EP0866953A1

EP0866953A1 - Method and apparatus for determining characteristics of a sample in the presence of ambient light

Info

Publication number: EP0866953A1
Application number: EP96928966A
Authority: EP
Inventors: Glenn F. Spaulding
Original assignee: Vivorx Pharmaceuticals Inc
Current assignee: Abraxis Bioscience LLC
Priority date: 1995-08-22
Filing date: 1996-08-22
Publication date: 1998-09-30
Also published as: EP0866953A4; AU738290B2; CA2229458A1; WO1997008523A1; JPH11511557A; AU6853696A

Abstract

A device (20) determines an optical intensity of light emanating from a sample (22) situated under periodically varying ambient light (34). The device (20) includes a selectively illuminating light source (26a, 26b) that can be switched between on and off states that illuminates the sample (22). A detector (36) detects the optical intensities of the sample (22) a first plurality of times with the effects of the illuminating light source (26a, 26b) to produce a plurality of collective light intensity measurements, and a second plurality of times without the effects of the light source (26a, 26b) to produce a plurality of ambient light intensity measurements. A processor quantitatively determines, based upon the multiple collective intensity measurements and the multiple ambient light intensity measurements, the intensity amount of each of the collective light intensity measurements that results from the illuminating source (26a, 26b).

Description

METHOD AND APPARATUS FOR DETERMINING CHARACTERISTICS OF A SAMPLE INTHE PRESENCE OFAMBIENT LIGHT

BACKGROUND OF THE INVENTION

The present invention relates generally to processes for detecting the intensity of light emanating from a sample in the presence of ambient light and, is useful in determining colorimetric or fluorescent light intensity of a sample, or the amount of a particulate in a sample based upon the detected intensity when the intensity is measured under ambient lighting conditions. Such light intensity measurements are useful in determining a large variety of properties of a large variety of samples.

Color has been used as a discriminator throughout our lives. Paint is chosen based on color. Fabrics and makeup are chosen based on color. Color printed material requires a matching of colors. In the laboratory, color is frequently used as an indirect measure of changed environmental parameters. The utility of color in many tests presently relies upon the eye's unique ability to discriminate subtle changes in intensity or wavelength under various and varying lighting conditions. However, human eyes are not always particularly precise or quantitative, and changes in ambient light can affect the eye's ability to differentiate color properly.

Microscopic and macroscopic particles contained in a sample can diffract, reflect, transmit, scatter, fluoresce, phosphoresce, or absorb light. In the case of fluorescence and phosphorescence, the emitted light is typically of a different wavelength than the incident light. The amount of light absorbed, scattered, transmitted, fluoresced or phosphoresced can provide an indication of certain physical characteristics of the substance into which an incident light is directed. The amount of light scattered, and the angle of the scattered light relative to the incident light, can be used as an index of particle concentration and size. Present instruments that measure light scatter and transmission properties are complex. These instruments typically use a non-solid state illumination source and have poor power efficiencies. Such instruments are useful in nephelometry, turbidity, bacterial suspension assays and biomass determinations.

Many laboratory and hospital tests might benefit from a photometric technique that could detect color intensity differences quantitatively under normal ambient laboratory lighting conditions. Many chemical tests that rely on a colorimetric indicator and colorimetric changes are related to the quantity of analyte in solution. Common colorimetric examples include ELISA (which is useful in clinical and biological tests) , test tubes coated with antibodies which can be used in clinical and environmental assays, direct determinations (for example pH, where the colorimetric indicator is directly sensitive to the concentration of the analyte oxygen content) , various clinical tests, immunological tests, tests for specific diseases, and classes of colorimetric tests that determine the concentration of individual components within a compound or material.

Photometric determinations have traditionally been made by spectrophoto eters. Illumination sources for spectrophotometers typically include either a tungsten element lamp or a rare gas arc lamp, which provide a wide bandwidth output and high intensity. However, the energy conversion efficiencies of these instruments are low, and they generally are powered by 120 volt ac sources, and dissipate tremendous heat. Additionally, the tungsten element illumination sources have a relatively short life, are susceptible to damage from vibration, and experience frequency and intensity drift. Circuitry required to stabilize such drift is relatively expensive and complex. The power requirements for most AC powered instruments are over 100 watts. Some instruments of this type incorporate a flashlight-style light bulb, which provides a wide band illumination and thus requires bandpass filtering to remove the wavelengths that would corrupt a particular photometric or colorimetric determination.

Generally, only narrowband illumination is desired in most standard testing. Thus, when a broadband illumination source is used, undesired wavelengths must be removed, for example using a diffraction gratings. This leads to difficulties, however, a diffraction gratings generally require collimated light from a thermally stable optical bench. Optical assemblies are also not flexible in design. Broadband light from tungsten element lamps generally can be filtered with a narrow bandpass dichroic filter. However, dichroic filters shift the bandpass wavelength as the angle of incident light changes. Unstable optical paths, movement of the tungsten element, frequency drift of the lamp, and the deposition of particulate from the heated element onto the inside surface of the glass lamp enclosure all contribute to an unstable system. Such unstable systems have a low resolving power.

