LUMINESCENCE LIFETIME

Cross-Refer ence to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 61/772,229 titled "METHODS AND SYSTEMS FOR IMPROVED ESTIMATION OF LUMINESCENCE LIFETIME", filed March 4, 2013, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

[0002] Embodiments described herein relate generally to methods and systems for improved estimation of luminescence lifetime.

Background

[0003] Some luminescent materials, and photoluminescent materials in particular, possess the property that, when stimulated or excited with light at the correct wavelengths, they will emit light at longer wavelengths, where the emitted light intensity correlates with a parameter such as, but not limited to, pH, temperature, oxygen concentration (hereinafter referred to as p0 ₂ ), and so on. These materials luminesce such that, when the excitation light is turned off, the rate of decay of the emitted light, Tau (τ), is exponential. This decay rate, τ (also interchangeably referred to as luminescence lifetime, or simply lifetime), correlates with a desired measurement parameter such as, but not limited to, p0 ₂ .

[0004] In general, there are two distinct ways to measure τ: time-domain measurements and frequency-domain measurements. Time-domain (TD) measurements typically attempt to measure τ by pulsed excitation followed by collection of luminescence decay information. An ideal luminescence intensity decay can be characterized by

[0005] Where I _t is the luminescence intensity at time t, I ₀ is the initial intensity (at time t = 0), and τ is the lifetime. At any time / that is greater than 0, solving for τ is: [0006] Accordingly, FIG. 1 A illustrates how τ can be calculated based on this linear relationship between t and In (— ), where the diamonds denote data points. A linear regression performed on

V/o

the data of FIG. 1 A (not shown) would be a straight line with a slope of -1/ r.

[0007] However, in most modern systems, there are sources of noise, random and/or otherwise, that can influence the measured signal I _t , such as electrical noise from amplifiers, digital noise from A D converters, and/or the like. For example, random noise (say n _t ) derived from analog circuitry can be additive to the true luminescence intensity I _t , and yield the following form:

I _t + n _t = I ₀ * e( ^_t ^)

[0008] FIG. IB illustrates how measuring n _t along with the true luminescence intensity I _t can result in erroneous Tau measurements, where the solid line represents a linear regression of the ideal data of FIG. 1A, and the dotted line is the best linear regression fit for the values of

In ( ^ ² ). As an exemplary illustration, if n _t is Gaussian-distributed noise with a one-sigma level of 5%, and Tau (ideal) is 14.00 μβεο as determined from the solid line of FIG. IB, the noise- influenced Tau (dotted line) is 12.25 μβεο. Further, applying such noise-influenced Tau measurements for determining oxygen levels (e.g. to determine p0 ₂ of an oxygen-sensitive luminescent material) results in as much as a 20% error in the p0 ₂ , for example reading from 250 mmHg to 300 mmHg.

[0009] While reducing noise in such systems is one way to handle this problem, for all practical purposes, there will always be noise in measurement systems, caused by these and other factors such as circuit design, outside interferences, or even low signal levels due to sensor aging. There is hence a need for improved estimation of luminescence lifetime, and particularly, a need to account for noise in systems measuring luminescence lifetime for improved estimation of lifetime.

Brief Description of the Drawings

[0010] FIG. 1A is a graph of ln(normalized intensity) vs. time for an ideal signal; [0011] FIG. IB is a graph of ln(normalized intensity) vs. time for the signal of FIG. 1A with (actual) and without (ideal) added random noise;

[0012] FIG. 1C is a graph of the difference between the ideal and actual measured ln(normalized intensity) values of FIG. IB vs. the ideal ln(normalized intensity);

[0013] FIG. 2 is a graph of ln(normalized intensity) vs. time for the signals of FIG. IB with and without added random noise, and with a weighting function applied to the signal with added random noise; and

[0014] FIG. 3 is a system of the invention, according to embodiments.

Detailed Description

[0015] As used in this specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "a detector" is intended to mean a single detector, multiple detectors, and/or a combination of detectors.

[0016] As used in this specification, the term 'luminescence' can refer to any form of cold body radiation including chemiluminescence, electroluminescence, photoluminescence, and/or the like. Photoluminescence can include fluorescence, phosphorescence, or both.

[0017] Methods and systems for improved estimation of luminescence lifetime are presented herein. Aspects of the invention analyze an optical detector's measured signal to determine Tau by weighting the measured signal, thereby compensating for noise(es) affecting Tau determination.

