WO2025217283A1

WO2025217283A1 - Systems and methods for fast fluorescence lifetime imaging

Info

Publication number: WO2025217283A1
Application number: PCT/US2025/023862
Authority: WO
Inventors: Anand Kumar; Murali KRISHNAMOORTHY
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 2024-04-09
Filing date: 2025-04-09
Publication date: 2025-10-16
Anticipated expiration: 2026-10-09

Abstract

A system and method for fast fluorescence lifetime imaging are presented. The system includes a light source that delivers periodically modulated light to tissue that has received a fluorescent compound. The system also includes a detector system that is configured to receive light fluoresced by the tissue and produce fluorescence lifetime (FLT) data. The FLT data includes continuous wave data and time gated data measured with a given frequency and phase. The system also includes a processor that is configured to analyze the FLT data to estimate a fluorescence lifetime at a plurality of locations across the tissue and to determine a presence or absence of cancer in the tissue based on the estimate of the fluorescence lifetime at the plurality of locations across the tissue.

Description

SYSTEMS AND METHODS FOR FAST FLUORESCENCE LIFETIME IMAGING

RELATED APPLICATIONS

[0001] This application is based on, claims priority to, and incorporates herein by reference for all purposes, U.S. Provisional Patent Application No. 63/631,697 filed on April 9, 2024.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 5R01CA211084- 06 and 5R01CA260857-03 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

[0003] The present disclosure relates generally to systems and methods for assessing tissue using fluorescence imaging. More particularly, the present disclosure provides systems and methods for intraoperative examination of tissue and identification of target cells in vivo to further guide the operative procedure in real time.

[0004] Interoperative imaging requires high speed imaging in order to make realtime clinical decisions based on the acquired images. Even in vitro and ex vivo imaging applications, such as microscopy, benefit from increased image acquisition speed (e.g., to increase experiment throughput, imaging fast biological processes, and so forth). Unfortunately, while frequency domain-based fluorescence imaging has several practical and clinical advantages, it is typically too slow for practical use. Thus, new systems and methods are desired to increase the acquisition speed of frequency domain fluorescence imaging while maintaining high quality signals.

SUMMARY OF THE DISCLOSURE [0005] The present disclosure addresses the aforementioned drawbacks by providing a system and method for fast fluorescence lifetime (FLT) imaging.

[0006] In accordance with one aspect of the present disclosure, a method is provided for estimating fluorescence lifetime of tissue. The method includes providing a periodically modulated light source that is configured to excite a fluorescent compound applied to tissue of a surgical patient. The method also includes acquiring frequency domain FLT data from the tissue in response to excitation by the periodically modulated light source. The method also includes processing the FLT data to determine a fluorescence lifetime of the fluorescent compound within the tissue at each of a plurality of locations across the tissue. Processing the FLT data also includes generating a report indicating the presence of cancer cells relative to the plurality of locations across the tissue.

[0007] In accordance with another aspect of the present disclosure, a medical imaging system is provided for fast fluorescence lifetime imaging. The system includes a light source that delivers periodically modulated light to tissue that has received a fluorescent compound. The system also includes a detector system that is configured to receive light fluoresced by the tissue and produce fluorescence lifetime (FLT) data. The FLT data includes continuous wave data and time gated data measured with a given frequency and phase. The system also includes a processor that is configured to analyze the FLT data to estimate a fluorescence lifetime at a plurality of locations across the tissue and to determine a presence or absence of cancer in the tissue based on the estimate of the fluorescence lifetime at the plurality of locations across the tissue.

[0008] These are but a few, non-limiting examples of aspects of the present disclosures. Other features, aspects and implementation details will be described hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Various objects, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.

[0010] FIG. 1A is diagram of one, non-limiting example of a system that may be used for fluorescence lifetime imaging in accordance with the present disclosure.

[0011] FIG. 1B is a diagram of another, non-limiting example of a system that may be used fluorescence lifetime imaging in accordance with the present disclosure.

[0012] FIG. 2 is a flow chart setting forth some, non-limiting example steps of a process in accordance with the present disclosure that may utilize a system such as described with respect to FIGS. 1A-1B.

[0013] FIG. 3A provides example simulated data demonstrating fluorescence excitation and emission signals.

[0014] FIG. 3B provides example simulated data demonstrating simulated lifetimes compare to those measured in accordance with the present disclosure.

[0015] FIG. 3C provides example simulated data demonstrating simulated lifetime concentration ratios of two lifetimes compared to those measured in accordance with the present disclosure.

