WO2013156820A1

WO2013156820A1 - Simultaneous multiple wavelength fluorescence measurement device, methods therefor and system therefrom

Info

Publication number: WO2013156820A1
Application number: PCT/IB2012/051989
Authority: WO
Inventors: Bala S. Manian
Original assignee: Reametrix Inc.
Priority date: 2012-04-20
Filing date: 2012-04-20
Publication date: 2013-10-24

Abstract

The invention relates generally to a fluorescence measurement device and more specifically to fluorescence measurement methods and devices utilizing two independent optics modules for simultaneous measurement of multiple fluorescence from a sample. The device (10) comprises: a sample assembly (12) configured for rotational motion and translational motion of a sample carrier that comprises at least one sample comprising at least one fluorophore. An optics assembly (24) comprises a first optic module (26) having a first focal point and a second optic module (28) having a second focal point, wherein the first optic module (26) and the second optic module (28) are placed spaced apart. A predetermined distance between the first focal point and the second focal point defines a reference line. An inversion point equidistant from the first focal and second focal point situated on the reference line defines a rotational axis for the rotational motion of the sample assembly (12). The predetermined distance defines a diameter for the rotational motion of the sample assembly (12). An axis of the translational motion of the sample assembly (12) is parallel to the reference line. The first optic module (26) comprises a light source for generating a first incident beam having a first excitation wavelength and a first focus diameter to impinge on the at least one sample to yield a first laser spot, and the second optic module comprises a light source for generating a second incident beam having a second excitation wavelength and a second focus diameter to impinge on the at least one sample to yield a second laser spot. The device (10) allows for spatial imaging of a given sample. The imaging occurs without any crosstalk between the light sources having different wavelengths.

Description

SIMULTANEOUS MULTIPLE WAVELENGTH FLUORESCENCE MEASUREMENT DEVICE, METHODS THEREFOR AND SYSTEM THEREFROM

TECHNICAL FIELD

[0001] The invention relates generally to a fluorescence measurement device and more specifically to fluorescence measurement methods and devices utilizing two independent optics modules for simultaneous measurement of multiple fluorescence from a sample.

BACKGROUND

[0002] The use of fluorescence measurements in different applications is known in the art, an exemplary application area includes measurements in biological diagnostics. However, in the making and using of devices for biological sample diagnostics and detection that are based on fluorescence measurements, several problems are encountered. Typically, the sample fluorescence is measured at a single wavelength. For multiple measurements at a plurality of wavelengths, sequential measurements are made over a period of time, during which time the sample may undergo changes. Further, such measurements do not allow for high throughput and high content sample diagnosis and detection as the time taken for each measurement at every wavelength is finite. Also, systems capable of simultaneous measurements such as flow cytometers tend to be quite complex in their construction and use.

[0003] All of the methods and devices mentioned herein suffer from the drawbacks that include at least one of being expensive, requiring considerable space and other requirements such as power consumption, using expensive reagents and consumable/ disposable parts, not capable of being used in a harsh and resource-scant environment, and are at best capable of very limited analysis, not conducive for adaptation for high throughput analysis, to name a few problems. Hence, there is a dire need to make available a device that can address all these drawbacks, and accordingly a method that can be adaptable to be used to obtain multiple vital information from a sample in a reasonable amount of time.

BRIEF DESCRIPTION

[0004] In one aspect, the invention provides a fluorescence measuring device comprising a sample assembly and an optics assembly. The sample assembly is configured for rotational motion and translational motion. The optics assembly comprises a first optic module having a first focal point and a second optic module having a second focal point. The first optic module and the second optic module are placed spaced apart at a predetermined distance. This predetermined distance between the first focal point and the second focal point defines a reference line. The predetermined distance also defines a diameter for a rotational axis for the rotational motion of the sample assembly. Also, an inversion point equidistant from the first focal and second focal point along is situated on the reference line that defines a rotational axis for the rotational motion of the sample assembly. The axis of rotation of the sample assembly parallel to the reference line defines a linear axis for the translational motion of the sample assembly.

[0005] In another aspect, the invention provides a method for generating a composite fluorescence image of a sample. The method comprises providing a sample assembly configured to receive a sample carrier that comprises at least one sample containing at least one fluorophore. The sample assembly is capable of rotational motion and translational motion. The method also comprises providing an optics assembly that comprises a first optic module and a second optic module. The first optic module and the second optic module are placed spaced apart at the predetermined distance. The method then comprises impinging the at least one sample simultaneously by the first optic module at a first focal point to generate a first fluorescence signal and the second optic module at a second focal point to generate a second fluorescence signal. The predetermined distance between the first focal point and the second focal point is such that it defines a reference line. The impinging is about an inversion point equidistant from the first focal and second focal point situated on the reference line. The method then includes detecting the first and the second fluorescence signals to provide a first image and a second image respectively. The method then involves superimposing the first image and the second image to generate a composite image, wherein the predetermined distance is used for superimposition. The method then involves translating the sample assembly along a linear axis parallel to the reference line while simultaneously rotating the sample assembly on a rotational axis having a diameter defined by the predetermined distance, and repeating steps of the impinging, detecting, and superimposing for each translating and rotating step to obtain the composite fluorescence image at each position of the sample assembly. [0006] In yet another aspect, the invention provides a fluorescence measurement system comprising the fluorescence measurement device of the invention.

