may be performed over a range of volumes wherein only the absorbance measurements are used to calculate the dispensed volume over a first portion of the range of volumes and only the fluorescence measurements are used over a second portion of the range of volumes. In an embodiment, a diluent may be added to each of the one or more wells prior to dispensing the sample solution. The diluent and the sample solution may contain a common chromophore.

[0009] The system of the invention includes a microtiter plate including a plurality of wells arranged to receive liquid dispensed therein by the liquid dispensing apparatus, a sample solution containing one or more fluorophores and one or more chromophores, a spectrometer arranged to measure fluorescence of the one or more fluorophores and to measure absorbance of the one or more chromophores in the plurality of wells having the sample solution dispensed therein, and a computing device configured to calculate a volume dispense size of the sample solution dispensed into one or more of the plurality of wells of the microtiter plate using the measured fluorescence and the measured absorbance. The system may also include a diluent containing a chromophore in common with one of the one or more chromophores of the sample solution. The system may include one or more of a calibration plate, a plate mixer, and an input device capable of reading and importing measurements from the spectrometer. The input device may be a barcode scanner, an RFID scanner or other device. In an embodiment, the spectrometer includes an absorbance spectrophotometer and a separate fluorometer.

[0010] The invention also includes a composition of matter used to calibrate a liquid dispensing apparatus with a spectrometer. The composition includes a liquid with one or more chromophores in the liquid and one or more fluorophores in the liquid. The one or more chromophores may be selected from Tartrazine, Ponceau S, and copper chloride. The one or more fluorophores may be selected from Fluorescein, fluorescein derivatives, Rhodamine, rhodamine derivatives, and Quantum dots.

[0011] In another embodiment of the invention, a method is provided for calibrating a dispensing apparatus through a range of volumes using absorbance and fluorescence measurements in a sample solution using a microtiter plate having a plurality of wells. The method includes the steps of dispensing with the dispensing apparatus the sample solution into at least a portion of the plurality of wells, wherein the sample solution includes a first absorbance dye and a fluorescence dye and wherein a concentration of the first absorbance dye is known, adding a diluent to the at least a portion of the plurality of wells including the dispensed sample solution, wherein the diluent includes a second absorbance dye, measuring absorbance and fluorescence for each of the at least a portion of the plurality of wells with a plate reader, and establishing a traceability of the fluorescence measurements through the range of volumes by correlating the fluorescence measurements with stable absorbance measurements. The sample solution may include the second absorbance dye. In an embodiment of this method, a second portion of the plurality of wells is filled only with the diluent, wherein the second portion of wells is used to provide baseline information about all of the plurality of wells of the microtiter plate to be used in the establishing traceability step. The step of measuring absorbance and fluorescence includes the steps of exciting the fluorescence dye at a first excitation wavelength and measuring fluorescence at an emission wavelength, exciting the fluorescence dye at a second excitation wavelength and measuring fluorescence at the emission wavelength, and measuring absorbance at an absorbance wavelength that is different from the first excitation wavelength, the second excitation wavelength, and the emission wavelength. The method may also include the generation of calibration curves for each of the two fluorescence measurements and the one absorbance measurement, wherein the calibration curves can be used to correlate accuracy and traceability of volume measurements through the range of volumes by moving from one calibration curve to another where the calibration curves overlap.

[0012] This method optionally includes exciting the fluorescence dye at a first excitation wavelength and measuring fluorescence at an emission wavelength, exciting the fluorescence dye at a second excitation wavelength and measuring fluorescence at the emission wavelength, and measuring absorbance at an absorbance wavelength that is different from the first excitation wavelength and the emission wavelength, and generating calibration curves for each of the two fluorescence measurements and the one absorbance measurement, wherein the calibration curves can be used to correlate accuracy and traceability of volume measurements through the range of volumes by moving from one calibration curve to another where the calibration curves overlap. [0013] The method of the invention can be used to provide daily monitoring and controlling instrument drift of the plate reader using an absorbance calibration plate without the need to use a fluorescence calibration plate using a version of the sample solution with three distinct spectral features by dispensing over a selectable time period a high volume of the sample solution into the at least a portion of the plurality of wells, wherein the first absorbance dye is of a well- controlled concentration and the fluorescence dye is of a relatively high concentration, measuring fluorescence and absorbance of the sample solutions, determining day-to-day instrument drift from any changes observed in the absorbance measurements, and correlating day-to-day instrument drift associated for the fluorescence measurements with the determinations made from absorbance measurement changes.

[0014] The hybrid fluorescence-absorbance system and method of the present invention enables accurate and reliable volume measurements at very low dispense volumes and across a wide dynamic range by blending the advantages and favorable aspects of both measurement methods and their associated dye systems. These and other advantages will be further understood upon review of the following detailed description, accompanying drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a simplified representation of the system of the present invention configured to carry out steps of the methods of the present invention.

[0016] FIG. 2 is a graph demonstrating the excitation and emission spectra for an example sample solution, as well as the absorbance spectra of the sample solution and diluent solution. [0017] FIG. 3 is a simplified diagram of a first embodiment of the method of the present invention.

[0018] FIG. 4 is a graph showing three separate calibration curves generated from dilutions of the same sample solution, measured as an absorbance signal for a larger volume range, an emitted fluorescence signal at an intermediate volume range, and a different emitted fluorescence signal at a low volume range.

[0019] FIG. 5 is a graph demonstrating the data from FIG. 4 in a log-log format to better demonstrate the overlap between the different calibration curves.

[0020] FIG. 6 is a graph representing the %CV, or reproducibility, of the individual data points used to create the calibration curve in FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

[0021] A system 10 of the present invention for measuring dispensed volumes over a wide range of low volumes is represented in FIG. 1. The system 10 includes a microtiter plate 12 including a plurality of wells 14 characterized by rows and columns that may be designated, such as Rows A-P and Columns 1-24, for example. Other well configurations are possible. The system 10 further includes a plurality of solutions 26 and a plate reader 18 capable of measuring both absorbance and fluorescence, a calibration plate 16 for maintaining control of the plate reader 18, a microtiter plate mixer 20, a computing device 22, and an input device 24. The computing device 22 is programmed to receive information from the input device 24 about the microtiter plate 12, the calibration plate 16, and the solutions 26. The computing device 22 is programmed to control the plate mixer 20, the input device 24, and the plate reader 18, and to carry out calculations as described herein to determine volumes of fluid dispensed by a liquid dispenser 16 into one or more of the plurality of wells 14. Note: the measurement device used to collect absorbance and/or fluorescence for samples contained in microtiter plates is often referred to as a “plate reader”. In general terms, a “plate reader” is a spectrometer, and may be a simple device capable of measuring absorbance at fixed wavelengths, or fluorescence at fixed wavelengths. A “plate reader” may also be much more complex and capable of measuring scanned absorbance spectra over a wide wavelength range as accomplished by a scanning spectrophotometer, or scanned emission spectra for fluorescent samples as accomplished by a scanning fluorometer. For this disclosure, “plate reader” may be used interchangeably for a device capable of measuring absorbance signals as well as fluorescence signals.