Photometers and spectrophotometers generally have a fixed design positioned on an optical bench. To change the design, the optical bench must be changed. Often the component values must change to accommodate changes in amplifier gain, and a thermal analysis must be completed to ensure that the new design will accommodate the heat dissipation. The enclosure also must be appropriately modified.

The resolving power of a spectrophotometer is, in part, dependent on the instrument's ability to reject stray ambient light. Ambient light from the surrounding environment can add an artifactual offset to the measured light signal. Manufacturers therefore have usually shielded the sample container and the optical path from ambient light. This shielding has been costly and cumbersome.

Some prior instruments have sought to subtract or integrate out the effects of high ambient light levels. For example, the light intensity from a sample with an impinging light source being turned on and off, the latter being an ambient light measurement, which is then subtracted from the former. The result, in those circumstances that the ambient light intensity is constant, equals the illuminating light intensity (the light intensity produced by the light source alone) . However, if the ambient light intensity is not constant, as happens with fluorescent or incandescent lighting that varies sinusoidally, the subtraction provides different results at different times and different phases.

Measuring the collective light intensity and ambient light intensity simultaneously requires two different sets of signal processing components. Each set is subject to its own imprecision and noise errors. Therefore, simple signal subtraction does not necessarily yield a precise indication of the level of the illuminating light intensity. A strategy to overcome this deficiency is to measure many signals from both sets of components over extended periods, thereby integrating the effect of noise. However, such integrating techniques are considered best suited for removing high and very low frequency noise, not for removing the ac component associated with standard ac power sources.

The commercial market for tissue culture supplies is very large. Yet optical pH detectors, pipette tip absorption readers, colorimetric flow cells, and fluorescent assay instruments are not generally commercially available for quantitative use under ambient light conditions. Most commercial instruments require a high power light source (typically greater than 1 watt) , special light shields, and/or a photomultiplier or an avalanche photodiode. Present acquisition systems typically require dual power supplies ranging from 5 to 15 volts.

Cancellation circuits, of the type that may be used to subtract one signal from another signal, are known in the art. Besar, et al., "Simple Fiber Optic Spectrophotometric Cell for pH Determination," J. Biomed. Eng. Vol. II, March 1988, describes a set of shielded photodiodes and two light-emitting diodes (at 565 and 635 nanometers) that may be used for phenol red pH determinations. The anode of one photodiode is connected to the inverting input terminal of an operational amplifier and the cathode of a second photodiode is connected to the operational amplifier's non-inverting input terminal. The opposite photodiode terminals are connected to ground. One photodiode detects light intensities, while the other photodiode normalizes for fluctuations in the light source. An instrumentation amplifier can be substituted for the above operational amplifier. The sensitivity of the isosbestic point to temperature and concentration changes, and the use thereof, are not described in the article. Nor does the article describe operation in a backscatter mode or under ambient light conditions. Imbalances in photodiode response and intrinsic noise in the Besar et al. system leads to large noise values and narrow dynamic range.

It should be appreciated from the forgoing description that there is a need for a device that can quantitatively measure the light intensity of an illuminated sample under ambient light conditions, where the intensity of the ambient light can vary

(typically sinusoidally at 50 or 60 Hz) . The present invention fulfills this need.

SUMMARY OF THE INVENTION The present invention relates to a device that determines an optical intensity of light emanating from a sample situated under a periodically varying ambient light. The device includes a selectively illuminating light source that can be switched between on and off states that illuminates the sample. A detector detects the optical intensities of the sample a first plurality of times with the effects of the illuminating light source to produce a plurality of collective light intensity measurements, and a second plurality of times without the effects of the light source to produce a plurality of ambient light intensity measurements. A processor quantitatively determines, based upon the multiple collective light intensity measurements and the multiple ambient light intensity measurements, the intensity amount of each of the collective light intensity measurements that results from said illuminating light source.

A feature of the present invention involves doing the above detecting in the presence of ambient light. The ambient light has a high frequency component, a 60 Hz component that results from the frequency of the power source, and a low frequency component. There are three filtering techniques that provide filtering of light at each of the prescribed frequencies. These filtering techniques include high pass filtering, low pass filtering, and notch filtering that filters out the effects of ambient light. The frequencies at which the filters function can be adjusted depending upon the specifics of the ambient light being provided and the optical intensity of the sample.

Another feature of the present invention is that the system functions effectively whether the sample is contained in a t-flask, pipette, cuvette, of any other well known type of container that the light can pass through. The present invention may be applied to measure the optical intensity of a sample by itself, a sample with an agent contained in it, a sample with particulates in it, or any other type of sample. The measured intensity can be a result of fluorescence, transmittance, particulate refraction, or other types of optical phenomenon.