DRAWINGS

[0007] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0008] FIG. 1 is a side view of an exemplary embodiment of the fluorescence measuring device of the invention; [0009] FIG. 2 shows another side view of the exemplary fluorescence measuring device of the invention; and

[0010] FIG. 3 is a flowchart representation of exemplary steps involved in the method of the invention.

DETAILED DESCRIPTION [0011] The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0012] As used in this specification and the appended claims, the singular forms "a",

"an", and "the" encompass embodiments having plural referents, unless the content clearly dictates otherwise. [0013] Unless otherwise indicated, all numbers expressing feature sizes, amounts, and physical properties used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein.

[0014] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. [0015] As used herein, sample means any substance that requires analysis for the purposes of either identification of one or more analytes, or measurement of properties, or quantification of one or more analytes, or the like, or combinations thereof. Sample may be in any given physical form, and this includes solution, suspension, emulsion, solid, and the like. In some embodiments, sample is an aqueous solution, and in other embodiments, sample is a suspension in a suitable medium, such as an aqueous medium. Samples are typically derived from any number of sources. In one instance, sample is derived from a body fluid. Body fluids may be derived from human or animal sources, such as primates, dogs, and the like. Body fluids include saliva, sweat, urine, sputum, mucous, semen, and the like. In another instance, sample may be derived from a fluid source, such as water from a reservoir. In yet another instance, sample may be derived from a location such as a cotton swab of a baggage at security checkpoints, which may be used as such or may be suspended in a suitable solvent for analysis. Other types of samples may include samples used in life science research, such as, but not limited to, cell lines. Further examples of samples include samples derived from manufacturing lines used for pharmaceutical or biopharmaceutical product lines.

[0016] Samples useful in the invention comprise at least one fluorophore.

Fluorophore as used herein means any moiety that is capable of being fluorescent upon excitation by a radiation corresponding to the excitation wavelength of the fluorophore, after which it emits radiation having a wavelength, which is referred to as emission wavelength. The fluorophore is attached to the remaining portion of the sample through physical linkages or through chemical linkages. Methods of incorporating fluorophores onto other materials are well-known to one of ordinary skill in the art, and can be arrived at without undue experimentation . [0017] Sample is generally made available for the aforementioned purposes in a suitable sample carrier. The nature of the sample carrier depends on the nature of the sample and analysis being performed. In some instances, sample carrier is a cuvette, in other instances, sample carrier is a well, in yet other instances, sample carrier is a plate. The nature of the sample carrier will also accordingly determine the characteristics of the sample carrier. Thus, a cuvette is characterized by a wall thickness, a depth, a volume, and the like, while a well is characterized by a depth and a volume, and a plate is characterized by width. Sample may be pipetted into the sample carrier, or may be poured in, or may be added as a solid and spread along the surface through application of shear force, or prepared in situ in the sample carrier in a suitable medium, or through any other means known to those of ordinary skill in the art.

