LIFETIME

Cross-Refer ence to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 61/772,236 titled "METHODS AND SYSTEM FOR MEASURING 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 accurate luminescence lifetime measurements.

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 presently two distinct ways to measure τ: time-domain measurements and frequency-domain measurements. Time-domain (TD) measurements typically involved expensive, large devices that were slow to acquire enough information to determine τ. The literature shows two main methods for measuring τ using TD: Time-Correlated Single- Photon Counting (TCSPC) and gated detection.

[0005] Frequency-domain (FD) measurements are generally performed by measuring the lag time of the luminescence signal to the excitation signal. The luminescent material is illuminated with a light source that is modulated typically with a sine wave. At a given excitation light source frequency ω, the measured phase angle, θ _ω , of the luminescent emission yields the luminescent lifetime τ:

This technique requires the frequency, ω, to be such that θ _ω be less than π/2 and ideally near π/4.

[0006] Irrespective of the technique employed for TD, one of the things that can corrupt the τ measurement is any offset in the measured signal. In any optoelectronic system, there are two major sources of offsets: electrical and ambient light. Electrical offsets are inherent in almost all electronic amplifiers. Ambient light can enter the system through the photodiode used for detection.

[0007] The decay equation for finding τ, where y _t is the total measured signal at time equals t is:

y _t = e( ^_t /

[0008] However, any offset(s) realistically change ^ to the following:

[0009] Where V _t is the unknown offset signal. The term V _t , if not equal to 0, causes nonlinearity in τ determination. Conversely, the closer V _t is to 0, τ determination approaches linearity. FIG. 1 A is a computational illustration of how a detector signal, when modified by an offset, can cause the normalized intensity measurement of the detector signal ('signal + offset') to deviate from the true signal ('signal'). FIG. IB is a computational illustration of how the differences in the measured signal illustrated in FIG. 1A can cause errors in Tau measurement. As shown in FIGS. 1A-1B, when there is no offset (signal indicated by diamond characters), the slope of the plot of time vs. ln(y _t /yo) is a straight line. When an offset is added (signal indicated by square characters), the plot of time vs. ln(y _t /yo) is non-linear, and results in an error in lifetime determination.

[0010] Prior approaches to eliminating and/or reducing offset attempt to eliminate the cause of the offsets, and/or to measure the offsets and remove them mathematically. For example, electrical offsets can be 'zeroed out' using potentiometers, and/or measured and subtracted. Drawbacks of these approaches include, but are not limited to, errors in electrical offset determination, sensitivity of measuring devices, and highly error prone results when working with signals with low signal noise ratio (SNR). Similarly, offset measurements to account for ambient light must be accurate, noise free, and not change. [0011] There is hence a need to be able to eliminate offset-based artifacts in Tau measurements in a more robust manner. Further, there is a particular need to be able to measure relatively long Tau values that takes advantage of the longer duration of these lifetimes to employ less optically, digitally and economically intensive resources while maintaining veracity of Tau measurement.

Brief Description of the Drawings

[0012] FIG. 1 A is a graph of normalized intensity vs. time for a signal, as well as for the signal with added offset;

[0013] FIG. IB is a graph of ln(normalized intensity) vs. time for the signal of FIG. 1A and its corresponding Tau value, as well as for the signal and its corresponding Tau value with added offset;

[0014] FIG. 2A is a graph of the normalized derivative of intensity vs. time for a signal, as well as for the signal with added offset;

[0015] FIG. 2B is a graph of ln(normalized derivative of intensity) vs. time for the signal of FIG. 2A and its corresponding Tau value, as well as for the signal and its corresponding Tau value with added offset; and

[0016] FIG. 3 is a system of the invention, according to embodiments.

Detailed Description

[0017] 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.

[0018] 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. [0019] Methods and systems for measuring luminescence lifetimes are presented herein. Aspects of the invention analyze an optical detector's measured signal to determine Tau regardless of any undesirable offset(s) in the output signal, and without measuring the undesirable offset(s). Aspects of the invention also enable direct determination of Tau by direct measurement of the entire exponential decay curve.

[0020] The offset may be of any suitable mathematical form (e.g. linear, non-linear, 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 offset takes the form V _t , and modified the detector's output signal as shown below:

where y _t is the total measured signal at time t. It is understood that while y _t is described here as the detector signal for ease of explanation, y _t can arise from any component associated with the emission signal. For example, in some embodiments, y _t is the signal from an amplification unit connected to the detector, or the 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. For example, the derivative dyldt (described below) could be taken using analog circuitry directly at a detector output in a strictly analog system, and/or prior to digitization by an A/D converter in a digital system.

[0021] In some embodiments, the y _t signal is not saturated; in other words, the optical detector is working in its linear response range, as is commonly understood in the art. In some embodiments, V _t is independent of time at least within the time frame of measurement of y _t . In other words, V _t is constant, or varying undetectably slowly, or varying at a slower rate than y _t within the time frame of measurement. In such embodiments, analysis of the detector's output signal includes taking the derivative of the y _t signal with respect to time, and yields the following equation:

[0022] In this manner, the V _t term is removed.

[0023] In some embodiments, analysis of the detector's output signal further includes normalizing the dy/dt to a positive value. It follows that in these embodiments, at time equals 0:

Derivative^ = - = - j = - _;

[0024] In some embodiments, analysis of the detector's output signal further includes dividing the derivative at any time t by the derivative at time equal 0 to yield the signal S _t as

Derivative 1 ₁

Derivative

[0025] In some embodiments, analysis of the detector's output signal further includes taking the natural log of S _t , which results in an equation of a line with a slope equal to— 1/τ: ln(S _t ) = - - * (t)

τ

[0026] FIGS. 2A-B are computational illustrations of the benefits of the invention, where the derivative of the pure signal, dy/dt and of the pure signal plus offset d(y + offset)/5t are co-linear (best seen in FIG. 2A). FIG. 2B further illustrates that the measured lifetime of the signal + offset is identical to the original lifetime of the signal alone, using the approach of the invention.

[0027] In some embodiments, analysis of the detector's output signal further includes determining τ from S _t . In some embodiments, τ is determined by solving for any time t as

[0028] In some embodiments, τ can be determined by an averaging approach. In such embodiments, τ is computed by: measuring and computing a plurality of S _t values corresponding to a plurality of values of t to yield a plurality of paired data of the form [t, ln(S _t )]; determining a plurality of τ values, each corresponding to one of the plurality of paired data; and averaging the plurality of τ values to determine an average τ value. In some embodiments, the plurality of t values are contiguous. In some embodiments, the plurality of t values are randomly chosen. In some embodiments, at least one t value is zero. In this manner, averaging can eliminate or eliminate noise associated with the detector's measured signal ^.

[0029] In some embodiments, τ is determined by a least squares approach. In such embodiments, τ is computed by: measuring and computing a plurality of S _t values corresponding to a plurality of values of t to yield a plurality of paired data of the form [t, \n(S _t )]; applying a least squares regression to find an optimum slope of the plurality of paired data, where the

1

optimum slope is equal to— ; determining the τ value using the optimum slope. In this manner, τ

additional noise rejection and/or removal can be achieved.

[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 include 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(II) meso-tetra( -methyl-4-pyridyl)porphyrin tetrachloride, platinum octaethylporphyrin_. Suitable Tau values corresponding to these fluorophores 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 analyzed to eliminate offset as discussed above. In some embodiments, the output of the amplification unit 310 is analyzed to eliminate offset as discussed above. In some embodiments, the output of the detector 308 is analyzed to eliminate offset 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.