Other features and advantages of the invention should become apparent from the following detailed description of the invention taken in conjunction with the accompanying drawings that illustrate, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of light intensity measuring apparatus of the present invention;

FIG. 2 is a partial cross-sectional view as taken through section lines 2-2 of FIG. 1, with a container holding a sample positioned above the light intensity measuring device;

FIG. 3 is a block diagram of one embodiment of light intensity measuring device of the present invention that may be applied to the FIGS. 1 and 2 light intensity-measuring apparatus;

FIG. 4 is a block diagram of an alternative embodiment of light intensity measuring device of the present invention that may alternately be applied to the FIGS. 1 and 2 light intensity measuring apparatus;

FIG. 5 is side elevational view illustrating light pathways in an alternative embodiment of light intensity measuring apparatus of the present invention;

FIG. 6 is a top elevational view illustrating light pathways in an alternative embodiment of the light intensity measuring apparatus of the present invention; and FIG. 7 is a block diagram illustrating the light pathways in a final embodiment of light intensity measuring apparatus of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As shown in the exemplary drawings, and particularly in FIGS. 1 and 2, there is shown a first embodiment of an apparatus 20 for measuring the intensity of light of a particular wavelength emitted from a liquid medium or sample 22. The sample is contained in a container 24, and is exposed to background, ambient light of varying intensity. A pair of light-emitting diodes (LEDs) 26a and 26b and a pair of photodiodes 28a and 28b are located on a printed circuit board 30, which is positioned immediately beneath the container (in this case, a t-flask) . In this specification, the term "LED" is used even though a laser diode may be substituted therefore, and the term used herein is intended to cover both types of light emitting diodes. Light from the LEDs is directed upwardly at the sample, which responds for example by reflecting a certain amount of light off a sample/air interface 32. The light that is reflected might be travel in multiple directions, one of which is toward the photodiodes. The amount of light that returns to the photodiode is a quantifiable physical characteristic of the sample that may be related to a variety of physical attributes of a variety of samples. The photodiodes are positioned such that they receive substantially equal amounts of background, ambient light, e.g., from a source 34. The photodiode 28a is positioned between the two LEDs, e.g., 1 to 5 millimeters (mm) away, and the photodiode 28b is spaced further away, e.g., 5 to 25 mm away, such that it receives relatively less light from the sample. Although two LEDs are illustrated in FIGS. 1 and 2, any number of LEDs may be used.

The apparatus 20 includes a light intensity detection circuit 36, one embodiment of which is depicted in FIG. 3. This circuit measures and stores the intensity of light impinging on the photodiodes 28a and 28b. The measurement made from the photodiode

28b closely corresponds to the intensity of background, ambient light, especially when the distance between the photodiode 28b and the LEDs 26a and 26b is increased. The circuit incorporates both analog and digital circuitry, including an operational amplifier

(op-amp) 38, an analog-to-digital converter (ADC) 40, and a microcontroller 44 that has an integrated digital signal processor (DSP) portion 42.

More particularly, the two photodiodes 28a and 28b are arranged in a parallel, reverse-biased relationship, with one terminal connected to the op-amp's inverting input terminal and the other terminal connected through a resistor 46 to an adjustable voltage reference provided by a first resistor divider 48. The resistor 46 preferably has a resistance of about 4 mega-ohms. A second resistor divider 50 is connected to the op-amp's non¬ inverting input terminal, and a second resistor 52 is connected between the op-amp's output and inverting input terminals. To calibrate the light intensity detection circuit 36, the two resistor dividers are adjusted to provide the desired dc voltage levels to the op-amp's two input terminals.

The resistances of the photodiodes 28a and 28b generally vary linearly with the intensity of incident light. Thus, if the two photodiodes receive equal amounts of light, no net electrical current will be produced. On the other hand, if one photodiode receives more incident light than does the other, a net positive or negative current will be produced, which the op-amp 38 converts into a voltage output signal of corresponding amplitude. The ADC 40 converts this voltage into a corresponding sequence of digital words, at a prescribed sample rate, and these words are supplied on lines to the microcontroller 44/DSP 42, for further processing. Operation of the LEDs 26a and 26b and the ADC are controlled by the microcontroller 44/DSP 42.

To maintain a nearly constant light intensity output from each LED 28a, 28b, despite temperature variations, an LED output compensation circuit (not shown) is included. A commercially available constant current source is connected from a voltage supply to a node, and provides a constant current to the node. The LED and a parallel-connected resistor are connected from the node to ground in parallel. The intensity of the light emitted by the LED varies as a function of both internal temperature and external temperature. As the intensity of the emitted light changes, the internal resistance of the LED also changes. The parallel-connected resistor, which preferably has a resistance between 10 - 5,000 ohms, functions as a current shunt. Thus, for example, if a temperature change causes the resistance of the LED to decrease, causing a decrease in its light intensity, current is shunted to the LED from the parallel-connected resistor. This increased current compensates for the temperature-dependent intensity change. Consequently, the LED does not require a separate servomechanism to regulate its intensity. Instead, the LED self-regulates via its own internal shunting mechanism, shunting current to and from the parallel-connected resistor, as needed.

In operation, the microcontroller 44/DSP 42 controls the LEDs 26a and 26b such that they receive pulses of electrical current, at a frequency of 2000 Hz and a duty cycle of 10%. The current preferably pulses between 2 and 200 milliamps. Other frequencies and duty cycles alternatively can be used. Depending on the particular nature of the sample 22 being tested, the LEDs preferably emit light having wavelengths between 420 and 3900 nm.

The more remote photodetector 28b typically receives a lower intensity of illuminating light from the sample than does the photodetector 28a. Since some illuminating light that emanates from the LEDs 26a, 26b is received by both photodetectors, the calculations associated with subtracting the ambient light appear to become very complex. However, calibrations can be used to compare the resultant output light intensities of an unknown sample with one or more resultant output light intensities of samples that have a known optical characteristic. Such known optical characteristics may result from, for example, using a control sample having a known pH. Improved accuracy can be obtained by adding additional calibration steps with a reference sample having a known pH. Other calibration techniques also can be used, for example, color changes present in a base tetramer and an acid tetramer, which can be ratioed to obtain a value related to pH.