[0018] As noted herein, in one aspect, the invention provides a fluorescence measuring device. Turning to Fig. 1, a side view of an exemplary embodiment of the fluorescence measuring device of the invention is shown and is generally depicted by numeral 10. In the exemplary embodiment of the fluorescence measuring device 10, the sample assembly 12 having a rectangular base and a circular top is depicted herein as an exemplary non-limiting embodiment. Other geometric shapes for the base and the top may be envisioned based on the requirements and is contemplated to be within the scope of the invention. The sample assembly 12 as noted herein is capable of translational motion and rotational motion. In the exemplary embodiment shown in Fig. 1, the sample assembly is capable of translational motion along an axis depicted by numeral 14, wherein position depicted by numeral 16 is the first position of the sample assembly 12, and position depicted by numeral 18 is the second position of the sample assembly 12 after undergoing a translational motion along the axis 14. In the exemplary embodiment shown herein, at least the top portion of the sample assembly shown by numeral 20 is capable of rotational motion about an axis depicted by numeral 22. As shown herein, the sample assembly is configured to receive multiple cuvette-type samples on its top portion that is capable of rotational motion about the axis 22. Other shapes and configurations of the sample assembly useful in the invention will become apparent to one skilled in the art and is contemplated to be within the scope of the invention. The manner in which translational and rotational motions of the sample assembly simultaneously can be achieved using methods known in the art. In one exemplary embodiment, such motions are achieved using a stepper motor. [0019] The fluorescence measuring device 10 also comprises an optics assembly, represented by numeral 24 in Fig. 1. The optics assembly 24 comprises a first optic module 26 and a second optic module 28. The first and second optic modules are placed parallel to each other in a mirror image configuration relative to each other. The first optic module comprises a first laser source capable of generating a first laser beam having a first excitation wavelength and a first predetermined diameter and a first focal point. Similarly, the second optic module comprises a second laser source capable of generating a second laser beam having a second excitation wavelength and a second predetermined diameter and a second focal point. The first excitation wavelength ranges from about 600 nanometers to about 800 nanometers, at which wavelength, the laser beam may sometimes be referred to in the art as "red laser". The second excitation wavelength ranges from about 300 nanometers to about 600 nanometers, at which wavelength, the laser beam may sometimes be referred to in the art as "blue laser". These lasers are used to excite the samples at the two different wavelengths by impinging the sample with the two laser spots simultaneously. The two laser spots are present at a predetermined distance from each other. A line joining the two laser spots is referred to as a reference line. A point on the reference line that is equidistant from the first and second laser spots is referred to herein as an inversion point. The inversion point may also be sometimes referred to as locus point or locus, and the impinging is done when the first and second focal point are equidistant from the inversion point. The first laser spot impinges the at least one sample on the sample assembly to excite the fluorophores, if and when present in the sample, to provide a first fluorescence signal. Similarly, the second laser spot is used to produce a second fluorescence signal. [0020] In some embodiments, each optic module comprises more than one laser source, each laser source capable of producing a corresponding laser spot that is impinged on the at least one sample. Each laser spot has a different wavelength such that each sample is capable of being exposed to multiple wavelengths from each optic module. In the instance wherein each optic module comprises more than one laser source, the focal point for the optic module may be the position of one of the laser spots, which then serves as the reference point, or alternately, the focal point may be a point different from the two laser spots, such as the mid-point of the two laser spots. Other variations to determine the focal points will become obvious to one skilled in the art, and is contemplated to be within the scope of the invention. Subsequently, the line joining the focal points will serve as the reference line, and a point on that line as described herein, such as the mid-point of the line may serve as the inversion point.

[0021] The first optic module 26 and the second optic module 28 are located at a spaced apart position from each other such that the first laser spot and the second laser spot are present at a predetermined distance. The predetermined distance defines a diameter for a rotational axis 22 for the rotational motion of the sample assembly. The inversion point serves as the center around which the axis of rotation for rotational motion of the sample assembly is defined. Further an axis parallel to the reference line defines the linear axis 14 for the translational motion of the sample assembly. In some embodiments, each optic module can be configured to include more than one laser source, wherein each laser source is associated with a wavelength. For example, a double laser configuration will have laser sources at four different wavelengths. Such configurations will allow for better spectral resolution of the image obtained from the sample, which also gives rise to more sample information. Further, the fluorescence measurement device of the invention still remains compact despite the presence of additional laser sources in the respective optic modules. This is advantageous over the existing sequential measurement devices such as the flow cytometers known in the art in that the information from the sample is obtained simultaneously in a rapid manner.

[0022] The fluorescence measurement device of the invention allows for configuration that is adaptable to a variety of situations in a facile manner as described herein. Further it allows for spatial imaging of a given sample due to the simplicity of the construction of the device. The imaging occurs without any crosstalk between the lasers having different wavelengths.

[0023] Referring to Fig. 2, wherein another side view of the exemplary fluorescence measuring device of the invention 10 is shown that comprises a detector assembly 30. The detector assembly is used to detect the first and the second fluorescence signals to provide a first image and a second image respectively. The opposite side of the fluorescence measuring device also comprises a second detector assembly (not shown herein). Each detector assembly is associated with an optic module in this exemplary embodiment. However, one skilled in the art will recognize that a single detector assembly may be used for all the optic modules in the fluorescence measuring device. In an exemplary embodiment, detection is achieved by splitting the emitted fluorescence signals into two or more spectral bands to enable more efficient detection to provide enhanced multi-parameter information may be derived for each sample. In one example, the emitted fluorescence signals are split into three spectral bands wherein the first spectral band has a wavelength that ranges from about 650 nm to about 690 nm, the second spectral band ranges from about 690 nm to about 740 nm, and the third spectral band ranges from about 740 nm to about 800 nm. The splitting of the fluorescence signals into spectral bands may be achieved in a facile manner using a suitable device such as beam splitter. [0024] The laser beams are allowed to impinge on the sample thereby causing the fluorophore portion of the sample to be excited. It may be noted that the incident laser beams may also sometimes be referred to as excitation beams. As mentioned herein the incident beam is characterized by the focus diameter, and when the incident beam impinges on the sample, the incident beam yields a laser spot that illuminates a defined volume of the sample. The defined volume of the sample is also referred herein as a sample volume. Thus it would be appreciated by those skilled in the art that the impinging of the incident beam on the sample defines the sample volume that has a defined relationship with the focus diameter of the incident beam. It would also be appreciated by those skilled in the art that the sample volume is a geometrical region comprised within the sample, the exact shape of the region depends on several factors, such as, but not limited to, thickness of the sample carrier, shape of the sample carrier, refractive index of the sample medium, material making up the sample carrier, and the like.