[0022] The solutions 26 contain one or more absorbance dyes and one or more fluorescent dyes. The absorbance/excitation band of the fluorescent dye(s) should not significantly interfere with the absorbance/excitation band of any other fluorescent dye, nor with the absorbance band of any other absorbance dye in the solution. The emission band of the fluorescent dye(s) should not significantly interfere with the absorbance band of the absorbance dye(s). The absorbance band of the absorbance dye(s) should not significantly interfere with any other absorbance or excitation band. Note: the physical portion of a fluorescent dye molecule that emits fluorescent light for measurement is commonly referred to as the “fluorophore”. Thus “fluorescent dye” and “fluorophore” are often interchangeably used throughout this disclosure. Similarly for absorbance dyes, the physical portion of an absorbance dye molecule is commonly referred to as the “chromophore”. “Absorbance dye” and “chromophore” are interchangeably used throughout this disclosure.

[0023] One sample solution of the solutions 26, Si, is manufactured to contain a high concentration of fluorescent dye to measure a relatively small test volume (e.g., 100s of picoliters). Another sample solution, S2, is manufactured to contain a lower concentration of fluorescent dye (for example, S2 may only have half the concentration of fluorescent dye as in Si) and is used to measure a relatively larger volume (e.g., 10s of nanoliters). S3 has an even lower fluorophore concentration and is used to measure an even larger volume (100s of nanoliters). In other words, to achieve a measurable signal at small dispense volumes, a high starting dye concentration is required. Conversely, large dispense volumes require less fluorescent dye. Si may also contain a relatively high concentration of an absorbance dye, S2 may contain a lower concentration of absorbance dye, and S3 may contain an even lower concentration. As with the fluorophore, this decreasing concentration may be used to test different test volume ranges. Conversely, the idea of measuring multiple spectral features, as described in US Patent No. 8,404,158 of the present applicant may be implemented herein to achieve a similar effect. The contents of that patent are incorporated herein by reference. It may also be noted that distinct from the notion of observing absorbances across multiple spectral features over a varying concentration of a starting sample solution, the present invention can take similar advantage of measuring multiple spectral features in a fluorescence emission spectrum. Furthermore, the present invention includes the option of exciting at multiple bands and detecting single or multiple emission features in a volume delivery analysis.

[0024] All sample solutions (Si, S2, S3,. . ., SN) may also contain a fixed concentration of a common absorbance dye (e.g., copper chloride). The common absorbance dye may be any dye provided in the present applicant’s MVS® multichannel liquid handler verification system including but not limited to the copper chloride dye. The MVS® system may be used in carrying out steps of the methods described herein but it is not required to be used. This common dye has an absorbance band that is far enough removed from any absorbance/excitation/emission band of any other dye in the sample solution to enable accurate and precise quantitation of this dye for pathlength and volume determination as necessary. The concentration of the common dye is the same among all sample solutions. All sample solutions are manufactured with a controlled buffer concentration to allow for fixing the solution pH to stabilize the dyes in solution. Sample solutions are manufactured to controlled specifications and measured. The concentrations of each absorbance dye are known for each bottle of sample solution and reported to the user (e.g., through a barcode label on the sample bottle) and is used for calculating final dispense volume. FIG. 2 demonstrates excitation, emission and absorbance spectra for an example sample solution and diluent solution. The dotted line with open triangles was collected by scanning the excitation light from 300nm to about 530 nm, while collecting light only at 530 nm. The dotted line with closed circles was gathered by exciting with light at 480 nm, while scanning and collecting the emission from 500 nm to 800 nm. Both the excitation and emission spectra were measured by the fluorescence and plotted as a secondary y-axis to the right. The solid line is the absorbance spectrum collected from 300-1000 nm for the sample solution. And the dashed line is the absorbance spectrum collected for a diluent solution. Both absorbance spectra are plotted on the primary y-axis to the left.

[0025] The system 10 further includes a diluent solution of the solutions 26. The diluent solution contains no fluorescent dye, but ONLY the common dye, as shown in FIG 2. The concentration of the common dye in the diluent solution is the same as the concentration in each of the sample solutions. This allows for mixture of any amount of any sample solution with any amount of the diluent solution, the result of which is no change in the concentration of the common dye. The purpose of the diluent solution is to allow for measurement of, and removal of total volume variability from the final sample volume measurement. The concentration of the common dye is known and may be reported to the user.

[0026] The system 10 also includes a baseline solution of the solutions 26. The baseline solution contains no fluorescent or absorbance dyes of any sort. It is a clear solution used to collect a baseline reading for the microtiter plate type being used. The baseline solution may be manufactured to identify certain types of ingredients common with the sample solutions. Characteristics about the baseline solution may be reported to the user.

[0027] The microtiter plate 12 has a selectable well density (e.g., 96-well, 384-well, 1536-well, etc.). The microtiter plate 12 may be characterized for dimensional characteristics such as well bottom diameter, side-wall taper angle, well-to-well differences, etc. It may also be characterized for well-to-well variability. Bottoms of the wells 14 may be optically clear or may be opaque. Characteristics about the plate 12 may be reported to the user.

[0028] The plate reader 18 may be embodied in a single spectrometer capable of measuring both absorbance and fluorescence signals from individual wells 14 of the microtiter plate 12. It is noted that absorbance measurements and fluorescence measurements may be carried out by two separate spectrometers, such as with an absorbance spectrophotometer and a fluorometer, or in a single device capable of measuring both absorbance and fluorescence as noted herein. The calibration plate 16 may optionally be used to control for daily drift inherent to plate readers and allow a method for extending traceability of the spectrometer measurements back to a reference spectrometer. The calibration plate 16 may contain absorbance standards, fluorescent standards, alignment standards, etc. Characteristics about the calibration plate 16 may be reported to the user.