FIG. 4 depicts an alternative embodiment of a light intensity detection circuit 56 for measuring the intensity of light emitted from the sample 22 in the presence of ambient light and light produced selectively from a LED 61. This embodiment incorporates only digital circuitry. The FIG. 4 circuit differs from the circuit 36 of FIG. 3 in that light emitted from the sample is not detected by two photodiodes, as described above, but rather by a single light-to-frequency converter 58, which produces digital signals whose frequencies vary substantially directly with the intensity of impinging light. The light-to-frequency converter includes a photodetector and a voltage-controlled oscillator integrated to the same substrate. One suitable light-to-frequency converter is available from Texas Instruments, under the part number TSL230.

The DSP portion 60 of the microcontroller 44 of FIG. 4 is configured to count the number of pulses produced by the light-to- frequency converter 58 for prescribed time periods, preferably over a period that lasts for an integral multiple of 1/60 of a second. This provides a measure of the intensity of light impinging on the converter. The light intensity detection circuit 56 makes optical measurements with the LED 61 biased OFF, to measure the contribution of ambient light. Also the circuit makes optical measurements with the LED biased ON, to measure the combined effects of ambient light and light from the LED.

Ambient Light Characteristics and Digital Filtering Techniques

This section describes signal processing techniques of the DSP portion 42 (FIG. 3) or the DSP portion 60 (FIG. 4) of the microcontroller. Both signal processing techniques can be used effectively to filter out the effects of ambient light, while accurately measuring the illuminating light produced by the LED 26a, 26b (FIG. 3), or 61 (FIG. 4). Ambient light typically varies substantially with time, with that variation including a low- frequency (or dc) component, an ac power line component (60 Hz in the U.S.), and a high-frequency component.

The preferred embodiment of the present invention uses three distinct DSP filtering techniques. Each filtering technique removes the effects of a separate component of ambient light. The signal can be transferred to a computer or a variety of electronic media to be stored and monitored, and to provide further system capabilities.

The first filtering technique implemented by the DSP 42 or 60, which is akin to high-pass filtering, involves modulating and demodulating the signal produced by the LED 61 synchronously with the sampling by the light-to-frequency converters in the FIG. 4 embodiment, and measuring the light intensity using the photodetectors of FIG. 3, and the light-to-frequency converters of FIG. 4, at a rate considerably higher than the periodic rate of the ac current. Thus, a first set of intensity measurements (in both the FIGS. 3 and 4 embodiments) are made while the LED is OFF, and a second set of intensity measurements are made while the LED is ON. This cycling of the LEDs on and off, and the synchronized measuring of the light intensity by the photodiodes or the light- to-frequency converter, preferably occurs at a very high rate such as over a hundred times in a fraction of l/60th of a second.

The second filtering technique, which is akin to notch filtering, removes abnormalities that are due to ambient light variations occurring at the frequency of ac power, and at harmonics of that ac power frequency. This technique involves sampling the signal produced by the op-amp 38 (FIG. 3) , or the light-to- frequency converter 58 (FIG. 4), over a l/60th second sampling period, or a multiple thereof (such as l/60th, 2/60th, 3/60th, . . . , 60/60th of a second, up to and including several seconds) The l/60th second sampling periods provide a 60 Hz notch filter effect.

The third filtering technique, which is akin to lowpass filtering, removes noise occurring at relatively high frequencies. It is accomplished by sampling the signal often within a relatively short duration, e.g., 128 times over a period of considerably less than 1/60 second, and then averaging the many samples. Momentary aberrations resulting from high-frequency noise components, thereby, are removed. Alternatively, this averaging can be substituted by digital lowpass filtering.

The preferred embodiments of the present invention combines the three filtering techniques described in the previous three paragraphs. The unique combination of these three filtering techniques effectively filters out the effects of ambient light from a signal representing the combined light intensities of ambient light and light resulting from an illuminating light source. This unique combination, for example, led to the discovery that the pH concentration and temperature errors could be determined and controlled in phenol red colorimeter determinators that are under near the isosbestic point of phenol red (approximately 460 to 480 nm) , as now described.

Alternative Configurations and Applications

FIGS. 1 to 4 show that the apparatus can measure a low level of illuminating light emanating from a medium or sample 22, which is exposed to a relatively high level of ambient light. In this section, alternative embodiments of the present invention are described. These embodiments are illustrated in FIGS. 5, 6 and 7, each being usable either with the analog light intensity detection circuit 36 of FIG. 3 or the digital light intensity detection circuit 56 of FIG. 4.