[0025] When the laser spot is focused on the sample volume, the fluorophores on the sample volume are excited giving rise to one or more fluorescence signals. The fluorescence signals of the fluorophore are generally associated with parameters such as an emission wavelength, amplitude, intensity, and the like. The fluorescence signals from the sample volume are then detected using the detector assembly 30. In the exemplary embodiment, the choice of possible wavelengths of fluorescence signals measurable by the fluorescence detector is specifically made such that the chosen spectral region is transparent or at least minimally interfering to other components of the sample that may otherwise severely interfere with the detection. Further, the choice of wavelengths of fluorescence signals allows the use of sample carriers that are made of plastic, which are significantly less expensive than those made of glass or other materials. [0026] After the first set of fluorescence signals from the two sample volumes that had been impinged by the laser beams have been detected, the sample assembly is allowed to translate along the axis described herein, and/or rotated along the rotational axis as described herein. Since the light source is stationary, a spiral scan of the sample carrier is achieved. The relative movement of the light sources and the sample carrier is advantageous since in absence of the relative motion, the laser beam spots are relatively small to interrogate an entire fluorescing reporter of the sample in a single scan. [0027] In this manner, a low resolution preview scan of the at least one sample is made available. The preview scan is conducted to define at least one individual volume of interest. This scan is also used to determine a thickness of the sample carrier based on empirical correlation between the fluorescence signals and the focus diameter. Alternately, the focus diameter may also be pre-defined to approximately match the thickness of the sample carrier. This advantageously allows for use of the sample carrier without the necessity for prior knowledge of accurate thickness value of the sample carrier.

[0028] Without being bound to any theory, when a sample comprising at least one fluorophore is present in a sample carrier, that particular region emits higher levels of fluorescence signals relative to other regions of the sample carrier, this particular region is referred herein as individual volume of interest. The preview scan is done to define at least one individual volume of interest within the sample volume based on the one or more fluorescence signals.

[0029] The preview scan also provides an opportunity to check the presence of the sample carrier, to find the approximate center of the sample carrier, confirm the proper positioning of the sample in the sample carrier, confirm the absence of bubbles, proper sample loading, and other such potential problems. Thus, the preview scan can reduce the most critical tolerance with respect to the fabrication of the sample carrier and other such manufacturing variations. This can have a measureable impact on cost reduction. [0030] As noted herein, in one exemplary embodiment, the preview scan is a Z- scan that is used to define at least one individual volume of interest. The optical scans of the individual volumes of interest in a sample carrier give rise to a distribution of emitted fluorescence signals based on the presence or absence of analytes, with respect to the direction of scan, for example in the depth direction. In one example, the distribution of emitted fluorescence signals is a Gaussian distribution. Thus the method involves processing the emitted fluorescence signals from the individual volumes of interest using a Gaussian curve-fitting method for each Z depth. The processed data in the exemplary embodiment represents Gaussian-fitted intensity maximum as a function of Z, width of the Gaussian maximum (i.e. the measure of the capillary thickness), and the location of the Gaussian maximum along the Z-position, also referred to as depth profile. It would be appreciated by those skilled in the art that an optimum depth or Z-position is useful for an R-theta scan to obtain event fluorescence measurements. [0031] In one exemplary embodiment, for a given dimension of the sample carrier and the focus diameter, a preview scan is conducted at a theta resolution of about 10,000 pixels per revolution and encompasses about a 3mm wide scan (to accommodate a positional error of about +/- 0.25 mm) at about 50 microns spatial resolution, resulting in 60 scans. After detecting the one or more fluorescence signals, individual volumes of interest are located, and the thickness of the sample carrier is obtained as described herein above. These scans provide for a normalized bulk fluorescence measurement of the sample using the depth profile and thickness of the sample carrier.