DETAILED DESCRIPTION

[0016] Before any aspects of the present disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising,” or "having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms "mounted," "connected,” "supported," and "coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, "connected" and "coupled” are not restricted to physical or mechanical connections or couplings.

[0017] As used herein in the context of computer implementation, unless otherwise specified or limited, the terms "component,” "system," "module," "controller," "framework," and the like are intended to encompass part or all of computer-related systems that include hardware, software, a combination of hardware and software, or software in execution. For example, a component may be, but is not limited to being, a processor device, a process being executed (or executable) by a processor device, an object, an executable, a thread of execution, a computer program, or a computer. By way of illustration, both an application running on a computer and the computer can be a component. One or more components (or system, module, and so on) may reside within a process or thread of execution, may be localized on one computer, may be distributed between two or more computers or other processor devices, or may be included within another component (or system, module, and so on).

[0018] The present disclosure provides systems and methods for measuring luminescence lifetimes, which can be applied in vivo or ex vivo. For example, lifetimes can be measured of administered fluorescence dyes, auto-fluorescence of tissues, phosphorescence, or other luminescent materials or phenomena. Moreover, the present disclosure recognizes that the fluorescence lifetimes (FLTs) of fluorescent dyes are significantly longer in tumor cells than the FLTs of the same dyes in healthy tissue. With this in mind, the present disclosure provides systems and methods for intraoperative tissue analysis that can distinguish tumors from healthy tissue with a consistency and specificity not realized in prior attempts to do so. Thus, the present disclosure recognizes that FLT, which can be measured in absolute units (typically nanoseconds), is a parameter that is robust to measurement conditions and can be used to alleviate many of the shortcomings of prior efforts at creating a robust, intraoperative tissue assessment tool. Additionally, systems and methods are provided for measuring concentrations when more than one lifetime are present within the same spatial location.

[0019] The systems and methods provided herein are flexible. The systems and methods provided herein do not require a specific paring of hardware with a particular dye or targeting agent. In one, non-limiting example, a fluorescently tagged EGFR- antibody can be used, but other dyes or fluorescent tagging mechanisms can be utilized.

As another non-limiting example, non-targeted dyes, such as indocyanine green (ICG), can be used. In accordance with one non-limiting aspect of the disclosure, the FLT imaging can utilize the near infrared (NIR) spectrum, but other wavelengths may also be used. The systems and methods provided herein can provide a dramatic specificity and sensitivity improvement over standard fluorescence intensity-based methods for distinguishing tumors from normal tissue in situ and in vivo.

[0020] The use of time domain (TD) -based FLT systems has been described in detail in W02023/076899, the entire contents of which is incorporated herein by reference. The present disclosure provides FLT systems and methods that can advantageously use frequency domain detection. In standard approaches, frequency domain detection is typically too slow for practical use. However, the systems and methods described herein provide frequency domain detection with increased speed, making frequency domain detection practical for intraoperative imaging and other applications. The use of frequency domain-based detection may also reduce costs of FLT systems. For example, the system may include a light source and camera that are cheaper than those used for time domain detection. Such cost reduction can improve system availability and reduce the financial burden on patients and the healthcare system.

[0021] Referring to Fig. 1A, one, non-limiting example of a system 100 that may be used in accordance with the present disclosure is illustrated. In the illustrated, nonlimiting example, the system 100 may be a frequency domain (FD) imaging platform configured for both in vivo imaging and ex vivo or in vitro imaging. In particular, the illustrated, non-limiting system 100 includes an in vivo imaging sub-system 102 and an in vitro or ex vivo imaging sub-system 104. In other implementations, the system may omit the in vivo imaging sub-system 102 or in vitro or ex vivo imaging sub-system 104.

[0022] In the illustrated configuration, the in vivo imaging sub-system 102 is designed to direct light from a surgical bed 106 to an optional fiber bundle 108, which is then collected by a relay lens (RL) and split via a dichroic mirror (D1) into a detector system 110. The detector system 110 may include one or more continuous wave cameras or other detectors configured to record fluorescence signal emitted from the sample. In some configurations, the detector system 110 can also include a red, green, and blue wavelength (RGB) camera.