An example of a property of certain samples, for which the illuminating light intensity of the samples provides important information, is pH. The pH of a sample may be tested by adding an agent (e.g., phenol red) that alters the color of the sample, and by then detecting the illuminating intensity emitted from the sample at prescribed wavelengths. Phenol red is applied at a preferred concentration of 5 ng/ml to 20 mg/ml, as is known in the medical testing field. Phenol red changes color based upon temperature and concentration, and it is only slightly influenced by the samples' pH at wavelengths of 470 nm. Therefore, phenol red is a unique reference since it acts as a control for concentration and temperature changes, and it can be used to normalize colorimetric pH determinations at various concentrations and temperatures. At wavelengths greater than 630 nm, the color intensity is sensitive to temperature or concentration changes, as described in Besar, S.S.A. et al., "Simple Fiber Optic Spectrophotometric Cell for pH Determination," J. Biomed. Eng. Vol. 11, March 1988. Consequently, these changes cannot be normalized. In the preferred embodiments, an averaged 630 nm was divided by an averaged 470 nm absorption signal reference to correct for temperature and concentration variations. The result is related to the samples' pH.

With reference again to the embodiment of FIG. 2, the apparatus 20 has particular utility for the sample 22 contained in the t-flask 24 used for the cell culture. The LED 26a emits light at a wavelength of 470 nm, and the LED 26b emits light at a wavelength of 630 nm. The 470 nm LED is used as a reference. Light from LEDs 26a, 26b is directed at (and through) a bottom wall 24 of the t-flask, into the liquid sample 22. A 25 ml t-flask having 2 to 25 ml of cell culture sample containing a prescribed amount of phenol red has been found suitable. The LED light from LEDs 26a, 26b follows respective paths 66a, 66b through the sample, reflected off a sample/air interface 32 and follows respective paths 70a, 70b back through the sample. The light following the paths 70a and 70b is backscattered such that the light intensity of each path is detected by the light intensity detection circuit 36. Phenol red, which is present in the sample, absorbs some light passing through the sample based upon Lambert's Law: T = 10^"αcx where T is transmittance, is the absorption coefficient, c is the concentration of absorbers, and x is the overall thickness of the absorbing sample. The light intensities received by the light intensity measuring device are processed as described in the signal processing section, to obtain the signal intensity under ambient overhead lighting conditions. The light generated by each LED 26a, 26b is processed in the same manner. The light generated by LED 26a is used to normalize for temperature and concentration variations of the agent in the sample. The light from the LED 26b is absorbed by the sample containing phenol red (wavelengths from 500 nm to 590 nm will work) in related to the resultant intensity of the sample. In the FIG. 5 embodiment, the light output of a 565 nm LED 74 is directed along at least one light path 76 through a pipette tip 78 into a light intensity detection circuit 80 (of the type illustrated either in FIGS. 3 or 4) . A 200 μl pipette tip (as produced by Sigma™, St. Louis, MO) is suitable. The pipette tip 78 containing water is interposed in the light path between the LED and the light intensity measuring device to act as a control. The number of pulses counted, after signal processing, is proportional to a 100% light transmission. A second pipette tip of similar size and design is filled with the sample (assume, for example, a 2% red dye) and interposed as before in the light path 76. The red dye absorbs green light of 565 nm, and therefore diminishes the green light received by the light intensity measuring device. After signal processing, the number of output pulses counted from the light intensity measuring device is less than the number counted by the 100% transmission reference. Diminution of the light received by the detector is related to the red dye concentration as governed by Lambert's Law. LEDs are selected based on how well the light output at the LED's wavelength is absorbed by the sample in the pipette tip, and a light output that is sufficiently intense for the voltage-to-frequency converter to generate pulses in response to the signal. This embodiment preferably uses a 68HC705P9 microcontroller (that incorporates the DSP portion) and a TSL230 light-to-frequency converter. LEDs at 420 n , 565 nm and 590 nm could be used with an associated four-digit display; or a 400 nm, 500 nm, or 700 nm LED could be used for the entire visible spectrum. The instrument is powered by a 9-volt battery and works under various and variant ambient light conditions. This embodiment is best suited to measure the optical effects of colorimetric indicators applied to a sample, and the diffraction of the sample.

In the FIG. 6 embodiment, light from a LED 80 (preferably 459 nm, though various wavelengths will work) is directed along light path 82 toward a sample 84 contained in a container 86 to measure fluorescence of the sample. The light intensity detection circuit 88 (of the type illustrated in FIGS. 3 or 4) is oriented at a right angle to the light path 82 facing the sample. Some light that encounters the sample causes the sample to emanate light based upon fluorescent principles. The fluorescent light follows light paths 90a, 90b, 90c, 90d, and other non-shown light paths spaced about the periphery of the container. The wavelength of the light following paths 90a, 90b, and 90c usually differ from the wavelength of the light following path 82. An absorbent filter 92 is interposed between the light intensity detection circuit 88 and the container 86. The filter filters out the light at the incident wavelengths, while permitting light at the fluorescent wavelengths to pass. The absorbent filter and the light intensity measuring device are oriented 90 degrees to the light path 82, to reduce the flux of excitation photons on the filter spatially. The absorbent filter may be a dichroic filter or an absorption filter. The absorbent filter is selected to allow light that is of the wavelength range(s) of the fluorescent emissions to pass, while absorbing the light that is of the wavelength range(s) produced directly from the LED 80. The only light that the light intensity measuring device receives is the ambient light and the filtered fluorescent light. This filter wavelength selection is generally known by those skilled in the art. The light received by the light intensity measuring device is translated into an output signal, which is processed by the DSP portion of the microcontroller as previously described. The level of fluorescent emissions relates to the number of pulses. Thus, most of the LED light does not reach the filter, but the spherically radiated fluorescent excitation light will reach the filter and pass through to the detector.