[0029] The plate mixer 20 is configured to homogeneously mix the dyes of the solutions 26 within the plate wells 14 necessary to perform accurate volume measurements. Alternatively, the system 10 may be used to determine adequacy of mixing steps for a customer defined mixing method. The computer device 22 is configured to be capable of being interfaced with the plate reader 18, the mixer 20 and an input device 24 capable of reading and importing all values from the various system components using one or more interface devices. The input device 24 may be a barcode scanner. In the barcode embodiment of the input device 24, all characteristics of each component will be contained in a barcode label affixed to the component. In this example, the user would use the barcode scanner to read and input this information to the computer device 22. Another type of the input device 24 is an RFID scanner, and the characteristics of each component of the system 10 is carried in an RFID tag affixed to the component.

[0030] The computer device 22 is configured to carry out steps of the method to generate output representative of the results of the analysis performed through either or both of the invention methods described herein. A system software is used to run the computer device 22 and all components. The software is arranged to guide the user through use of all components of the system 10. It incorporates any input information about component characteristics. It uses all input information, and all collected measurements to calculate dispense sample volumes and any other calculation necessary.

[0031] In a first embodiment of a hybrid method of the invention represented in FIG. 3 for determining dispensed liquid volume, an average dispense volume can be calculated using only absorbance readings of the plate reader 18 carried out on dispensed liquid in filled wells of the microtiter plate 12, and the reproducibility of dispenses can be calculated using only fluorescence readings of the plate reader 18 carried out on the same filled wells of the microtiter plate 12. The absorbance may not be measurable for individual dispenses because it may be too low (i.e., the uncertainty of the absorbance measurement for individual dispenses will be too large to be useful). However, a summation of multiple (N) dispenses can produce an accurate absorbance measurement, which can be divided by the number of dispenses to calculate the average dispense size. Thus, the accuracy of individual dispenses is not directly measured with absorbance, but an average dispense volume can be calculated. At the same time, individual dispenses are measured using fluorescent reads after each dispense. The variability of multiple dispenses into any given well as measured by fluorescence can be used to determine reproducibility of the N dispenses.

[0032] As an example of the hybrid method, a sample solution is created with selectable concentrations of both an absorbance dye (e.g., Tartrazine, Ponceau S, copper chloride, etc.) and a fluorescent dye (e.g., Fluorescein, fluorescein derivatives, Rhodamine, Rhodamine derivatives, Quantum dots, etc.). The dyes of both types are chosen so they do not interact chemically with each other, and the absorbance peaks or excitation peaks of the absorbance and fluorescent dyes do not significantly interfere with quantitative assessment. The fluorescent dye is chosen such that there is sufficient separation between the excitation peak and the emission peak (i.e., acceptably large Stokes Shift) to allow for quantitative assessment. Also, the absorbance peak of the absorbance dye should not overlap significantly with the emission peak of the fluorescent dye. It is possible that an absorbance peak (i.e., the excitation peak) of the fluorescent dye can serve as the peak for an absorbance measurement.

[0033] With continuing reference to FIG. 3, an empty form of the microtiter plate 12, which may be any of 96, 384, 1536 or other microtiter plate types, has the sample liquid dispensed by the dispenser 16 into the wells 14 in a defined pattern, which may be tailored to suit the purposes of the calibration. There are several patterns that would work, depending on the dispense volumes under test and the geometry and operation of the dispenser 16 (user and application defined). Consideration may be given to evaporation that can occur with time (if considered significant to total volume in the well), and photobleaching that may occur with multiple reads. An example dispense configuration for method 100 as depicted in FIG. 2 is as follows: o Row A - The wells in this row are filled with baseline solution or diluent at a working final volume. The wells in this row will provide baseline information for both fluorescence and absorbance measurements. o Row B - A volume of sample solution, Vi, is dispensed into all wells in this row. Either a baseline solution or diluent is also added (before or after sample dispense) to the wells to create a final working volume. The sample and baseline (or diluent) are mixed. This Vi volume and resulting concentration may be small enough so that it cannot be measured using an absorbance method and can only be measured using a fluorescence method. o Row C - This row contains multiple dispenses (N times) of volume Vi into each well in the row with an appropriate working final volume of baseline solution or diluent. N must contain enough Vi dispenses to produce a large enough absorbance to allow an accurate measurement of volume using an absorbance measurement but may be small enough to allow fluorescence-based detection as well. There may exist an area of correlation (collinear or other) for the two types of measurements (absorbance and fluorescence), which would allow for an expanded functional dynamic range for this embodiment of the hybrid technique of the present invention. For all the results which contain N dispenses, the final values are divided by N to get the average value of a single dispense. Also, all absorbance measurements can provide traceability of the volume results back to recognized standards. Note: in this example, a description of the hybrid method defines filling all wells in a plate row. However, this method also applies when fewer wells are filled. In a generic sense, M wells are filled with N dispenses of Vi volumes. It should also be noted that this example defines volumes dispensed in a row format. However, dispensing in a column format, or in a random format across the plate, is also applicable. Modem liquid handling devices are capable of dispensing in rows, in columns, in quadrants, or in wells scattered across a microtiter plate. Thus, this method is not confined to dispensing in any set pattern, so long as the dispense pattern is known and measured accordingly. [0034] One option for the order of dispense into Row C is to make one dispense per well into each column (e.g., one dispense in wells Ci thru C12 for a 96 well plate, or Ci thru C24 for a 384 well plate) with absorbance and fluorescence reads collected after each dispense, and then repeating that pattern N times, collecting the absorbance and fluorescence reads after each dispense. Each well in the row will get N total dispenses, and the last absorbance read will correspond to the total volume dispensed (VTD) in each well. Dividing VTD by the total number of dispense N provides the average volume of each individual dispense, regardless of potential delivery dispense drift over the course of the N dispenses. The individual fluorescence and absorbance reads will show dispense volume drift and can be used to detect these drift characteristics from the delivery system used.

[0035] Another pattern for carrying out the hybrid method is to make all N dispenses into Row C, Column 1 (Well Ci) followed by N dispenses into Row C, Column 2 (Well C2), etc., with absorbance and fluorescence reads after each dispense, until all the wells in Row C are filled. This dispense pattern may allow different quantification of how much the dispenser volume is drifting, for instance if it is dispensing less liquid at the start of the run time than at the end. For all of the results which contain N dispenses, the results must be divided by N to get the average value of a single dispense.