[0032] Then the depth profile is used to determine at least one microvolume of interest. As already noted herein, the region comprising the at least one fluorophore of the sample would exhibit higher intensity of one or more fluorescence signals. The microvolume of interest would typically be the region exhibiting the Gaussian maximum. The incident beams are focused on the microvolume of interest and translating the laser beam spots in the depth direction to obtain concentrated emitted fluorescence signals from the at least one fluorophores present within the microvolume of interest. Detection of the emitted concentrated emitted fluorescence signals may be achieved in an efficient and facile manner by splitting the concentrated emitted fluorescence signals into two or more spectral bands. In one example, the concentrated emitted fluorescence signals are split into three spectral bands wherein the first spectral band has a wavelength that ranges from about 650 nm to about 690 nm, the second spectral band ranges from about 690 nm to about 740 nm, and the third spectral band ranges from about 740 nm to about 800 nm. In another example, the concentrated emitted fluorescence signals are split into two spectral bands wherein the first spectral band has a wavelength that ranges from about 650 nm to about 690 nm, and the second spectral band ranges from about 690 nm and above. The splitting of the fluorescence signals into spectral bands may be achieved in a facile manner using a suitable device such as beam splitter. It would be appreciated by those skilled in the art that directing incident beams onto the sample with a focal spot size having a generally constant diameter provides uniform illumination along the depth dimension of the sample carrier. This leads to a defined relationship between the spot size of the incident beam, and the depth dimension of the sample carrier. Similar spectral bands splitting for the blue wavelength region are known and may be split into 490 nm to 540 nm and 540 nm and above. [0033] Thus one or more event fluorescence measurements for the sample using the concentrated emitted fluorescence signals are obtained. In one exemplary embodiment, a further R-theta scan is conducted to obtain bead and cell analysis. In another example, three or more R-theta scans were performed at the appropriate microvolume of interest. It will be understood by one skilled in the art that the different scans measured herein may be obtained by a single optical scan or it may be a composite of more than one scan. More than one scan, whether it is a R-Theta scan or a Z-Theta scan, may be conducted as the situation demands, such as when it has been determined that the whole scan sequence does not fall into one Z band due to non-flatness of the sample carrier. In an exemplary embodiment, the R-theta scan for a microvolume of interest can encompass about a 2 mm wide scan, resulting in 500 scan lines.

[0034] One skilled in the art will be able to appreciate that the two (or more in case more than one laser source is present in each optic module) laser spots may be used to impinge two different regions of the sample assembly at a given time. Subsequently, the sample assembly may be moved translationally and rotationally such that the position that was impinged by the first laser spot initially is impinged by the second laser spot and vice versa. In this manner, the entire sample assembly may be analyzed using two laser beams with two different wavelengths, thus giving rise to a greater amount of information in a relatively short period of time. [0035] The fluorescence measuring device of the invention further comprises a processor module (not shown) for superimposing the first image and the second image to generate a composite image using the predetermined distance for superimposition. The processor module is an electronic circuit in the form of, for example, a PROM chip that has been programmed to execute commands to achieve the results described herein. The accurate superimposing is made possible by the fact that the location of the fluorescence signals at any time point is accurately known based on the predetermined distance between the laser spots, the inversion point, the position of the reference line, the diameter of the rotational axis of the sample assembly and the linear axis for translation of the sample assembly. In one exemplary embodiment, the data processing can include: generation of one image from each scan; generation of one superimposed image to establish spatial locations of events; determination of the local background from both emitted beams through pixel window spatial averaging to smooth out the effects of noise and events; subtraction of the background plus a noise floor to highlight events; using matched filter convolution to detect events; fitting a 2-D Gaussian function to characterize the events; and generating an event parameter table. At the end of any one ofthe scanning sequences or all of the scanning sequences, an application- specific image processing software of a suitable programmable analysis device can be used to stitch or knit together all of the rotational passes over the sample to produce a final sample data image.

[0036] In general, the above described fluorescence measuring device provides high- sensitivity fluorescence measurements from relatively small samples. The fluorescence measuring device of the invention functions effectively as a cell function biology system, and have uses in situations that include: viewing breast cancer cells, tumor cells, drug development, and the like. These attributes further render the device to be adapted for use when and where critical decisions are needed to be made, such as, emergency rooms, ICUs, operating rooms, and the like.

[0037] Thus the fluorescence measuring device as described herein advantageously provides for the simultaneous detection of normalized bulk fluorescence and event fluorescence for the sample at two different wavelengths, and superimposing the images from both scans to obtain multiple information about the sample. Such a device allows for rapid and accurate analysis of samples that is inexpensive in its operation and maintenance. One skilled in the art would also appreciate the simplicity and versatility of the applicability of the device as described herein above. Thus, in one embodiment, the device is used for assaying samples, immunoassays, assaying cell lines, assays based on GFP, apoptosis testing, and the like, and combinations thereof. Assay methods as used herein include any in vitro testing methods. Assays may also include testing of substances, for example, presence of bacteria in water. Immunoassays as used herein include sandwich immunoassays, competitive immunoassays, and the like. In yet another embodiment, the device as described herein is used for cell and bead assays. In a further embodiment, the device is used for chemical detection, such as explosive detection, drug detection, and the like. Further examples of the use of the device include, but not limited to, the detection of the presence or absence of an antigen associated with an antibody; the detection of presence or absence of microorganism contamination in water; the quantification of amount of glucose present in a blood; detection of the presence or absence of a narcotic in a urine sample; and the like, and combinations thereof. Currently, different devices are used for the different applications enumerated herein, whereas the fluorescence measuring device as described herein provides the capability of having a single device that can be used for all of the various applications described herein.