In an alternative embodiment from that shown in FIG. 6, multiple photodetectors are placed around a pipette such that the detector is not in the direct light path of the LED. Each detector faces toward the sample, and has an absorbent optical filter (similar to 92 above) interposed between the detector and the pipette tip. Each filter has different band pass properties so that each detector views the intensity of the light provided by a different color fluorescent light. Using this design, and with the signal processing features previously described, several fluorescent colors intensities can be simultaneously collected from the same sample. One or more fluorescent dyes can also be used in the sample contained in the pipette tip.

In the FIG. 7 embodiment, multiple light intensity detection circuits 94a, 94b, and 94c are placed at different positions about a container 96 that holds a sample 98 to measure the diffraction/transmission characteristics of the sample. A normal 106 to the light intensity detection circuit 94a is parallel to, and coincident with, the normal 104 in the middle of a sample insertion hole 108. A container that contains a sample with particulate matter is positioned within the sample insertion hole. Light entering the sample container that is scattered approximately 90°, and to the left as illustrated in FIG. 7, from the incident light is collected by the 90° light intensity detection circuit 94b. The intensity of light scattered by particles in the sample, and thereupon received by the light intensity detection circuit 94c is related to the particulate size and concentration of the particulate matter, and can be compared with the other light intensities detected by the light intensity detection circuits 94a, 94b. By placing one or more light-to-frequency converters 94a, 94b, 94c at various angles, an estimate of the concentration and size of particles in solution can be obtained. Scatter and absorbance information is useful in calculating turbidity, colorimetric densities, numbers of bacteria in solution, concentration of pollutes, biomass, and concentration of molecules or compounds.

The sample aperture is configured to hold a sample container, and can be either round for test tubes or square for cuvettes, for example. An insert can alternatively be placed into the sample aperture to hold containers having various sizes and shapes as would be generally understood by those skilled in the art. A square cuvette sample guide may be used. A cuvette sample guide is molded into the top of a standard commercially available enclosure by methods generally known in the art. The sample aperture can extend approximately 2 cm down from the wall through a sample insertion hole formed in the printed circuit board. Displaceable prongs (not shown) might extend toward an opposite wall such that they are contacted, displaced, and an associated electrical contact is made or broken when the cuvette is inserted into the sample insertion hole. In this manner, the light intensity detection circuit becomes activated when the cuvette is inserted into the sample hole. Light paths in sample guide walls are placed to allow light to pass from the LED (See FIG. 1) to one or more light-to-frequency converters. Light paths can be positioned anywhere in the sample guide wall.

Certain embodiments of the present invention may be applied to a variety of applications. For example, for ELISA testing, a commercially available TMB™ is used as the chromogen. A sample reacts in a cuvette using horseradish peroxidase as the catalyst.

For a positive reaction the TMB™ turned color and was stopped.

Colorimetric determinations were made in the apparatus of FIG. 7.

Changes were quantified and displayed on the LCD display. The wavelength tested was 450 nm, one or more other wavelengths could be used instead.

The light intensity measuring devices shown herein can be used in other, non-pH environmental assays as well. For example, an antibody to a known toxin (e. g. , PCB) is coated onto the inner walls of a test tube. A solution containing an unknown amount of PCB is then added to the test tube. An enzyme labeled antibody is then added to the test tube and allowed to react. After the reaction, the non-bound antibody is removed and a chromogen added. The chromogen produces a color change that is related to the PCB concentration. Colorimetric determinations (which may use the embodiments illustrated in FIGS. 1, 2, 5, and 7) can be made by inserting the sample container into the sample guide of the present invention, and comparing how much light is absorbed by the reacted chromogen to known standards. The result would provide an accurate indication of the unknown concentration of PCB in the solution. The colorimetric determinations may be calibrated against other samples with known PCB concentrations. Other colorimetric indicators include, but are not limited to, Alamar Blue™ for metabolic and cell mass determinations, TMB™ for ELISAs, and

BCIP/NBT™ for ELISAs. Fluorescent and phosphorescent compounds

(such as measured by the FIG. 6 embodiment) can be used for oxygen,

DNA and RNA determinations. Light scatter (such as measured by the

FIG. 7 embodiment) can be taken at a narrow angle (<90°) and a large angle (approximately 90°), to be used for size, concentration, bacterial suspensions, turbidity and biomass determinations.

Bacterial suspensions could be used for antibiotic and toxicity testing. Bacterial suspensions of known concentration can be suspended in a solution and analyzed for 630 nm light scatter.

After a base line of light scatter is established, a toxic analyte can be added to the bacterial suspension. As the bacteria die from exposure to the toxin, the solution clears and a lower light intensity is read by the photodiode or the light-to-frequency converter which is positioned perpendicular to the LED normal. The reduction of the light scatter is related to the concentration of the toxin. The bacteria can be preselected for intolerance to a specific substance or genetically altered to become intolerant to a specific substance. Toxins include a variety of organic and inorganic materials. The toxins can include toxic bacterial or cells, inorganic pollutants, antibiotics, peptides, proteins, hydrophobic materials and temperature sensitive materials.