[0036] Still another pattern for carrying out the hybrid method is to make one dispense of Vi into each well of Row C, then make two dispenses of Vi into each well of the next row, which may be referred to herein as Row D, then make three dispenses of Vi into each well of the row after that, which may be referred to herein as Row E, then continue this pattern with incremental increases in dispenses for all wells in subsequent rows. Once baseline or diluent has been added and the solutions mixed, absorbance reads, and fluorescence reads of the whole plate can be made. This approach can be used to circumvent photobleaching that can occur with multiple reads.

[0037] Some dispensers have an adjustable dispense volume, and it may be desired to test several different volumes of dispense in one plate. For example: o In this example, the row referred to as Row D contains other volume dispensed at a second test volume (V2) and an appropriate working final volume of baseline solution or diluent. This V2 volume and resulting concentration may be small enough so that it cannot be measured using an absorbance method and can only be measured using a fluorescence method. o The next row referred to as Row E would then follow the same procedures as outlined above in regard to Row C. It may be that smaller multiple dispenses are used to allow an accurate absorbance measurement.

[0038] A final working volume will be achieved either by diluting with baseline solution or a specialty diluent solution. The baseline solution, or specialty diluent solution, may be added before or after sample solution has been dispensed; the order of dispense is not important. The baseline or specialty diluent is required to create a total working volume that allows for absorbance and fluorescence measurements. All contents in each well should be completely mixed before absorbance and fluorescence measurements can be made. The diluent solution may contain a chromophore which is chosen so it does not interfere with quantitation by either the absorbance of the sample or the emission signal of the fluorescence, nor chemically with the sample. Copper ions may be suitable for this purpose. They absorb light at 730 nm which is well beyond the absorbance bands of most fluorophores, such as tartrazine or fluorescein and derivatives. Also, copper absorbs very minimally at the absorbance/excitation bands of those chromophores.

[0039] Each dispense/dilution should be mixed prior to reading the plate 12, and centrifuging may be required to remove potentially trapped air bubbles commonly introduced when dispensing. The fully dispensed and mixed plate 12 may be placed into a multimode reader comprising the plate reader 18 as both the spectrometer and fluorometer, for both absorbance and fluorescence readings taken for all wells 14 containing liquid. There may exist an area of statistical correlation (e.g., collinearity, or other) for the two types of spectroscopic measurements (absorbance and fluorescence), which would allow for an expanded functional dynamic range for this hybrid technique. Expanding the functional dynamic range may be achievable to correlating a crossover volume or region from one measurement method used for one volume range, to the other measurement method used for a neighboring volume range. Additionally, the absorbance of the diluent may be used for a total volume and pathlength correction, which accounts for variability of the bulk dispenser used to backfill the wells.

[0040] Upon completion of the well readings, the computing device 22 may be used to carry out volume dispense determinations. Specifically, Row A provides baseline information for both fluorescence and absorbance measurements for all rows. If final working volumes are not equal, additional Rows may be used for baseline information or individual columns in Row A may be filled with different final volumes. If diluent with a chromophore is used, an additional row (identified as Row I herein, for example) would be used for diluent information. Row B may provide information only from the fluorescence readings, since the absorbance readings will likely be too small to measure for Vi dispenses. The results of fluorescent readings of Row B provide information about the reproducibility of the dispenser when dispensing volume Vi. The variation in the measured volume, as calculated by the standard deviation in the fluorescent signal, has contributions not only from the dispenser, but also from the reader, microtiter plate, the completeness of the mixing, environmental conditions, and other sources of uncertainty. [0041] Uncertainty calculations may be used to evaluate dominant source(s) of error (both systematic and random). Understanding and controlling sources of error in the measurement procedure (reader, plates, mixing, environmental conditions, and calibration solutions) enable the user to determine the imprecision of the dispenser 16. Separate testing may be used to determine the magnitude of these other sources of variability and to assure the user that the dominant source of imprecision comes from the dispenser 16. When these other sources are accounted for, the standard deviation of the volume measurements when divided by the average volume measurement is an estimate of the coefficient variation (CV) of dispensed volume only. This estimate is not dependent on the concentration of fluorophore or fluorescence accuracy, and thus requires no calibration but this estimate does require precise and reproducible measurement of signal. The present invention controls or accounts for variability of the reader, the plates, mixing, and environmental conditions, resulting in a signal that reports only the variability of the liquid delivery device under test.

[0042] Finally with respect to the hybrid method, Rows C, E, etc., can provide information from both absorbance and fluorescence readings; however, the absorbance or fluorescence of some reads may not be used if they are outside the functional dynamic range of the measuring method. Additionally, absorbance readings at multiple wavelengths for different chromophores and/or different spectral features associated with particular dyes may be used to calculate the average dispense volume using a method that follows that of the Artel MVS methodology. Such methodology is at least described in Artel US Patent No. 8,404,158 .

[0043] A second embodiment of the hybrid method of the present invention provides an alternative way to fully characterize liquid dispensation at very low volumes. In this embodiment, absorbance and fluorescence results are combined. One example of how this combination may provide useful information is the following. Suppose that the dispenser under test is being tested to see if the dispense volume is drifting over relatively short time intervals, such as while the N dispenses are being made. In this instance, it may be useful to get information about the absolute volume of dispense at total volumes too small for the absorbance method to be applied in the way that is done under the first hybrid method as described above. However, in this embodiment of the method of the invention, the functional dynamic range may be expanded by combining the absorbance and fluorescence results, and the individual volumes dispensed may be quantified.

[0044] In the second hybrid method, data are gathered as described with respect to the first hybrid method, however it is analyzed differently. The fluorescence results and absorbance results of individual dispenses are combined to provide a calibration for fluorescent measurements using a correlation between them. For one approach, it can be assumed that the fluorescent intensity Qis linear with concentration of fluorophore over a wide enough range that the relationship I_F =K_FC_F holds, where K_F is an empirically determined fluorescence response constant that accounts for the inherent relativity of fluorescence measurements based on excitation intensity, the fluorophore quantum yield, the environment, detector sensitivity, and the optical configuration of the plate reader. Qis the concentration of fluorophore in the well as determined by absorbance measurements, or gravimetric measurements that are quantitative and traceable to NIST standards.