[0023] The camera can be configured to collect continuous wave intensity data from the sample. For example, the camera may preferably include a continuous wave camera that is not time gated and collects intensity images in real-time. Use of a continuous wave camera may be preferable, as CW cameras are typically less expensive and simpler as compared to time gated cameras. For example, a standard smart phone video camera may be sufficient for the CW camera. As another example, the camera may be time gated and collect continuous wave data by summing the time gated data, cumulatively. As another example, the camera may be a time gated camera configured with an infinitely large gate width. In some non-limiting examples, this may be between 0 and 12.5 ns, or similar. In this way, the detection system can be configured to measure a summation of multiple or all phases (e.g., 0°-360°) of the periodically modulated fluorescence signal.

[0024] The detection system also includes a second camera or detector configured for time gated imaging. As a non-limiting example, the time gated camera may include an intensified charge-coupled device (CCD) camera. As another non-limiting example, a complementary metal-oxide-semiconductor (CMOS) or other camera can be used with a gate intensifier, which can provide temporal resolution. As a non-limiting example, the time gated camera may be configured with a time window described by a delay (e.g., 1 ns after stimulation) and gate width (e.g., 0.5 ns). In some non-limiting examples, the gate width may be between 400 ps and 1,000 ps, or similar. In some implementations, the delay or gate width can be varied to measure varying phases of the emission signal. As will be described, such time gated camera can be used to acquire fluorescence data measured at one or more phases. As only one phase measurement is required, the time gated camera may be configured with relaxed constraints compared to time gated cameras employed in standard fluorescence time domain (TD) imaging. For example, the time gated camera may not be required to provide as sharp of gate width modulation (e.g., increases and decreases) that are typically required for standard fluorescence TD imaging.

[0025] In some implementations, the cameras may be designed for wavelengths less than a threshold, for example, 650 nm. For example, an intensified camera (CCD/Intensifier) designed for wavelengths over the threshold (e.g., 650 nm) may be included.

[0026] The system 100 can also include an optical light source 112 configured to excite the sample (e.g., surgical field) with a modulated excitation light. As one nonlimiting example, the light source may include light-emitting diodes or laser diodes that are electrically modulated in the MHz range. As one non-limiting example, the light source may include a laser diode driver, a laser diode, and a modulator (e.g., acousto-optic modulator, electro-optic modulator) that provides periodic modulation of the laser light. As another example, the light source may include a pulsed laser system (e.g., Ti-Sa lasers), which may be further modulated.

[0027] The light source 112 is configured to generate a periodic excitation light source with a given frequency (e.g., MHz range). As a non-limiting example, the excitation light may be modulated using a sine function described through time (t) by l_ex(t) = A + B sin(ωt), where A describes the average excitation intensity, B describes the waveform amplitude, and describes the frequency of the sine waveform. Similarly, the excitation light can be modulated using a cosine function. As another non-limiting example, the excitation light may be modulated with a periodic square waveform, or other periodic waveform with a frequency ω. In some implementations, the excitation frequency can be configured between 10-100 MHz, or even between 1 KHz-500 MHz.

[0028] A mirror housing (M) may be attached to the Intensifier/CCD and may be configured to be remotely switched to receive light from the fiber bundle 108 or from the specimen stage. Fluorescence or NIR excitation can be collected using, for example, a filter wheel (F) attached to the ICCD. A fiber delivers light (for example, 780 nm light) into both a digital light projector (DLP) via a dichroic (D2) (for example, 800 nm) for specimen illumination, and to the surgical bed 106 via a port in an objective lens (B). These wavelengths are simply examples, and other wavelengths can be utilized. In particular, as will be described, the near infrared (NIR) spectrum maybe utilized. In this non-limiting example of NIR light, the light can penetrate up to 5-10 cm into the tissue, which can be advantageous for assessing even tumor that is beneath several cm thick tissue layer.

[0029] The system 100 can be configured for a wide field of view (FOV) while providing micron resolution. The fiber bundle is mounted on a flexible articulating arm (A) attached to a portable stand (C). The arm A can be positioned for a desired view of the surgical bed 106. In this way, an in vivo probe is provided that can be hand-directed or hand-held for manipulation about the surgical site. In some configurations, the in vivo probe can be integrated with a confocal endomicroscopy system.

[0030] As illustrated, the system 100 can be integrated into a cart or rack 120, which can include the in vitro or ex vivo imaging sub-system 104. The in vitro or ex vivo imaging sub-system 104 can be controlled by a stepping motor driver that can control positioning of a sample chamber 122. The system 100 may also include, as illustrated, a laser diode driver, a modulator, a picosecond (PS) or other delay unit, and an HRI controller configured to coordinate delivery of the laser illumination. The system 100 may also include a computer system or processor that is configured for data acquisition, data processing, and report generation in accordance with the present disclosure.

