WO2007131055A2

WO2007131055A2 - Fluorescence measurement and optical imaging method and apparatus

Info

Publication number: WO2007131055A2
Application number: PCT/US2007/068061
Authority: WO
Inventors: John F. Heanue; Joseph A. Heanue; Brian P. Wilfley; Augustus P. Lowell
Original assignee: Ocela Technologies
Priority date: 2006-05-03
Filing date: 2007-05-02
Publication date: 2007-11-15
Also published as: WO2007131055A3; EP2021777A2

Abstract

A system and method for measuring fluorescence lifetimes utilizing a light source modulated with a code sequence to interrogate a sample of interest. The system is useful for studying the interaction of chemicals, biomolecules, and other substances. The fluorescence lifetime is used as an indicator of chemical binding and chemical environment. A system and method for measuring photons. The system produces an estimate of the distribution of flight times for photons traveling from the source to the detector. A system and method for optical lymph node mapping. A correlation of a photo-detector signal and a digital code sequence, used to modulated light signal, is calculated to produce an estimate of the distribution of flight times for photons traveling from a given source to a given detector. These distributions are used along with the measured amplitudes to reconstruct a map of contrast agent location within the tissue.

Description

Fluorescence Measurement and Optical Imaging Method and Apparatus

Field of the Invention

[0001] The field of the present invention pertains generally to fluorescence techniques used in the measurement of chemical interactions and chemical properties, including, more specifically, the measurement of fluorescence lifetime for determining the degree of binding of chemical substances or for determining the properties of the chemical environment of a substance; to optical imaging using near-infrared light, including more specifically, to the optical detection of sentinel lymph node location in order to guide surgical procedures.; to systems and methods for detecting and measuring photons, including, more specifically, the measurement of the time-of-fiight of photons traveling through a scattering media such as tissue.

Background

[0002] Fluorescence techniques are known for studying chemical properties and processes. Fluorescence emission is a process in which a fluorophore is excited to a higher energy state by absorption of a photon at some excitation wavelength. The molecule decays via emission of a fluorescence photon on time-scales on the order of lOOps to lμs. The fluorescence lifetime is defined as the average time the fluorophore spends in the excited state. Various scientific applications involve the measurement of fluorescence lifetime because it represents an intrinsic molecular property of the fluorophore and can be affected by small changes in the fluorophore's direct environment. For example, fluorescence lifetime measurements are used in high- throughput screening for drug discovery. If one substance is labeled with a fluorophore and combined in solution with a second substance, the lifetime of the fluorophore typically will change if the two substances interact. Affinity analysis can be performed by measuring the variation in lifetime with the relative concentration of the two substances. Alternatively, kinetic studies can be carried out by monitoring the lifetime as a function of time after the two substances are mixed. In some cases, the substances being studied may exhibit intrinsic fluorescence, thereby eliminating the need for labeling. One example is the study of protein-protein interactions using the intrinsic fluorescence of tryptophan, tyrosine, or phenylalanine, three aromatic amino acid residues contained in most proteins. Turconi, et al. give an overview of fluorescence lifetime techniques for drug discovery in "Developments in fluorescence lifetime-based analysis for ultra-HTS", Drug Discovery Today, Vol. 6, No. 12 (Suppl.) 2001 and in the references therein.

[0003] Measurements of fluorescence lifetime have been carried out using either a time- domain or a frequency-domain technique. In the time-domain technique, the sample is typically excited with a pulse of light from a pulsed laser and the fluorescence light is measured using a detector with single-photon sensitivity. The detector measures the time delay between the excitation pulse and the first detected photon. The fluorescence lifetime distribution is usually determined by using many repeated pulses and building up a histogram of the measured time delays. Unfortunately, the pulsed laser sources and single-photon detectors are relatively expensive. Because detection is typically done at the single-photon level, it can require a significant amount of time to build-up enough data to approximate the fluorescence lifetime distribution. One disadvantage of the frequency-domain approach is that it is not a direct measurement of the fluorescence lifetime distribution. Rather, it provides an estimate of the mean lifetime based on the phase shift between a detected signal and the excitation signal. When the fluorophore exhibits multi-exponential time decay, extrapolation of the lifetime from the phase shift data is more difficult. Usually this requires measurements at more than one modulation frequency. In some cases, a complete measurement of the lifetime distribution yields evidence of particular chemical interactions that is not evident in a measurement of the mean lifetime alone. This data is not readily obtained with frequency-domain instrumentation. A further disadvantage of the frequency-domain approach is the need for accurate high-frequency analog electronics. An overview of both the time-domain and frequency-domain techniques can be found in the above-referenced article by Turconi, et al.

[0004] Sentinel lymph node biopsy is a surgical procedure that involves removing a small sample of lymph tissue and examining it for signs of cancer. As an alternative to conventional full lymph node dissection, it is increasingly used as the standard of care in the staging of breast cancer and melanoma. The sentinel lymph node (SLN) is the first node, or group of nodes, in the lymphatic network to come into contact with metastatic cancer cells that have spread from the primary tumor site. SLN biopsy allows a physician to obtain information about the other lymph nodes in the network without exposing the patient to the risks of conventional surgery. Further surgery to remove other lymph nodes may be avoided if no cancer cells are found in the sentinel lymph nodes.