[0045] If the fluorescent signal is not sufficiently linear but correlates with absorbance measurements for the solution, and is nonetheless reproducible, then calibration curves can be provided, or the user can be instructed on a method that will generate the necessary calibration curves using suitable dilutions and volumes of the sample solution, mixed and measured at the time of utilization. Assuming that the signal is adequately linear, we can find the fluorescence response constant by using the absorbance data generated from row C to find the average volume of dispense V_abs for the N deliveries as averaged over the entire row. This fluorescence response constant in essence ties the fluorescent signal at the time of measurement to an absorbance signal. As previously discussed, absorbance measurements can be tied to recognized absorbance standards which provide traceability and certainty to absorbance measurements. Thus, at the time of measurement, this fluorescence response constant allows the fluorescence signal to be tied to a traceable standard, which allows for accuracy calculations to be performed.

[0046] By way of example, and not intending to be limiting, the following experimental protocol summarizes a proof of concept experiment based on a system using a sample solution composed of 4 mM Rhodamine 110 in DMSO. A volume range of approximately 0.5nL - 550 nL was tested using 39 independent dilutions accurately made using a large volume gravimetric method. Each of the 39 dilutions was prepared using a large volume gravimetric dilution wherein a measured quantity of Rhodamine 110 sample solution was accurately weighed and mixed with an accurately weighed amount of copper diluent (a copper diluent that contained 1.1 grams Copper chloride dihydrate per liter as available in the applicant’s MVS® system). The various dilution points were used to establish three independent calibration curves, as shown in FIG. 4, whose responses were measured with absorbance or fluorescence signals. In essence, three different calibration curves were established for using this 4mM Rhodamine 110 to measure volumes over this wide volume range.

[0047] The 39 sample dilutions were prepared gravimetrically using calibrated balances (Sartorius Cubis analytical balance,). A known amount of Rhodamine 110 was weighed and dissolved in a weighed amount of dimethyl sulfoxide (DMSO) or copper diluent or combination of the two. The weight of solution was converted to volume using the measured relative density of each solution, and accounting for temperature, humidity, and barometric pressure. The exact concentration of the Rhodamine 110 in solution was calculated and this solution was called Stock 1. Stock 1 was then diluted in copper diluent to prepare standards at target concentrations covering the concentration ranges in Table 1 by weighing the solutions (stock, diluent, and/or standards in the case of serial dilutions) and using relative density measurements to calculate exact volumes and concentrations. For example, to prepare a standard representative of a 547 nL droplet, 50.54 grams of copper diluent was weighed and added to a bottle, followed by 1.47 grams of a separately prepared 0.4 mM dilution of the Rhodamine 110 sample solution; relative density measurements were used to calculate the exact concentration of this large volume dilution.

[0048] Each large volume dilution was then used to fill a whole plate (i.e., all wells in an entire plate were filled with a defined working volume) using a 96-tip Cybio liquid handler (96-tip head CyBi-Well, Analytik Jena, Jena, Germany). A working volume of 200 pL was dispensed with the CyBi-Well into each plate well. The total volume of dilution dispensed into the plate was determined by weighing the plate, and the average working volume per well determined by dividing by 96. The volume of Stock 1 per well was then calculated from the average working volume, the measured concentration of Rhodamine, and the known concentration of Rhodamine in Stock 1.

[0049] A Molecular Devices M2e plate reader (Molecular Devices LLC, San Jose, CA) was used to collect fluorescence measurements (both top read and bottom read) and absorbance measurements for every well in the test plate, as specified in Table 1 : Table 1

Fluorophore

Test Volume Range Concentration

(nL) (nM) Measurement

0.5 - 11.7 10 - 244 Fluorescence emission at 530 nm with excitation wavelength at 480 nm

7 - 156 145 - 3270 Fluorescence emission at 530 nm with excitation wavelength at 332 nm

90 - 550 1,900 - 11,500 Absorbance at 496 nm

With reference to FIG. 4, multiple calibration curves were generated using: excitation at 480 nm with emission at 530 nm (calibration curve for -0.5-11.5 nL; 10-244 nM); excitation at 332 nm with emission at 530 nm (calibration curve for -7-156 nL; 145-3270 nM); and absorbance at 496 nm (calibration curve for -90-550 nL; 1,900-11,500 nM). The absorbance of copper diluent at 730 nm was used to create pathlength corrected calibration curves. Linear least squares regression fits were applied to all the calibration curve data. The signal of the individual samples and calibration curve equations were used to calculate the concentration of the samples, and their representative volumes. The accuracy of the samples and methods was assessed by comparing the calculated sample volumes to the gravimetric sample volumes. FIG. 4. demonstrates the inaccuracy of the data points from each of the calibration curves over the volume range of 0.5nL - 550 nL (lOnM - 11,500 nM). FIG. 5. displays each of the calibration curves in log-log format and more easily demonstrates the linear overlap of each curve. FIG. 6 shows the %CV of the signals and how each test point can be used to assess reproducibility.

[0050] Each of the calibration regions overlapped its neighboring curve at the edges, by design. This overlap corresponds to the correlation (collinear or other) described previously and can be used to extend the accuracy and traceability from the absorbance region (e.g., 90nL - 550 nL) to the neighboring fluorescence region (e.g., 7 nL - 156 nL), which is subsequently extended to the next neighboring fluorescence region (e.g., 0.5 nL - 11.5 nL). With reference to FIG. 2, multiple excitation wavelengths were utilized for Rhodamine 110 to cover a wider volume range with fluorescence. Excitation at 332 nm in this example provides approximately 10% the excitation power as compared to excitation power at 480 nm, which by design bridges the volumes covered with absorbance measurements and fluorescence measurements.

[0051] To clarify, the calibration curves shown in FIG. 4 and FIG. 5 were collected as follows: Absorbance curve (e.g., 90nL - 550 nL) was collected by measuring the absorbance at 496 nm, the intermediate fluorescence (e.g., 7 nL - 156 nL) was collected by exciting at 332 nm and collecting at 530 nm, and the low-end fluorescence (e.g., 0.5 nL - 11.5 nL) was collected by exciting at 480 nm and collecting at 530 nm. The difference between the intermediate fluorescence solution and low-end was a significant concentration increase in fluorophore. To achieve measurable fluorescence, the intermediate sample was measured at the little peak at 332 nm (which provides only about 10% of the excitation power). Had the intermediate solution been excited at 480 nm, the emission would have been too strong and unmeasurable because too many dye molecules would have been excited by the excitation light, the response would have been unusable for quantitation purposes.