[0018] The noise may be of any suitable mathematical form (e.g. linear, non-linear, analog, digital, and/or the like), and may arise from any influence (e.g. electrical effects, optical, thermal, instrumentation, and/or the like) on the system. In some embodiments, the noise includes electrical noise from amplifiers in analog circuitry of a luminescence lifetime measuring system. In some embodiments, the noise includes digital noise caused by the limited resolution of the A/D, math processing, pixelization, and/or the like. In some embodiments, the noise can include an offset. In such embodiments, the offset can be removed, such as by calculating a derivative of the signal.

[0019] In some embodiments, the noise takes the form n _t , and modifies the detector's output signal as:

I _t + n _t = I ₀ * e( ^_t ^)

where I _t + n _t is the measured signal ("measured signal" hereon). It is understood that while the measured signal is described here as the detector signal for ease of explanation, the measured signal can arise from any component associated with the emission signal. For example, in some embodiments, the measured signal is the analog signal from an amplification unit connected to the detector, or the measured signal is the digital signal from an A/D converter connected to the detector and/or the amplification unit, and/or the like. Further, the analysis presented below may be applied in part or whole to any of the detector and/or other components downstream of the detector, including the A/D converter, the amplification unit, and/or the like. In some embodiments, the measured signal is not saturated; in other words, the detector is working in its linear response range, as is commonly understood in the art.

[0020] In some embodiments, Tau is determined as discussed earlier. The nature of the logarithm transformation, however, reduces a small percentage change in a large measured signal while exaggerating a larger percentage change in a smaller measured signal. Using an example of 5% noise, a full-scale IV signal would have 50 mV of noise (5%), while a 200 mV signal would also have 50 mV of noise (25%) riding on it. The magnification of the noise with respect to the signal is best illustrated in FIG. 1C, which plots the log transformation with ideal, noise- free data versus the difference in the log transformation using the same ideal data plus noise. As the measured signal, (/ in this case) gets smaller compared to Io (i.e., the In (— ) gets more negative), the error due to the added random noise increases.

[0021] This error divergence from the ideal (i.e. from the 0.0 line) results in increasing error when linear regression, or a variant thereof, is used to determine τ from the In (— ) data. In a standard linear regression calculation, each data pair has equal weighting, effect, and/or is otherwise given equal consideration when determining where to "draw" the line that best describes the data set. With the "noisy" data on the right side of FIG. 1C (i.e. data points that deviate significantly from the 0.0 line), the linear regression calculates a slope for that gets "pulled" by these noisy points, resulting in an incorrect τ value, as illustrated in Figure IB.

[0022] Aspects of the invention overcome these drawbacks by differential weighting, effect, and/or consideration to data points corresponding to an exponential decay data depending on which part of the exponential decay curve the data point(s) lies on. In other words, data points corresponding to 'earlier' portions of an exponential decay curve (e.g. closer to the time of an excitation pulse, closer to t=0, having higher signal-to-noise ratio, and so on) are weighted differently than data points that correspond to later portions of the same decay curve.

[0023] Described again with respect to FIG. 1C, Applicants discovered that the "earliest" data points on the exponential decay, i.e., the In (— ) values towards the left in FIG. 1 C, provided the

Ό

most accurate Tau estimates upon linear regression; the data to the right, though noisier, still contributed to the regression analysis, albeit to a lesser extent. Said another way, the data on the left had higher "information content" compared to the data on the right.

[0024] Aspects of the invention are hence directed to weighting of exponential decay data for purposes of improving lifetime determination. In some embodiments, a weighting function is applied to the decay curve, and may be of any suitable form to achieve the benefits of the invention. For example, the weighting function may be a straight line with a negative slope, a step function, an exponential decay, and/or the like. Further, the weighting function may be a constant (e.g. a line with a predetermined slope) or a function of any suitable parameter (e.g. a line with a slope that is a function of time). In some embodiments, the suitable parameter is derived from any portion of the exponential decay curve, including, but not limited to, intensity at each data point, time at each data point, a data point number in a sequence of data points, and/or the like.

[0025] Further, the weighting function may be added, subtracted, multiplied and/or otherwise combined with the exponential curve data by any suitable mathematical operation. In some embodiments, the weighting function is analog, while in other embodiments, the weight function is discrete and/or digital in nature. [0026] In some embodiments, the weighting function is a weighting parameter that is applied to each data point (i.e. each data pair of I _t and time t). In some embodiments, the weighting function w _t is of the form: w _t = I _t * I _t

[0027] In other words, w _t is an exponential decay curve. In this manner, w _t can influence the regression analysis by over-exaggerating the data pairs with the largest I _t values and correspondingly, the largest w _t values.