[0038] The analysis data obtained from the fluorescence measuring device of the invention may be used for arriving at informed decisions regarding the source of the sample, such as quality of water in a region to determine if the water is potable; identifying a disease condition, for example, the amount of CD-4 cells measured may be used to determine the susceptibility of a patient to any immunodeficiency related afflictions, or blood glucose concentration, the determination of a disease condition, namely, diabetic or not, can be made. Further, the determination of the disease condition may be made to determine a course of a suitable treatment. This may include administration of drugs such as insulin to the patient, the dosage being determined based on several factors such as, but not limited to, medical history, medical condition, diet, weight, physical condition, and the like. The disease condition may further be classified as being one of onset, a progression, a regression, stable, and an advanced condition.

[0039] The fluorescent measuring device further comprises a controller module (not shown) coupled to the sample assembly, the optics assembly, the processor module and the detector assembly, wherein the controller module is configured to issue instructions for operation of the sample assembly, the optics assembly, the processor module and the detector assembly based on a selected menu option. The control module is made available as an electronic circuit, such as an EPROM chip configured to execute commands appropriately.

[0040] The fluorescent measuring device also includes a graphical user interface to display a plurality of menu options, to receive inputs from an operator and to display results for the sample. [0041] In another aspect, the invention provides a method for generating a composite fluorescence image of a sample. Fig. 3 shows exemplary steps of a flowchart representing the method of the invention that is generally depicted by numeral 32. The method comprises providing a sample assembly configured for rotational motion and translational motion, represented by numeral 34 in Fig. 3, and providing an optics assembly and depicted by numeral 36 in Fig. 3. The optics assembly, as described herein, comprises a first optic module and a second optic module, wherein the first optic module and the second optic module are placed spaced apart.

[0042] The method then involves impinging at least one sample on the sample assembly simultaneously by the first optic module at a first focal point and the second optic module at a second focal point, as shown in Fig. 3 by numeral 38. As already described, the first and second optic modules are placed parallel to each other and are in a mirror image configuration relative to each other about the inversion point. As noted herein, the first and second optic modules comprise light sources of two different excitation wavelengths. Further, as already noted, each optic module may comprise more than one laser sources. Also, as noted herein, a predetermined distance between the first focal point and the second focal point is used to define a reference line, and the impinging is about an inversion point situated on the reference line and equidistant from the first focal and second focal point. The impinging of the sample with the laser spots from the first optic module and the second optic module generates a first fluorescence signal and a second fluorescence signal respectively from the sample. It must also be noted that each fluorescence signal may be the combination of multiple signals emanating from multiple excitation wavelengths when each optic module comprises more than one laser source.

[0043] The first and the second fluorescence signals are then detected using the detector assembly as described herein to provide a first image and a second image respectively, shown in Fig. 3 by numeral 40. The two images obtained by impinging the sample at the same position with lasers of two different wavelengths are then superimposed with each other to obtain a composite image, shown in Fig. 3 by numeral 42. This composite image is generated based on the known predetermined distance between the laser spots on the sample such that the exact position from which the images from each laser spot are obtained is known.

[0044] Then the sample assembly is translated by a known distance along a linear axis that is parallel to the reference line, shown in Fig. 3 by numeral 44, and simultaneously rotating the sample assembly by a known angle on a rotational axis having a diameter that is defined by the predetermined distance between the two laser spots, depicted by numeral 46 in Fig. 3. [0045] The steps of impinging, detecting, and superimposing for each translating and rotating step are repeated to obtain the composite fluorescence image for the at least one sample present on the sample assembly, referenced in Fig. 3 by numeral 48. It would be appreciated by those skilled in the art that the known distance, and known angle for each translation and rotation of the sample assembly are based on a suitable coordinate system, for example the Cartesian coordinate system and the Polar coordinate system are used in the exemplary and non-limiting embodiment. Thus, the complete fluorescence image at two different wavelengths of the at least one sample on the sample assembly may be obtained using the method of the invention. The method of the invention allows for gleaning a variety of useful information from a sample in a relatively short period of time, which opens up the possibility for high-throughput diagnostics.