[0052] For this example, each calibration region is adequately modeled with a line, defined by the following equations: y_l=m_l x_l+ b_l (0.1) y_2=m_2 x_2+ b_2 (0.2) y_3=m_3 x_3+ b_3 (0.3) where equation (0.1) represents the linear equation for the calibration data collected via absorbance measurements at 496 nm, equation (0.2) represents the calibration data collected via fluorescence at 332 nm excitation (530 nm emission), and equation (0.3) represents the calibration data collected via fluorescence at 480 nm excitation (530 nm emission). Traceability from the 496 nm absorbance calibration curve to the fluorescence curve with excitation at 332 nm is accomplished by algebraic substitution of xl for x2, which results in: y_2=m_2/m_l (y_l-b_l )+ b_2 (0.4)

Correlation between the fluorescence curve with excitation at 332 nm to the fluorescence curve with excitation at 480 nm is accomplished by further algebraic substitution of x2 for x3: y_3=m_3/m_2 (y_2-b_2 )+ b_3 (0.5) Substituting y2 from equation (0.4) into equation (0.5) results in equation (0.6) which demonstrates the extension of traceability of the absorbance calibration curve through both fluorescence calibration curves. y_3=m_3/m_l (y_l-b_l )+ b_3 (0.6)

The percent differences from predicted values did not exceed 2.5% demonstrating the good accuracy of this approach to achieving traceability of fluorescence measurements through absorbance measurements. This method demonstrates how a fluorescence measurement collected for a low test volume can be correlated to a stable, traceable absorbance measurement. This correlation in effect extends traceability to ultra-low test volumes only directly measurable via absorbance measurements.

[0053] In yet another application of the current invention, a sample solution is used to combine the traceability of an absorbance measurement with the sensitivity and dynamic range of a fluorescent measurement to measure liquid volumes over a large volume range. This method uses a single sample solution and a diluent to cover the volume range from 500 nL down to 0.5 nL. The sample solution includes three dyes in solution; two of the dyes are fluorescent and one is absorbent.

[0054] A general overview of how a user would use this invention is as follows: i) the user puts the sample solution in a suitable container for access by the dispenser under test, ii) the dispenser under test is used to dispense programmed amounts of the sample solution into various wells of a microtiter plate, iii) a diluent is also added to the wells either before or after the sample volumes have been dispensed (note: the diluent volume is not critical and the diluent does not have to be dispensed by the device under test), iv) the sample and diluent solutions are mixed and the plate is centrifuged if necessary to remove bubbles, and v) both absorbance and fluorescence readings are read in a multimode reader. The absorbance and fluorescence readings are used to calculate the actual dispense volumes for all wells using software integrated with the system. A report is prepared which includes the precision of dispense results, the accuracy of dispense results, data trends and a heat map of results. [0055] For this application, the sample solution is composed of an accurate concentration of an absorbance dye. The sample solution also contains an accurate concentration of at least one fluorescence dye but may also contain a second fluorescence dye. The diluent contains a second absorbance dye, which may or may not be present in the sample solution. The absorbance dye provides traceability, reproducibility, and accuracy of results. It is present in sufficient quantity to allow an accurate absorbance reading at a first wavelength XI when delivered at volume Vcall and diluted with volume Vdil of diluent. This absorbance reading is combined with a second absorbance reading at a fourth wavelength X4, which corresponds to either a fourth dye present in the diluent or the water band of an aqueous diluent. Between these two absorbance readings, an accurate and traceable volume is calculated using calculations provided through the MVS® system as previously described in the ratiometric methodology also in association with the MVS® system.

[0056] An example implementation for this method is as follows:

• The wells of a column of a microtiter plate are filled with only diluent to a defined working volume (e.g., 55 uL for a 384-well plate). Any column could be chosen, but for this example we will choose column 1 for the diluent. The wells in column 1 will provide baseline information for both fluorescence and absorbance measurements collected for the rest of the plate.

• A volume of sample Vcall is dispensed into the wells of another column in the plate; for this example, we will use column 2. Enough diluent is also added to the wells of column 2 such that the total volume of sample plus diluent is approximately equal to the defined working volume (e.g., 55 uL for a 384-well plate).

• The volume Vcall needs to be sufficient to give an accurate absorbance reading of that dye (when mixed with the diluent also in the well). The sample solution contains an accurate concentration of an absorbance dye. A second absorbance dye present in the diluent (or else the water band of the solvent) provides a second absorbance reading needed to calculate an accurate and traceable volume of sample dispensed into the wells in column 2. The exact volume Vcall of this dispense is calculated from the absorbance readings.

• The sample also contains a first fluorophore at a concentration suitable for larger volume dispenses. It is important that the fluorophore concentration is within the linear range of its fluorescence response. When the plate is read, both absorbance and fluorescence readings will be collected for all wells in both column 1 and column 2. Using both the volume Vcall results calculated from the absorbance measurements from above and the fluorescence intensity of the first fluorophore I_F1 we can calculate a calibration factor SF- = Vcall / I_F1 which relates the actual volume dispensed into this column, as determined by the absorbance measurements, to the fluorescence intensity. The calibration factor SFi establishes a traceable connection between the measured volume Vcall and the fluorescence intensity I_F1 that is independent of the exact concentration of this fluorophore in the sample solution, or its quantum yield, or the extent to which the fluorophore is quenched or bleached.

• A second calibration point may be established by filling the wells of a column with volume Vcal2 of sample solution, plus the requisite volume of diluent. For these wells, two fluorescence readings are taken. The reading of the first fluorophore yields an accurate value for Vcal2 as described above, while the fluorescence intensity of the second fluorophore allows calculation of a second calibration factor: SF₂ = Vcal2 / I_F2 .

• Once the calibration factors SFi and SF2 are established, test volumes of unknown volume can be determined. A smaller volume may be dispensed, and a traceable volume calculated using SFi and SF2. Volumes less than Vcall but greater than a second calibration volume Vcah will be quantified using fluorescence readings of the first fluorophore multiplied by the calibration factor SFi. Volumes smaller than Vcal2 can now be accurately measured by multiplying the second fluorescence intensity for the well in question by SF2.

• The lowest volume which can be measured using this approach is limited by the sensitivity of detection of the second fluorophore emission and by the linearity of the dye’s emission at the low end of concentration.