Alternatively, the present invention can be used to adjust inoculum density against a McFarland Standard. The present invention can be calibrated with McFarland standards. Light scatter from an unknown concentration of bacteria can be compared to controls that have known bacterial concentrations. The bacterial suspension is then adjusted, by adding additional bacteria or diluting the solution, to achieve the desired light scatter.

For scatter testing, an apparatus was constructed as illustrated in FIG. 7, with a light intensity detection circuit located at 90° to the LED normal and a second light intensity detection circuit juxtaposed to the LED normal. Commercially available nephelometry standards or calibration standards for turbidity measurements are placed in round test tubes and placed into the apparatus. Light scatter is measured and displayed on the LCD display. Additionally, commercially available McFarland standards are obtained to establish bacterial suspension calibration. The standards are placed in round test tubes and scatter measured. Results are displayed on the LCD display. The resultant resolution is sufficient to resolve 0.1 McFarland units in a range of 0.1 to 5.0 Alternatively, the light collection from photodetectors (FIG. 3) or light-to-frequency converters (FIG. 4) that are directed perpendicular to the incident light can be used to acquire sample florescence or phosphorescence. The wavelengths tested were 590 nm, 630 nm, and 660 nm, one or more other wavelengths could be used including 625 nm.

The synchronous modulation and demodulation of the LED with each light intensity detection circuit is controlled by the microcontroller. The power to the LED is turned on/off by the microcontroller to synchronously modulate and demodulate the signal. Light from LED is directed at, and controlled by, the light intensity detection circuit to determine how much light is absorbed, scattered, transmitted, or fluoresced by the contents of the sample container. Sample containers are held in place by the sample guide positioned within the sample insertion hole. The microcontroller turns the power to the light intensity measuring circuits on when the signal is collected and off when the light intensity measuring circuit not required, thereby conserving battery power. The light-to-frequency converters provide a 50% duty cycle at a frequency that is proportional to the incident light. Additionally, the microcontroller collects the output signal from the light intensity detection circuit. The output of the light intensity detection circuit has a frequency that proportionally ranges to the input from 0 Hz to 500,000 Hz. The signal is acquired when the microcontroller counts the number of pulses generated during a fixed time interval. The number of pulses counted is related to the wavelengths of the incident light, and the resultant light intensity emitted from the sample.

Alternatively, the sample in the pipette tip, in the FIG. 5 embodiment, is replaced by a sample containing cells. These cells are stained with colorimetric stain or fluorescent stain. Staining intensity, fluorescence, or intensity of color, is related to the function for which the stain is associated. An example of a colorimetric indicator is AlamarBlue™ (produced by Alamar Biosciences, Inc., CA) , which shifts color absorption of the cells to somewhere between the wavelenghts of 570 nm and 600 nm, the particular wavelenght depends on the viability of the cells contained in the sample. AlamarBlue also has an excitation/emission spectra change as a function of cell viability in the sample to somewhere between the wavelenghts of 560 nm and 590 nm, respectively. Another example of applying cells to the present apparatus is to detect sperm viability using two color fluorescence by replacing the sample with sperm mixed with live/dead FertLight™ (produced by Molecular Probes, Inc., OR).

In an alternative embodiment, two or more LEDs, or laser diodes, formed within an integrated housing, can be substituted for any of the above described LEDs having a single wavelength. The ratio of transmittance using simple ratios, or the quadratic equation, can be used to detect humidity or to detect the presence of water, carbon dioxide, oxygen, or other absorbing materials at wavelengths in the visible and near infrared spectra. These detections can occur under ambient light conditions. Water has an absorption band at approximately 970 nm i 40 nm. A LED that emits light in the 970 nm absorption band can be ratioed with absorption at a lower wavelength, outside the water absorption band, to obtain an index of water that is present. A similar approach can be used to detect the level of carbon dioxide or other gasses.

A variety of containers can contain the sample for any of the above embodiments. These containers include, but not limited to, cuvettes, pipettes, t-flasks, test tubes, and virtually any container that light can pass through such that the optical characteristics of the sample can be measured. In addition, different configurations may be used to measure light emanating from sample under different mechanisms> such as fluorescence colorimetric measurements, diffraction, transmittance, and reflectance. Colorimetric and fluorescent measurements, associated with special signal processing methods, can be used to detect pH of the sample under ambient light optically, without external temperature regulation. In an alternative embodiment, a container having static fluid can be replaced by a tube through which fluid flows. Colorimetric, fluorescent, diffraction, transmittance and reflectance changes over time can be monitored.

Preferred embodiments of the present invention resolve at least to 0.5 parts in 1000, have a dynamic range beyond 3.0 optical densities, have a 1% variation from linearity over the entire range, and have a battery life of 2 years that is sufficient for thousands of tests. The produced apparatus is solid state, is pocket-calculator in size, does not have an optical assembly, displays a 4 digit result, and does not require ambient light shielding. These characteristics make the present system easier to use accurately than the prior art systems. The LED illumination source is preferably solid state, is efficient, is narrow band, consumes low power because of the intrinsic high efficiency, and does not require a warm-up period. The preferred embodiment uses a microcontroller that allows the apparatus to always be at least partially on and to turn the desired components on or off as needed. The present invention uses a LED on a standard printed circuit board without an optical bench, collimating optics, focusing optics or diffraction gratings.