[0046] In a further aspect, the invention provides a system that comprises the fluorescence measuring device of the invention that further uses the method as described herein. The system is useful in a variety of situations that include, for example, but not limited to, cell analysis, cell cycle analysis, apoptosis analysis, secondary antibody analysis, in vitro screening, life science research, drug discovery research, and the like, and combinations thereof. The system of the invention is a compact configuration that can provide more information for a given sample in a relatively short period of time within a given laboratory setting, without having to depend on "centralized core facilities". The system may comprise other units and modules that advantageously utilize the fluorescence measuring device, which may include, for example, a personal computer with a suitable operating and a software program that is capable of processing the information from the device and displaying it in a suitable manner. The system may also comprise a communications module that communicates the processed information through suitable means to a user located nearby or at a remote location, such as through electronic mail. Communication means may include, for example, infrared, Bluetooth, wired network connection, wireless network, and the like, and combinations thereof. Exact mode of communication will depend on various factors such as, but not limited to, file size, data transfer rate, bandwidth and the like, and combinations thereof. Choice of the mode of communication will become obvious to one of ordinary skill in the art without undue experimentation. Other components and features useful in the system of the invention will also become obvious to one skilled in the art, and is contemplated to be within the scope of the invention. [0047] While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

I Claim:

1. A fluorescence measuring device comprising: a sample assembly configured for rotational motion and translational motion; and an optics assembly comprising a first optic module having a first focal point and a second optic module having a second focal point, wherein the first optic module and the second optic module are placed spaced apart, wherein a predetermined distance between the first focal point and the second focal point defines a reference line; wherein an inversion point equidistant from the first focal and second focal point situated on the reference line defines a rotational axis for the rotational motion of the sample assembly, wherein the rotational motion of the sample assembly is about the rotational axis such that the predetermined distance further defines a diameter for the rotational axis for the rotational motion of the sample assembly, and wherein an axis parallel to the reference line defines a linear axis for the translational motion of the sample assembly.

2. The fluorescence measuring device of claim 1 wherein the first optic module and the second optic module are placed parallel to each other.

3. The fluorescence measuring device of claim 1 wherein the first optic module and the second optic module are in a mirror image configuration relative to each other about the inversion point.

4. The fluorescent measuring device of claim 1 wherein the each optic module comprises at least one laser source.

5. The fluorescent measuring device of claim 1 wherein the each optic module comprises two laser sources.

6. The fluorescence measuring device of claim 1 wherein the sample assembly is configured for receiving a sample carrier that comprises at least one sample, wherein the sample comprises at least one fluorophore.

7. The fluorescent measuring device of claim 6 wherein the first optic module and the second optic module impinge the at least one sample simultaneously to generate a first fluorescence signal and a second fluorescence signal respectively.

8. The fluorescent measuring device of claim 7 further comprising a detector assembly to detect the first and the second fluorescence signals to provide a first image and a second image respectively.

9. The fluorescent measuring device of claim 8 further comprising a processor module for superimposing the first image and the second image about the inversion point to generate a composite image.

10. The fluorescent measuring device of claim 9 further comprising a controller module coupled to the sample assembly, the optics assembly, the processor module and the detector assembly, wherein the controller module is configured to issue instructions for operation of the sample assembly, the optics assembly, the processor module and the detector assembly based on a selected menu option.

11. The fluorescent measuring device of claim 1 further comprising a graphical user interface to display a plurality of menu options, to receive inputs from an operator and to display results for the sample.

12. The fluorescent measuring device of claim 1 wherein the first optic module has a first excitation wavelength that ranges from about 600 nanometers to about 800 nanometers and the second optic module has a second excitation wavelength that ranges from about 300 nanometers to about 600 nanometers.

13. A method for generating a composite fluorescence image of a sample, wherein the method comprises: providing a sample assembly configured for rotational motion and translational motion, wherein the sample assembly is configured for receiving a sample carrier that comprises at least one sample, wherein the sample comprises at least one fluorophore; providing an optics assembly, wherein the optics assembly comprises a first optic module and a second optic module, wherein the first optic module and the second optic module are placed spaced apart; impinging the at least one sample simultaneously by the first optic module at a first focal point and the second optic module at a second focal point, such that a predetermined distance between the first focal point and the second focal point defines a reference line to generate a first fluorescence signal and a second fluorescence signal respectively, wherein the impinging is about an inversion point situated on the reference line that is equidistant from the first focal and second focal point; detecting the first and the second fluorescence signals to provide a first image and a second image respectively; and translating the sample assembly along a linear axis parallel to the reference line by a known distance; rotating simultaneously the sample assembly on a rotational axis around the inversion point having a diameter defined by the predetermined distance, and the rotating is by a known angle; superimposing the first image and the second image about the inversion point to generate a composite image; and repeating steps of the impinging, detecting, and superimposing for each translating and rotating step to obtain the composite fluorescence image.