[0057] For this method, the absorbance dye provides traceability, reproducibility, and accuracy of results. It is present in sufficient quantity to allow an accurate absorbance reading at a first wavelength I when delivered at volume Vcall and diluted with volume Vdil of diluent. This absorbance reading will be combined with a second absorbance reading at a fourth wavelength X4, which will correspond to either a fourth dye present in the diluent or the water band of an aqueous diluent. Between these two absorbance readings an accurate and traceable volume is calculated using determinations employed in the applicant’s MVS® system specifically the ratiometric methodology disclosed in the applicant’s materials for the MVS® system, which is available from the applicant or more generally through the internet.

[0058] As mentioned, the calibration factor (SFi, SF2) accounts for variable factors commonly associated with a fluorescence methodology, namely quantum yield, quenching, and photobleaching. These factors can cause drift in fluorescence dye over time. However, for this approach, so long as the absorbance dye remains stable in solution, the calibration factors (SFi, SF2) provide a traceable link that accounts for drifting fluorescence signals, so long as those drifts do not occur on the timescale of the test protocol. For example, the fluorescence signal needs to be stable for hours at a minimum.

[0059] A common practice in spectroscopy is to monitor and control daily instrument drift in absorbance and fluorescence readers using an artifact such as a solid-state calibration plate. While an absorbance-based calibration plate is still needed to provide needed traceability and account for absorbance drift, a fluorescence-based calibration plate is not necessary because a daily correlation ties these signals to the absorbance signal. Establishing the calibration factors (SFi, SF2) on a daily basis can be used in place of a fluorescence calibration artifact. A fluorescence validation plate may still be used as part of the system or installation.

[0060] A common liquid handling system that this methodology could be applied to is the acoustic droplet ejection system found in the Beckman Coulter LabCyte Echo, which is reported to provide accurate and previse volume delivery down to 2.5 nl and possibly below. Calibration of the Echo system will happen at 500 nL, which is 200 dispenses of 2.5 nl each. The absorbance produced by this 500 nL volume of sample solution will be -0.45 OD in a 96-well plate. This 500 nL total test volume need not be a quantitative dispense (i.e., the Echo may drift during the 200 dispenses and it will not matter). The calibration described above may take place within every test plate or perhaps every few plates, or perhaps one every day or longer. The frequency needs be as often as necessary to account for either dye or instrument drifting during the test time (e.g., drift in fluorescent output over time due to quenching of dye by O2 in the environment). Absorbance measurements need to be stable, however experience shows this to be easily achievable for hours. Note: the above test protocol need not be confined to the Echo, but it could also be done with a low volume multichannel pipette. [0061] The sample solution contains three dyes, or three distinct spectral features. Calibration of the system is provided internally by dye A, which is purely an absorbance measurement. There is no need for any separate fluorescence calibration solutions, and really no need for a fluorescence calibration plate. As with the MVS® system, there may be need for an absorbance calibration plate. We calibrate the system using a high volume (e.g., 500 nL ) of sample solution. In this model, internal calibration is built into the sample via a well-controlled concentration of dye A, whose absorbance signal needs to be stable, have a good shelf life, etc. The next dye is fluorophore B which is present in relatively high concentration. Its concentration is great enough that at the lowest dispense volume 2.5 nL there is sufficient emission intensity to get a good reading.

[0062] While the present invention has been described with respect to specific examples, it is not intended to be limited to such specific examples. Instead, the present invention is defined by the following claims and reasonable equivalents.

Claims

What Is Claimed Is:

1. A method for calibrating a liquid dispensing apparatus by measuring a volume of a sample solution through a selectable range of volumes dispensed by the liquid dispensing apparatus into one or more wells of a microtiter plate, the method comprising the steps of: dispensing the sample solution into each of the one or more of the wells of the microtiter plate, wherein the sample solution includes one or more chromophores and one or more fluorophores; measuring with a spectrometer fluorescence of the sample solution in the one or more wells to obtain one or more fluorescence measurements; measuring with the spectrometer absorbance of the sample solution to obtain one or more absorbance measurements; and calculating a volume dispense size of the dispensed sample solution for each of the one or more wells using the one or more fluorescence measurements and the one or more absorbance measurements.

2. The method of Claim 1 wherein the step of calculating a volume includes the step of calculating an average dispense of the liquid dispensing apparatus using only the one or more absorbance measurements.

3. The method of Claim 2 further comprising the steps of: dispensing N dispenses of volume VI of the sample solution into one or more wells of the one or more wells of the microtiter plate; measuring the absorbance to obtain absorbance measurements for each of the wells into which the sample solution has bene dispensed after all N dispenses of volume VI have been made; and dividing the absorbance measurements for each of the wells into which the sample solution has been dispensed by N to obtain an average volume VI dispense size for the N dispenses in each of the wells having the sample solution.

4. The method of Claim 1 wherein the step of calculating a volume dispense size includes the step of calculating a reproducibility of dispenses of the liquid dispensing apparatus using only the one or more fluorescence measurements.

5. The method of Claim 4 further comprising the steps of: dispensing N dispenses of volume VI of the sample solution into M wells of the microtiter plate; measuring the fluorescence to obtain fluorescence measurements for each of the M wells after each individual dispense of volume VI; determining a variability of the N dispenses using the fluorescence measurements for each of the VI volumes dispensed into the M wells; and calculating a reproducibility of the N dispenses from the variability determination.

6. The method of Claim 1 wherein the step of calculating a volume dispense size includes the step of correlating the fluorescence and absorbance measurements.

7. The method of Claim 6 wherein the step of correlating includes the steps of: assuming that a fluorescent intensity / . is linear with concentration of fluorophore over a wide enough range that a relationship I_F =K_FC_F holds, where K_F is an empirically determined fluorescence response constant based on excitation intensity, fluorophore quantum yield, the environment, detector sensitivity, and optical configuration of the plate reader, and is a concentration of the one or more fluorophores in the one or more wells as determined by absorbance measurements, or gravimetric measurements that are quantitative and traceable to NIST standards; providing calibration curves if the fluorescence measurements are not sufficiently linear but are reproducible and correlate with the absorbance measurements for the solution; and calculating the volume dispense size using the calibration curves.

8. The method of Claim 1 wherein the step of calculating includes using multiple spectral features of either or both of the fluorescence measurements and the absorbance measurements.