[0028] While described here with respect to a single weighting function applied to an entire exponential decay curve for simplicity, it is understood that variants of this approach, such as using multiple weight functions each independently selected from the other, applying weighting functions in an overlapping and/or mutually exclusive manner, applying weighting function(s) to a portion of the decay curve, and/or the like, may be employed.

[0029] FIG. 2 illustrates benefits of the invention by applying the weighting function w _t = I _t * I _t to the linear regression illustrated in FIG. IB. A closer match between the ideal (solid line) and actual/measured (dotted line) Tau values is observed in FIG. 2 as compared to FIG. IB. Consistent with the exemplary illustration provided earlier, if the ideal line has a Tau of 14.00 μβεο, the actual line in FIG. 2 has a Tau of 13.88 μβεο (vs. a Tau of 12.25 μβεο for FIG. IB). Applying the same calibration coefficients described earlier, the p0 ₂ reading is now determined to be 255 mmHg, a factor of 10 reduction in error prior to weighting.

[0030] FIG. 3 illustrates an environment and/or system 300 within which aspects of the invention may be implemented. The system 300 can be a stand-alone system or, in some embodiments, be part of and/or otherwise integrated with any suitable optical analysis system including, but not limited to, an in vivo system, an ex vivo system, an in vitro system, a spectroscopy system, a microscopy system, and/or the like. The system 300 includes a computing apparatus 302, a light control 304, a light source 306, a detector 308, an amplification unit 310, an analog-to-digital (A D) converter 312, and a timing unit 314. A sample holder 318 is also illustrated, although it is understood that the sample holder need not be part of the system 300, and does not affect operation of the system 300. Interconnections shown between these components by solid lines may be electrical, optical, wireless, and/or the like. Further, it is understood that some of these components may be combined. For example, the light control 304 may be integral to the light source 306 in terms of design and/or function, the amplification unit 310 may be combined with the detector 308, the A/D converter 312 may be combined with the amplification unit, and so on.

[0031] It is understood that appropriate coupling optics (not shown) may be employed for coupling the excitation light from the source 306 to the sample holder 318, and for coupling the emission light from the sample holder to the detector 308. The coupling optics can include, but are not limited to, one or more of filters, mirrors, prisms, lens, shutters, polarizers, fiber optics/other transmission media, and/or the like.

[0032] The light source 306 can be any suitable light source for analyzing the sample for fluorescence lifetime, and can include, but is not limited to, one or more of an incandescent light source such as halogen lamps, a light-emitting diode, a gas discharge lamp, a CW or pulsed laser and/or other suitable monochromatic source, and/or the like. In some embodiments, the light source 306 is a pulsed laser source. The light control 304 can be any suitable electronic component controllable by the computer 302 and/or the timing unit 314, and can control aspects of operation of the light source 306, including, but not limited to, triggering, output intensity, gating, and/or the like.

[0033] The detector 308 can be any suitable detector for detecting one or more optical signals from the sample holder 316 and/or portions of the sample holder, and can include, but is not limited to, one or more of a phototube, a photo multiplier tube (PMT), a photodiode, a charge- coupled device (CCD) sensor or camera, a complementary metal-oxide-semiconductor (CMOS) sensor, and/or the like. In some embodiments, the detector detects luminescence. In some embodiments, the detector is a silicon PIN photodiode such as, but not limited to, the Hamamatsu S5973-01.

[0034] The amplification unit 310 can be any suitable component capable of amplifying the output of the detector 308, and/or any aspect thereof, such as specific frequency-dependent components of the output, a subset of all pixels (when the detector output is a digital image, for example). The amplification unit 310 can be controllable by the computer 302 and/or the timing unit 314. The amplification unit 310 can be a single amplifier, or a string of amplifiers. In some embodiments, the amplification unit includes one or more operational amplifiers such as, but not limited to, the Texas Instruments OPA657N, the Texas Instruments OPA820, and the Linear Technology LT6230.

[0035] The A/D converter is operable for converting any suitable output of the detector 308 into a digital signal. Desirably, the A/D converter is capable of digitizing the detector 308 output at a rate significantly faster than the exponential decay associated with the luminescent material being measured. In some embodiments, the A/D converter is capable of digitizing a luminescent decay with a Tau on the order of microseconds and higher. In some embodiments, the luminescent decay is associated with a p0 ₂ measurement.