[0031] Another non-limiting example system 150 is provided in FIG. 1B. As FIG. 1B shows, the system 150 includes a light source that provides a modulated excitation signal (e.g., sine wave). Such excitation light can excite the sample, causing a modulated emission fluorescence signal. In some implementations, the sample may be a surgical field. In other implementations, the sample may be an ex vivo or in vitro sample placed in the focal plane of the camera(s). The system 150 also includes a continuous wave (CW) camera directed toward the sample. The continuous wave camera can detect the fluorescent signal emitted by the sample. In some implementations, the continuous wave camera may include a gated camera configured with a long gate width or configured to cumulatively sum gated data through time. In some implementations, the system 150 can include a second, camera. Such secondary camera may include a time gated camera (e.g., intensified CCD camera) configured with an acquisition start time and gate width that can be used to acquire the emission signal at one or more phases.

[0032] As will be described below, the system (e.g., 100 or 150) can be used for fluorescence lifetime imaging to estimate a lifetime of a fluorophore or species. Such imaging may be spatially localized such that the sample can be characterized by varying fluorescence lifetimes in space. For example, the system (e.g., 100 or 150) can be used to distinguish tumor from healthy tissue within a surgical field based on the varied fluorescence lifetimes of tumor and healthy tissue. In some implementations, the system

(e.g., 100 or 150) can also be used to characterize concentration distribution of multiple fluorescent lifetimes present.

[0033] In some implementations, the system 100 can be used to acquire time domain data. Advantageously, the system 100 can also be used to acquire frequency domain data at one or more phases. In some implementations, only a single phase is required, reducing the acquisition time required for lifetime estimation and enhancing signal quality. In this way, system 100 can be used for real time fluorescence lifetime imaging to provide intraoperative imaging. For example, in some implementations, system 100 can provide fluorescence lifetime imaging with a frame rate of 10 frames or more per second.

[0034] Referring to Fig. 2, a process 200 is provided for in vivo or ex vivo analysis in accordance with the present disclosure. Process 200 may use a system (e.g., 100 or

150) such as described with respect to Fig. 1A or 1B. As will be described, process 200 can be used to acquire and process frequency domain data. Process 200 can be used with traditional frequency domain data in which frequency domain data are acquired with multiple detection phases, frequencies, or a combination thereof to estimate the lifetimes of molecules. However, process 200 can also, advantageously, be used with the acquisition of just a single phase measurement along continuous wave (CW) fluorescence intensity data. In this way, process 200 can provide faster acquisition times compared to standard frequency domain fluorescence imaging. As speed is a key factor in the practical use of fluorescence lifetime imaging for real time fluorescence lifetime-based surgery, process 200 can be used for intraoperative imaging or other applications. Moreover, the CW measurements provide high signal to noise ratio, enhancing the speed without losing signal quality and allowing for reduced system complexity. Additionally, CW detection in the frequency domain can be achieved at high speed with relatively simple equipment, thereby simplifying the acquisition process and reducing system complexity. In this way, process 200 can be used with lower-cost systems.

[0035] Process 200 can be used to measure fluorescence lifetimes measured from tissue that has received a fluorescent compound. While the lifetimes describe a physical characteristic of the fluorophore or fluorescent compound (e.g., fluorescent dye], lifetimes are also influenced by the tissue in which the dye is present. In this way, in various discussions, the present disclosure describes a fluorescence lifetime of the tissue while recognizing that the lifetime describes a characteristic of the fluorescent dye within the tissue. Likewise, the present disclosure describes concentrations of lifetimes recognizing that such concentrations characterize a concentration of a fluorescent compound with a given lifetime.

[0036] The lifetimes measured in process 200 can be localized in space to produce a lifetime map. In this way, a single fluorescence dye can be administered to produce a map of the fluorescence lifetimes of the various tissue types (e.g., cancer and normal tissue) spatially distributed throughout the imaging field of view. In other aspects, process 200 also provides a method for measuring concentrations of two or more lifetimes colocalized in space. For example, process 200 can characterize the in vivo bio distribution of multiple dyes when the lifetimes are known or estimated a priori.

[0037] Process 200 may begin at process block 202 with the acquisition of fluorescence lifetime (FLT) data. The FLT data may include fluorescence imaging data received from the fluorescence emission of an excited fluorescent compound with a given fluorescence lifetime. Thus, process block 202 may include the application or administration of a fluorescent compound, such as by injection of or other treatment by one or more fluorescent dyes. Moreover, process block 202 may include excitation of such fluorescent compound using an optical light source. For example, process block 202 may include exciting the fluorescent compound with an excitation light having a periodically modulated amplitude [e.g., characterized by a sine wave function).