It will be apparent to those skilled in the art that various changes can be made in the invention without departing from the spirit and scope thereof, and therefore the invention is not limited by that which is shown in drawings and specifications as preferred embodiments of the present invention, but only as indicated in the appended claims.

Claims

What is claimed is:

1. A method for detecting the intensity of light emanating from a sample, wherein the sample is in the presence of periodically varying ambient light, the method comprising: illuminating the sample with light from an illuminating light source; detecting the intensity of light from the sample a first plurality of times to provide a plurality of collective light intensity measurements, while the sample is being illuminated with light from the illuminating light source; detecting the intensity of light from the sample a second plurality of times to produce a plurality of ambient light intensity measurements, while the sample is not being illuminated with light from the illuminating light source; and filtering out the effects of the periodically varying ambient light from the collective light intensity measurements to provide a quantitative measurement of how much of each collective light intensity measurement results from the illuminating light source.

2. The method as defined in claim 1, wherein certain of the collective light intensity measurements at least partially overlap in time with certain of the ambient light intensity measurements.

3. The method as defined in claim 1, wherein said filtering technique comprises removing the ambient light intensity measurement from the collective light intensity.

4. The method as defined in claim 1, wherein said first plurality of times and said second plurality of times are each sampled at a predetermined sampling rate.

5. The method as defined in claim 1, wherein the varying ambient light intensity varies at a predetermined frequency and the first and second sampling rates are selected to provide characteristic notch filtration of photonic radiation at the predetermined frequency.

6. The method as defined in claim 4, wherein the sampling rate is l/60th of a second, or a harmonic thereof.

7. The method as defined in claim 1, further comprising averaging the collective light intensity measurements and the ambient light intensity measurements over a prescribed time period.

8. The method as defined in claim 7, wherein said averaging provides low pass filtration.

9. The method as defined in claim 1, wherein the range of the optical intensity has a range spanning three optical densities.

10. The method as defined in claim 1, wherein said illuminating light source occurs at a prescribed frequency, wherein said optically detecting the sample occurs a first plurality of times; and said optically detecting the sample a second plurality of times each occur at a prescribed frequency range that includes said prescribed frequency.

11. The method as defined in claim 1, wherein said optically detecting steps provides high pass filtration.

12. An method as defined in claim 1, further comprising determining a desired parameter of said sample.

13. The method as defined in claim 1, wherein the desired parameter is pH.

14. The method as defined in claim 1, wherein the desired parameter relies upon laboratory assays that have colorimetric indicators.

15. The method as defined in claim 1, wherein the desired parameter is relies upon laboratory assays that have fluorescent indicators.

16. An apparatus that determines an optical intensity of light emanating from a sample that is situated under a periodically varying ambient light, comprising: a selectively illuminating light source which may be switched between on and off states that illuminates the sample; a detector that detects optical intensities of the sample a first plurality of times with the effects of the illuminating light source to produce a plurality of collective light intensity measurements, and a second plurality of times without the effects of the light source to produce a plurality of ambient light intensity measurements; and a processor that quantitatively determines, based upon said collective light intensity measurements and said plurality of ambient light intensity measurements, the intensity amount of the plurality of collective light intensity measurements that results from said illuminating light source.

17. The apparatus as defined in claim 16, wherein the detector that detects the optical intensities of the sample a first plurality of times is taken at a different location with respect to the illuminating light source than the detector that detects the optical intensities of the sample a second plurality of times, such that the intensities of the periodically varying ambient light are similar during both the first plurality of times and the second plurality of times, so that the detector functions as a high pass filter.

18. The apparatus as defined in claim 16, wherein the illuminating light source is turned on and off synchronously with the detector, so that the detector functions as a high pass filter.

19. The apparatus as defined in claim 16, wherein the determining device acts as a low pass filter to filter certain of the high frequency effects of ambient noise.

20. The apparatus as defined in claim 19, wherein the low pass filter function relies upon a sampling rate of the detector during the first plurality of times and the second plurality of times.

21. The apparatus as defined in claim 16, wherein the determining device functions as a notch filter to filter out certain effects of ambient noise.

22. The apparatus as defined in claim 21, wherein the notch filter function relies upon a sampling rate of both the detector the first plurality of times and the second plurality of times.

23. The apparatus as defined in claim 16, further comprising a pipette tip, wherein said sample is contained in the pipette tip, and said first detector is directed through a wall of the pipette tip.

24. The apparatus as defined in claim 16, further comprising a cuvette, wherein said sample is located in the cuvette, and sai detector is directed at a wall of the cuvette.

25. The apparatus as defined in claim 16, further comprisin a test tube, wherein said sample is located in the test tube, an said detector is directed at a wall of the test tube.

26. The apparatus as defined in claim 16, further comprising a t-flask, wherein said sample is located in the t-flask, and said first detector is directed at a wall of the t-flask.

27. The apparatus as defined in claim 16, further comprising agents that are added to said sample.

28. The apparatus as defined in claim 27, wherein said agent is phenol red.

29. The apparatus as defined in claim 16, wherein the determining means acts as a high pass filter to filtering out the dc components of the ambient light.