[0036] The timing unit 314 can be any suitable component capable of receiving, generating, and/or otherwise outputting timing signals for controlling the other components of the system 300 as illustrated. In some embodiments, the timing unit 314 controls at least the turning on, the turning off, and the duration of excitation of the light source 306 via the light control 304. In some embodiments, the timing unit 314 controls the rate of A/D conversions by the A/D converter 312. In some embodiments, the timing unit 314 synchronizes operation of the light source 306 (via light control) and the operation of the A/D converter 312 during at least one of the following time periods: a dark period before the light source is turned on; an excitation period when the light source is turned on; and an emission period (also referred to as the luminescent decay period) when the light source is turned off.

[0037] In some embodiments, the computing apparatus 302 is configurable to analyze the output from the A/D converter to determine the Tau or lifetime as discussed above. In some embodiments, the timing unit synchronizes Tau determination by the computing apparatus 302 to period(s) with the light source off; i.e. during the emission period, as described above.

[0038] In some embodiments, the system 300 is optimized for measuring luminescent materials with Tau values in the microseconds range. In some embodiments, the system 300 is optimized for measuring oxygen-sensitive luminescent materials. Examples of such luminescent materials include, but are not limited to tris(2,2'-bipyridine)ruthenium dichloride, Pt(II) meso- Tetra(pentafluorophenyl)porphine, Tris (4,7-diphenyl-l ,10-phenanthroline)ruthenium (II) chloride, Pt(H) meso-tetra( -methyl-4-pyridyl)porphyrin tetrachloride, platinum octaethylporphyrin. Suitable Tau values corresponding to these fiuorophores can be microseconds to milliseconds. Suitable A/D conversion rates can be selected based on an estimate of the Tau value being determined; for example, in some embodiments, the A/D conversion rate is selected such that at least two data points acquired for an expoential decay curve are separated by about Tau. In some embodiments, at least three data points are acquired for an exponential decay curve within time Tau.

[0039] When Tau is on the order of microseconds, the detector 308, the amplification unit 310, and the A/D converter can directly measure the entire exponential decay curve from a single excitation pulse by the light source 306. The timing unit is then operable to synchronize the various components of the system 300 as described above to analyze the exponential decay fast enough to determine Tau. In some embodiments, the exponential decay obtained from the A/D converter 312 is weighted to improve Tau estimation as discussed above. In some embodiments, the output of the amplification unit 310 is weighted to improve Tau estimation as discussed above. In some embodiments, the output of the detector 308 is weighted to improve Tau estimation as discussed above. Benefits of this approach allow the use of fewer optical components when emission detection occurs with the light source 306 off, thereby eliminating or otherwise alleviating the need for optical filters to block excitation light from reaching the detector 308. Additionally, since the light source can be on for a short duration of time (e.g. a single excitation pulse), photodegradation of the luminescent material is greatly reduced and/or eliminated.

[0040] In some embodiments, the computer 302 can constitute at least a processor (not shown) and a memory (not shown). The processor of the computer 302 can be any suitable processing device configured to run and/or execute a set of instructions or code. For example, the processor can be a general purpose processor, a central processing unit (CPU), an accelerated processing unit (APU), an application-specific integrated circuit (ASIC), a field programmable gate array (FPGA) and/or the like. As described above, the processor can be configured to run and/or execute a set of instructions or code stored in the memory associated with using a personal computer application, mobile application, an internet web browser, and/or the like. Additionally, in some embodiments, the processor can run and/or execute a set of instructions associated with performing numerical methods to control the system 300, to determine Tau, and/or the like. [0041] The memory can be any memory (e.g., a RAM, a ROM, a hard disk drive, an optical drive, other removable media) configured to store information (e.g., one or more software applications, training course/task information, user account information, media, text, etc.). The memory can include one or more modules performing the functions described herein. In some embodiments, the functions described herein can be performed by any number of modules. For example, in some embodiments, the functions described herein can be performed by a single module. The memory can also alternatively store one or more resources (e.g., software resources such as drivers, code libraries, etc.) associated with one or more of the modules.

[0042] Some embodiments described herein relate to a computer storage product with a non- transitory computer-readable medium (also referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer- implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include transitory propagating signals (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as space or a cable). The media and computer code (also referred to herein as code) may be those designed and constructed for the specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to: magnetic storage media such as hard disks, optical storage media such as Compact Disc/Digital Video Discs (CD/DVDs), Compact Disc-Read Only Memories (CD-ROMs), magneto-optical storage media such as optical disks, carrier wave signal processing modules, and hardware devices that are specially configured to store and execute program code, such as Application- Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM) devices.

[0043] Examples of computer code include, but are not limited to, micro-code or microinstructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. For example, embodiments may be implemented using Java, C++, or other programming languages and/or other development tools. [0044] The various embodiments described herein should not to be construed as limiting this disclosure in scope or spirit. It is to be understood that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.

[0045] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.