[0038] The FLT data acquired in process block 202 includes continuous wave data. Such continuous wave data may include either "sequential” data that is retrospectively summed together over time or "cumulative” data that is measured over time using a continuous wave detector. For example, the continuous wave data can be measured cumulatively from the tissue beginning at a chosen time origin to an end point or multiple end points in time.

[0039] The FLT data acquired in process block 202 can also include time gated or single-phase frequency domain data acquired at one or more phases and frequencies (e.g., using gated detection). In some implementations, the time gated frequency domain data may be acquired at multiple phase delays, however, additional phases are advantageously not required. Thus, continuous wave data are acquired with a single phase, time gated frequency domain data are acquired with a single phase, and time gated frequency domain data are optionally acquired at multiple phases.

[0040] In process block 202, the FLT data are acquired in response to excitation light modulated with a frequency ω in the presence of a fluorophore. The excitation light can be described with an intensity modulation as: l_ex(t) = A + B sin(ωt) (1).

[0041] The corresponding emission intensity of the fluorophore is given by: l_em(t) = α + β sin(ωt + Ψ) = I₀ (1 + M sin(ωt + Ψ)) (2),

[0042] where

[0043] Here, denote the apparent lifetime and modulated lifetime, respectively. For mono-exponential decays,

[0044] Traditional frequency domain detection includes sequentially measuring the emission intensity (l_em) with several (e.g., four or more) phase delays or detection frequencies.

[0045] However, process 200 can be used to increase acquisition speed by measuring CW fluorescence in parallel with the frequency domain measurement. The continuous wave nature of the measurement causes the frequency information to be lost or averaged out. Thus, an expression for CW fluorescence can be obtained by setting ω = 0, leading to l_CW = l₀(1 + M sin(Ψ)) . Note that s Thus, I_CW = l₀. In other words, CW camera integrates and averages the information across all phases, which can be described as Ψ))dΦ, where Φ = ωt, which also leads to which can then be scaled (e.g., averaged by dividing by 2π) to obtain I_CW. Thus, process block 202 can include measuring continuous wave intensity data ( l_CW ) independently, so that l₀ can be determined experimentally. Replacing I₀ with I_CW, Equation (2) can be rearranged as:

[0046] where

[0047] Thus, the FLT data can be described in terms of continuous wave data ( l_CW), time gated emission data ( l_em ), and characterization of the excitation signal

Advantageously, can be characterized by measuring I_CW and I_em at a single time point, t_i. Thus, using the expressions above, measured at time t_i can be written as

[0048] Here, is expressed entirely in terms of measurable experimental quantities, including the emission intensity I_em at a time t_i, modulation frequency of the excitation light ω, and the CW fluorescence, I_CW. Hence, for a given frequency ω, the lifetime can be estimated by measuring for one phase. In this way, the data can be processed in process block 204 to determine the fluorescence lifetime

[0049] In some implementations, processing the data to estimate the fluorescence lifetime includes fitting or solving Equation (8) for based on the FLT data described by Equation (7). Such fitting may be accomplished using a linear least squares approach, a lookup table, or other fitting algorithm. Frequency (ω) and measurement phase (ωt_i ) can be selected to avoid excessive phase shifts, which could lead to non-unique lifetime measurements.

[0050] In some implementations, process block 204 may include processing pixel- wise or voxel-wise data in order to generate a lifetime map of the sample, which may be included in a report in process block 208.

[0051] In some implementations, process 200 also includes additional data analysis in process block 206. Such analysis may provide additional data (e.g., clinically relevant) based on the lifetime data processed in process block 204. As one non-limiting example, the lifetime maps can be analyzed to distinguish tumor from normal tissue. In some implementations, such distinction may be based on a lifetime threshold. In some implementations, such distinction may include processing the data using additional computer vision or machine learning algorithms. In some implementations, analyzing the data may also include generating estimates of the certainty of cancer. For example, a map can be generated that provides a percentage of certainty of cancer that is based on the estimated lifetimes.

[0052] As another non-limiting example, process block 206 may include manual or automated generation of region of interest maps based on the lifetime data. As another non-limiting example, tumor statistics can be analyzed (e.g., total tumor volume, change in tumor volume, tumor homogeneity, tumor boundary characteristics, and so forth). As another non-limiting example, analysis can include further characterization of the tissue (e.g., cancer type) based on the lifetime data.