14. The method of claim 13 wherein the first optic module and the second optic module are placed parallel to each other.

15. The method of claim 13 wherein the first optic module and the second optic module are in a mirror image configuration relative to each other about the inversion point.

16. The method of claim 13 wherein the each optic module comprises at least one laser source.

17. The method of claim 13 wherein the first optic module has a first excitation wavelength that ranges from about 600 nanometers to about 800 nanometers and the second optic module has a second excitation wavelength that ranges from about 300 nanometers to about 600 nanometers.

18. A fluorescence measuring device comprising: a sample assembly configured for rotational motion and translational motion, wherein the sample assembly is configured for receiving a sample carrier that comprises at least one sample, wherein the sample comprises at least one fluorophore; and an optics assembly comprising a first optic module having a first focal point and a second optic module having a second focal point, wherein the first optic module and the second optic module are placed spaced apart, wherein a predetermined distance between the first focal point and the second focal point defines a reference line; wherein an inversion point equidistant from the first focal and second focal point situated on the reference line defines a rotational axis for the rotational motion of the sample assembly, wherein the predetermined distance defines a diameter for the rotational motion of the sample assembly, and wherein an axis of the translational motion of the sample assembly is parallel to the reference line, and wherein the first optic module comprises a light source for generating a first incident beam having a first excitation wavelength and a first focus diameter to impinge on the at least one sample to yield a first laser spot, and the second optic module comprises a light source for generating a second incident beam having a second excitation wavelength and a second focus diameter to impinge on the at least one sample to yield a second laser spot, wherein the first laser spot defines a first sample volume, and the second laser spot defines a second sample volume.

19. The fluorescence measuring device of claim 18 wherein each optic module comprises at least one laser source.

20. The fluorescent measuring device of claim 18 wherein the first laser spot and the second laser spot are impinged on the at least one sample simultaneously.

21. The fluorescent measuring device of claim 18 wherein the first laser spot and the second laser spot are at a mirror image position about the inversion point with respect to each other.

22. The fluorescent measuring device of claim 18 further comprising a means for displacing the first laser spot and the second laser spot relative to the respective first sample volume and second sample volume in a three dimensional space defined by the respective first sample volume and second sample volume, wherein each sample volume comprises at least one individual volume of interest.

23. The fluorescent measuring device of claim 22 wherein the means further comprises translating means to translate the first laser spot and second laser spot across the first sample volume and the second sample volume respectively in a depth direction and to obtain one or more emitted fluorescence signals.

24. The fluorescent measuring device of claim 23 further comprising a processor module to superimpose the one or more emitted fluorescent signals about the inversion point, from the first and second sample volume to provide a superimposed image.

25. The fluorescent measuring device of claim 24 wherein the processing module is also used for obtaining a depth profile and a thickness of the sample carrier from the one or more emitted fluorescence signals from each sample volume, wherein the depth profile comprises the at least one individual volume of interest.

26. The fluorescent measuring device of claim 24 wherein the processor module uses the depth profile and the thickness for measuring normalized bulk fluorescence for each sample volume.

27. The fluorescent measuring device of claim 24 wherein the processor module is configured to determine at least one microvolume of interest from the depth profile for each sample volume.

28. The fluorescent measuring device of claim 27 further comprising a controller module to trigger the first light source and second light source to focus the respective incident beams on the at least one microvolume of interest to obtain at least one concentrated emitted fluorescence signal.

29. The fluorescent measuring device of claim 28 wherein the processor module uses the at least one concentrated emitted fluorescence signal to measure one or more event fluorescences for the sample.

30. The fluorescent measuring device of claim 28 further comprising a detector assembly to detect the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal.

31. The fluorescent measuring device of claim 30 wherein the detector assembly further comprises two or more beam splitters for splitting the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal into two or more spectral bands.

32. The fluorescent measuring device of claim 30 wherein the processor module is configured for receiving data representative of the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal from the detector assembly, and for measuring one or more fluorescence events for the individual volume of interest and normalized bulk fluorescence for the sample, based on the data.

33. The fluorescent measuring device of claim 18 further comprising a graphical user interface to display a plurality of menu options, to receive inputs from an operator and to display results for the sample.

34. The fluorescent measuring device of claim 28 wherein the controller module is coupled to the sample assembly, the optics assembly, and the detector assembly, wherein the controller module is configured to issue instructions for operation of the sample assembly, the optics assembly, and the detector assembly based on a selected menu option.

35. The fluorescent measuring device of claim 18 wherein the first optic module has a first excitation wavelength that ranges from about 600 nanometers to about 800 nanometers and the second optic module has a second excitation wavelength that ranges from about 300 nanometers to about 600 nanometers.

36. A fluorescence measurement system comprising the fluorescent measurement device of claim 1 or 18.