9. The method of Claim 1 wherein the one or more chromophores are selected to have absorbance characteristics substantially distinct from excitation and emission characteristics of the one or more fluorophores.

10. The method of Claim 1 wherein an excitation band used to elicit one or more emissions of the one or more fluorophores is selected based on operational characteristics of the spectrometer.

11. The method of Claim 1 wherein the spectrometer may be a plurality of spectrometers.

12. The method of Claim 1 wherein the sample solution in each of the one or more wells may be excited with the spectrometer at a first wavelength, the absorbance measurements may be carried out at a second wavelength, and the fluorescence measurements may be carried out at a third wavelength.

13. The method of Claim 1 wherein the step of calculating a volume dispense size may be performed over a range of volumes wherein only the absorbance measurements are used to calculate the dispensed volume over a first portion of the range of volumes and only the fluorescence measurements are used over a second portion of the range of volumes.

14. The method of Claim 13 wherein volumes of the first portion are greater than volumes of the second portion.

15. The method of Claim 13 wherein the range of volumes is about 2.5nl to about 500nl and the step of calculating the dispensed volume is accomplished over the range of volumes with only one dispensation of the sample solution into the one or more wells.

16. The method of Claim 1 further comprising the step of adding a diluent to each of the one or more wells prior to the step of dispensing the sample solution.

17. The method of Claim 16 wherein the diluent and the sample solution contain a common chromophore.

18. A system for calibrating a liquid dispensing apparatus by measuring a volume of sample solution dispensed by the liquid dispensing apparatus into one or more wells of a microtiter plate, the system comprising: a microtiter plate including a plurality of wells arranged to receive liquid dispensed therein by the liquid dispensing apparatus; a sample solution containing one or more fluorophores and one or more chromophores; a spectrometer arranged to measure fluorescence of the one or more fluorophores and to measure absorbance of the one or more chromophores in the plurality of wells having the sample solution dispensed therein; and a computing device configured to calculate a volume dispense size of the sample solution dispensed into one or more of the plurality of wells of the microtiter plate using the measured fluorescence and the measured absorbance.

19. The system of Claim 18 further comprising a diluent containing a chromophore in common with one of the one or more chromophores of the sample solution.

20. The system of Claim 18 wherein the one or more fluorophores include two or more fluorophore concentrations.

21. The system of Claim 20 wherein the one or more chromophores include two or more chromophore concentrations.

22. The system of Claim 18 further comprising a calibration plate.

23. The system of Claim 18 further comprising a plate mixer.

24. The system of Claim 18 further comprising an input device capable of reading and importing measurements from the spectrometer.

25. The system of Claim 24 wherein the input device is a barcode scanner.

26. The system of Claim 24 wherein the input device is an RFID scanner.

27. The system of Claim 18, wherein the spectrometer includes an absorbance spectrophotometer and a separate fluorometer.

28. A composition of matter used to calibrate a liquid dispensing apparatus with a spectrometer, the composition comprising: a liquid; one or more chromophores in the liquid; and one or more fluorophores in the liquid.

29. The composition of matter of Claim 28 wherein the one or more chromophores are selected from Tartrazine, Ponceau S, and copper chloride.

30. The composition of matter of Claim 28 wherein the one or more fluorophores are selected from Fluorescein, fluorescein derivatives, Rhodamine, rhodamine derivatives, and Quantum dots.

31. A method for calibrating a dispensing apparatus through a range of volumes using absorbance and fluorescence measurements in a sample solution using a microtiter plate having a plurality of wells, the method comprising the steps of: dispensing with the dispensing apparatus the sample solution into at least a portion of the plurality of wells, wherein the sample solution includes a first absorbance dye and a fluorescence dye and wherein a concentration of the first absorbance dye is known; adding a diluent to the at least a portion of the plurality of wells including the dispensed sample solution, wherein the diluent includes a second absorbance dye; measuring absorbance and fluorescence for each of the at least a portion of the plurality of wells with a plate reader; and establishing a traceability of the fluorescence measurements through the range of volumes by correlating the fluorescence measurements with stable absorbance measurements.

32. The method of Claim 31 wherein the sample solution includes the second absorbance dye.

33. The method of Claim 31 wherein a second portion of the plurality of wells is filled only with the diluent, wherein the second portion of wells is used to provide baseline information about all of the plurality of wells of the microtiter plate to be used in the establishing traceability step.

34. The method of Claim 31 wherein the step of measuring absorbance and fluorescence includes the steps of: exciting the fluorescence dye at a first excitation wavelength and measuring fluorescence at an emission wavelength; exciting the fluorescence dye at a second excitation wavelength and measuring fluorescence at the emission wavelength; and measuring absorbance at an absorbance wavelength that is different from the first excitation wavelength, the second excitation wavelength, and the emission wavelength.

35. The method of Claim 34 further comprising the step of generating calibration curves for each of the two fluorescence measurements and the one absorbance measurement, wherein the calibration curves can be used to correlate accuracy and traceability of volume measurements through the range of volumes by moving from one calibration curve to another where the calibration curves overlap.

36. The method of Claim 31 wherein the step of measuring absorbance and fluorescence includes the steps of: exciting the fluorescence dye at a first excitation wavelength and measuring fluorescence at an emission wavelength; exciting the fluorescence dye at a second excitation wavelength and measuring fluorescence at the emission wavelength; and measuring absorbance at an absorbance wavelength that is different from the first excitation wavelength and the emission wavelength.

37. The method of Claim 36 further comprising the step of generating calibration curves for each of the two fluorescence measurements and the one absorbance measurement, wherein the calibration curves can be used to correlate accuracy and traceability of volume measurements through the range of volumes by moving from one calibration curve to another where the calibration curves overlap.

38. The method of Claim 31 further comprising the step of providing daily monitoring and controlling instrument drift of the plate reader using an absorbance calibration plate without the need to use a fluorescence calibration plate using a version of the sample solution with three distinct spectral features, the method comprising the steps of: dispensing over a selectable time period a high volume of the sample solution into the at least a portion of the plurality of wells, wherein the first absorbance dye is of a well-controlled concentration and the fluorescence dye is of a relatively high concentration; measuring fluorescence and absorbance of the sample solutions; determining day-to-day instrument drift from any changes observed in the absorbance measurements; and correlating day-to-day instrument drift associated for the fluorescence measurements with the determinations made from absorbance measurement changes.