[0053] Regardless of the particular analysis process, the overall processing of the data can be helpful to enhance tumor contrast, without requiring specific chemicals designed to target cancer. The design of chemical probes with cancer specificity has been challenging and has not been successful to date since cancer-specific markers are also expressed in normal tissue. On the other hand, the present disclosure can use fluorescence lifetime distinctions between the dyes taken up by cancer cells versus the lifetimes of the dyes in healthy tissue, allowing enhanced sensitivity and specificity compared to traditional fluorescence intensity-based detection. In this regard, a threshold may be used that distinguishes cancerous tissue. For example, images may be reconstructed, which can then be analyzed against one or more thresholds at process block 206.

[0054] In one non-limiting example, the threshold may be selected to delineate tumor from normal tissue. This threshold may be selected to make discrimination applicable to multiple patients, at least for a given type of cancer. In other words, different types of primary and metastatic cancer (e.g., oral, brain, skin, breast, liver, melanomas, and sarcomas) may be discriminated using a different FLT threshold, but a given cancer type can be discerned using the FLT threshold across multiple patients, independent of the measurement system or other variables. In this way, the threshold may provide a cutoff lifetime selected for a given cancer type or anatomical region.

[0055] The present disclosure also provides the ability to quantify concentrations of multiple lifetimes. When more than one lifetime is present at the same location in space, a similar approach can be used to recover the decay amplitudes for multiple lifetime components, assuming the lifetimes are known a priori. This is applicable in scenarios involving multiplexing with more than one fluorescent dye with distinct lifetimes. For example, the fluorescence lifetimes of two dyes can be known or estimated a priori based on prior experimentation or measurement. In other scenarios, the two lifetimes may represent two distinct lifetimes of a single dye in two types of tissue (e.g., tumor and healthy tissue). Thus, process block 202 may include imaging data acquisition in the presence of two lifetimes (e.g., The two dyes or tissue types may have varying concentrations, described by amplitudes a₁ and a₂, which can be measured (e.g., in process block 204) as described below.

[0056] First, for a single fluorophore with lifetime where a refers to the decay amplitude, which depends on the fluorophore concentration. Using this in

Equation [2], the frequency domain emission signal for a bi-exponential decay of the form can be written as:

[0057] The CW intensity can be written as

[0058] Assuming that the lifetimes are known, the two unknown amplitudes a₁ and a₂ can be recovered by solving the linear simultaneous equations:

[0059] where

[0060] Note that only one measurement, l(t₁), which is measured at a given frequency (ω) and phase (ωt₁ ), is required in the above equation, along with continuous wave intensity data l_CW. The results of the proposed method are presented in FIG. 3C, which shows the ratio of measured amplitudes (a₁/a₂).

[0061] The processed data generated in process block 204 or result of the analysis performed at process block 206 can be used in process block 208 to generate a report. In some implementations, the report may include a spatial map of fluorescent lifetimes or statistics of the fluorescent lifetimes in varying regions of interest. In some implementations, such as in a surgical setting, the FLT threshold for a particular cancer can be used to define a tumor/normal boundary that may be included in the report at process block 208. Furthermore, the processed data may be spatially registered to the tissue. For example, the lifetime map or tumor/normal boundary may be overlaid to an anatomical image or projected onto the surgical bed to guide the operating surgeon on where to begin resection and how much tissue to cut. If processed, amplitude data (e.g.,a₁ or a₂) can be provided in the report. For example, a₁/a₂ can be displayed as a color map to indicate the ratio of healthy tissue to tumor in a given voxel. Thus, the report generated at process block 208 may take a variety of forms.

[0062] As described, process 200 provides a method for providing FLT imaging using frequency domain detection with high acquisition speed. Using traditional FLT techniques typically relies on phase differences, which necessitates the collection of all phase information to pinpoint phase peaks and subsequently derive phase differences. Thus, using standard FLT imaging, multiple phases are systematically measured in order to fully characterize the modulation wave (e.g., sine wave) of the emitted signal. In contrast, the systems and methods described herein provide the ability to measure fluorescence lifetimes within a sample with measurement of a single phase in conjunction with continuous wave data. In this way, the present disclosure recognizes that full characterization of the phase information is not required. Such reduced requirement can allow for much faster acquisition speeds that enable several practical applications previously unachievable. For example, a frame rate of 10 frames per second or even better can be achieved. As one non-limiting example, this increased acquisition speed can enable real time intraoperative imaging, producing fluorescence lifetime maps of a surgical field.

[0063] Even more, the reduced reliance on multiphase data can allow for a reduction in the technical requirements of the imaging system, if desired. For example, CW cameras are typically inexpensive and readily available. CW cameras also operate more simply than time gated cameras (e.g., without requiring precise gate width timing or wasting time switching gate widths). Moreover, as only a single phase measurement is required, the time gated camera of the system may also have relaxed technical specification requirements. For example, the time gated camera may be configured with a single well-tuned gate width to circumvent the need to precisely change gate widths on a very fast timescale.

[0064] Moreover, the use of frequency domain imaging may allow for relaxed light source requirements. For example, the light source may be configured to provide periodically amplitude-modulated light with a frequency on the order of MHz. In contrast, time domain detection typically uses a pulsed or delta light source (e.g., femtosecond laser) with a very short pulse width (e.g., 140 fs) at some repetition frequency (e.g., 80 MHz). Such high pulsed light sources are typically more expensive than light sources configured for periodically modulated light (e.g., sine wave with MHz frequency).

[0065] Examples

[0066] FIGS. 3A-C provide example simulation data. An 80 MHz sinusoidal signal was assumed, and the emission signal was computed using Equation (2), as shown in FIG. 3A. At a specific phase or measurement time measured from the excitation time, t = Φ, where the signal is the lifetime was estimated by solving Equation (8). The simulation was repeated for a range of lifetimes between 0.4-1.4 nanoseconds. FIG. 3B shows the estimated and actual lifetimes according to simulation, demonstrating their comparability. The simulation was repeated for two known lifetimes at varying levels of concentrations (a₁/a₂). The ratio of amplitudes, a₁/a₂, was estimated using Equation (11) and is plotted compared to the simulated a₁/a₂ in FIG. 3C.

[0067] In some implementations, devices or systems disclosed herein can be utilized or installed using methods embodying aspects of the disclosure. Correspondingly, description herein of particular features, capabilities, or intended purposes of a device or system is generally intended to inherently include disclosure of a method of using such features for the intended purposes, a method of implementing such capabilities, and a method of installing disclosed (or otherwise known) components to support these purposes or capabilities. Similarly, unless otherwise indicated or limited, discussion herein of any method of manufacturing or using a particular device or system, including installing the device or system, is intended to inherently include disclosure, as embodiments of the disclosure, of the utilized features and implemented capabilities of such device or system.

[0068] As used herein, the phrase "at least one of A, B, and C" means at least one of A, at least one of B, and/or at least one of C, or any one of A, B, or C or combination of A, B, or C. A, B, and C are elements of a list, and A, B, and C may be anything contained in the Specification.

[0069] The present disclosure has described one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.

Claims

1. A method of estimating fluorescence lifetime of tissue, the method comprising: providing a periodically modulated light source configured to excite a fluorescent compound applied to tissue of a surgical patient; in response to excitation by the periodically modulated light source, acquiring frequency domain fluorescence lifetime (FLT) data from the tissue having received the fluorescent compound; and processing the FLT data to determine a fluorescence lifetime of the fluorescent compound within the tissue at each of a plurality of locations across the tissue and generate a report indicating the presence of cancer cells relative to the plurality of locations across the tissue.

2. The method of claim 1, wherein the periodically modulated light source is modulated by a sine wave function.

3. The method of claim 1, wherein the FLT data comprises continuous wave data and time gated data measured with a given phase delay and frequency.

4. The method of claim 3, wherein the periodically modulated light source is described by I_ex = A + B sin(ωt), and the FLT data are given as wherein I_em is the time gated data measured with a phase delay, l_CW is the continuous wave data, and

5. The method of claim 4, wherein the FLT data are given by where is a lifetime of the fluorescent compound and t_i is a measurement time.

6. The method of claim 5, wherein determining the fluorescence lifetime at each of the plurality of locations across the tissue comprises estimating based on the continuous wave data and time gated data using at least one of a linear least squares approach or a lookup table.

7. The method of claim 3, wherein acquiring the continuous wave data includes acquiring fluorescence data cumulatively from the tissue beginning at a chosen time origin to an end point or to multiple time points in time.

8. The method of claim 3, wherein the acquiring the time gated data includes acquiring fluorescence data from tissue at a single or multiple modulation frequencies and phases.

9. The method of claim 1, wherein processing the FLT data to generate a report indicating the presence of cancer cells comprises determining FLT data at any of the plurality of locations above a threshold indicative of a presence of cancer cells.

10. The method of claim 9, wherein the threshold is a cutoff lifetime selected for one of a selected cancer type or an anatomical region.

11. The method of claim 1, wherein generating the report further includes producing a fluorescence lifetime map spatially registered to the tissue.

12. The method of claim 1, wherein generating the report further includes producing a map of a percentage certainty of cancer based on the estimated lifetimes, the map being spatially registered to the tissue.

13. The method of claim 1, wherein the tissue is characterized by two distinct fluorescence lifetimes; and wherein the method further comprises estimating an amplitude of a concentration of each of the two fluorescence lifetimes being known a priori.

14. The method of claim 13, wherein the two distinct fluorescence lifetimes correspond to two distinct fluorescent compounds received by the tissue.

15. The method of claim 13, wherein the two distinct fluorescence lifetimes correspond to two types of tissue having received one fluorescent compound.

16. The method of claim 13, wherein estimating the concentrations of and comprises solving a linear system described by: where

A and B describe the periodically modulated light source according to I_ex = A + B sin(ωt), l_em(t) describes time gated data measured with a given frequency and phase at time t, and I_CW describes continuous wave data.

17. The method of claim 1, wherein the tissue is located in an in vivo surgical site.

18. A medical imaging system comprising: a light source configured to deliver periodically modulated light to tissue, the tissue having received a fluorescent compound; a detector system configured to receive light fluoresced by the tissue and produce fluorescence lifetime (FLT) data, the FLT data comprising continuous wave data and time gated data measured with a given frequency and phase; and a processor configured to analyze the FLT data to estimate a fluorescence lifetime at a plurality of locations across the tissue and determine a presence or absence of cancer in the tissue using the estimate of the fluorescence lifetime at the plurality of locations across the tissue.

19. The system of claim 18, wherein the detector system comprises a continuous wave camera and a time gated camera.

20. The system of claim 18, wherein the periodically modulated light is characterized by a sine wave.

21. The system of claim 18, wherein the periodically modulated light is described by I_ex = A + B sin(ωt), and the FLT data are given as where I_em is the time gated data measured with a phase delay, I_CW is the continuous wave data, and

22. The system of claim 21, wherein the FLT data are given by where is a lifetime of the fluorescent compound and t_i is a measurement time.

23. The system of claim 22, wherein the processor is configured to analyze the FLT data to estimate the fluorescence lifetime by estimating based on the continuous wave data and time gated data using at least one of a linear least squares approach or a lookup table.

24. The system of claim 18, wherein the detector system is configured to receive light cumulatively from the tissue beginning at a chosen time origin to an end point or to multiple time points to produce the continuous wave data of the FLT data.

25. The system of claim 18, wherein the detector system is configured to receive light at a single or multiple modulation frequencies and phases to produce the time gated data of the FLT data.

26. The system of claim 18, wherein the processor is configured to analyze the FLT data to determine the presence or absence of cancer by determining fluorescence lifetimes at any of the plurality of locations above a threshold indicative of a presence of cancer cells.

27. The system of claim 26, wherein the threshold is a cutoff lifetime selected for one of a selected cancer type or an anatomical region.

28. The system of claim 18, wherein the processor is further configured to generate a report indicating the presence or absence of cancer in the tissue at the plurality of locations across the tissue.

29. The system of claim 28, wherein the processor is further configured to generate a fluorescence lifetime map spatially registered to the tissue, and the report further includes the fluorescence lifetime map.

30. The system of claim 28, wherein the processor is further configured to generate a map of a percentage certainty of cancer based on the estimated lifetimes, the map being spatially registered to the tissue; and wherein the report further includes the map.

31. The system of claim 18, wherein the tissue is characterized by two distinct fluorescence lifetimes; and wherein the processor is further configured to estimate an amplitude of a concentration of each of the two fluorescence lifetimes being known a priori.

32. The system of claim 31, wherein the two distinct fluorescence lifetimes correspond to two distinct fluorescent compounds received by the tissue.

33. The system of claim 31, wherein the two distinct fluorescence lifetimes correspond to two types of tissue having received one fluorescent compound.

34. The system of claim 31, wherein estimating the concentrations of comprises solving a linear system described by:

A and B describe the periodically modulated light source according to I_ex = A +

B sin(ωt), l_em(t) describes time gated data measured with a given frequency and phase at time t, and I_CW describes continuous wave data.

35. The system of claim 18, wherein the tissue is located in an in vivo surgical site.