[0005] SLN biopsy usually begins with the injection of a radioactive tracer (technetium- 99 sulfur colloid), a blue dye, or both into the area around the original cancer site. Lymphatic vessels carry the tracer to the sentinel node (or nodes); this is the lymph node most likely to contain cancer cells. Prior to surgery, a wide field-of-view gamma camera is typically used to image the location of the radiotracer. Images are generally taken from multiple positions and perspectives, resulting in a map of the drainage pattern of lymphatic fluid from the skin to the lymph nodes. By showing where the cancer is likely to have spread, the map enables the surgeon to plan the full procedure prior to the first incision. During surgery, the surgeon achieves further guidance either through direct visualization of the injected blue dye or by detecting the radioactive tracer with a handheld gamma probe. After surgery, the lymph node is sent for pathological examination that can include microscopic inspection, tissue culture, or immunological tests.

[0006] The current approach of using radioisotopes for SLN mapping has several drawbacks. First, while the radiation risk to patients and medical practitioners is relatively low compared to other medical procedures, the handling of radioisotopes still requires special precautions. Second, the procedure requires the coordination of both surgical and nuclear medicine personnel, resulting in both scheduling issues and increased cost. Lastly, the time required for the radiotracer to travel through the lymphatic system can be as long as several hours. It is highly desirable to have an alternative system that could be used without radiotracers and that a surgeon could utilize without the involvement of other specialists. It is also desirable to have a system that uses a contrast agent with more rapid kinetics.

[0007] Diffuse optical imaging techniques are known in medical and biological applications. Overviews of diffuse optical imaging techniques can be found in "Recent Advances in Diffusion Optical Imaging" by Gibson, et al, Phys. Med. Biology, vol. 50 (2005), R1-R43 and in "Near-infrared Diffuse Optical Tomography," by Hielscher, et al, Disease Markers, Vol. 18 (2002), 313-337. Briefly, diffuse optical imaging involves the use of near-infrared light incident upon a sample of interest. An example in the medical and biological field is optical mammography where near infrared light is used to illuminate breast tissue. A detector is placed on the opposite side of the breast from the incident light some distance away and collects scattered light from the breast tissue. The scattered light of interest that is detected may be directly scattered incident light or scattered fluorescence light caused by the excitation of an injected fluorescing material that fluoresces when exposed to the incident light. By measuring the amplitude of the light of interest at the detector and the distribution of photon arrival times at the detector for various source and detector positions, a reconstruction of the underlying tissue optical properties can be made. An overview of image reconstruction techniques can be found in the citations given in the aforementioned review articles. [0008] Measurements of the photon flight-time distributions are typically carried out using either a time-domain or a frequency-domain technique. In the time-domain technique, the sample is excited with pulse of light from a pulsed laser and the scattered light is measured using a detector with single-photon sensitivity. The detector measures the time delay between the excitation pulse and the first detected photon. The flight-time distribution is determined by using many repeated pulses and building up a histogram of the measured time delays. Unfortunately, the pulsed laser sources and single-photon detectors are relatively expensive. Because detection is typically done at the single- photon level, it can require a significant amount of time to build-up enough data to approximate the flight-time distribution. One disadvantage of the frequency-domain approach is that it is not a direct measurement of the photon flight time. Rather, it provides an estimate of the mean flight time based on the phase shift between a detected signal and the excitation signal. In some cases, more accurate image reconstructions can be obtained using more complete measurements of the flight-time distributions. This data is not readily obtained with frequency-domain instrumentation. A further disadvantage of the frequency-domain approach is the need for accurate high-frequency analog electronics. An overview of both the time-domain and frequency-domain techniques can be found in the above-referenced article by Hielscher, et al.

[0009] U.S. Pat. No. 5,565,982 discloses a time-resolved spectroscopy system using digital processing techniques and two low power, continuous wave light sources. The disclosed system requires two light transmitters of different wavelengths modulated with separate codes for interrogating a sample of interest. Properties of the sample are inferred by differential comparison of the return signals from each of the two light sources. It is undesirable to have two distinct light sources due to the cost and complexity involved. Furthermore, the noise level associated with a measurement made with two separate light sources will be higher than with a single source even if the codes used to drive the two sources are orthogonal.

[0010] A system and method capable of addressing these disadvantages while providing acceptable photon measurements or fluorescence lifetime measurements for whatever application the measurement is being used is needed.

[0011] It is also desirable to have a means of interrogating a particular tissue volume with a single light source at one wavelength in order to obtain temporal information. What is needed is an imaging system capable of sentinel lymph node mapping that does not require the use of radiotracers. Furthermore, the system should be implemented with low-power continuous- wave light sources and digital electronics.

Summary of the Inventions

[0012] The inventions presented herein provide for direct measurements of fluorescence lifetime or photon flight-time using any light source modulated with a known digital pattern. A preferred system uses a low-power continuous-wave light source and low-cost detector. Preferably the measurement system is implemented with digital electronics. One embodiment of the system and methods disclosed comprises a continuous-wave light source modulated with a digital waveform for interrogating a sample or a tissue volume, a photo-sensitive detector for measuring the fluorescence light or scattered light from the sample, and electronics for sampling the detector output and performing a correlation of the output signal with the modulation waveform. Other embodiments include electronics and software for calculating the parameters of the fluorescence lifetime of the photon flight-time distribution from the measured correlation.

[0013] The inventions further comprise a system with multiple sources for interrogating different sections of tissue volume, multiple detectors for detecting light scattered by the different sections of tissue volume, and software means for converting the detected signals into a reconstructed image of the underlying volume. Another embodiment includes a means of imaging the location of fluorescent dye within tissue in order to construct a map of the lymph nodes.

Brief Description of the Drawings

[0014] Fig. 1 is a functional block diagram of the major components of preferred fluorescence or photon measurement systems of the present invention.

[0015] Fig. 2 is a diagram of preferred Analog-to-Digital converters and their interface to the signal detector.

[0016] Fig. 3 is a functional block diagram of a preferred signal generator.

[0017] Fig. 4 depicts an implementation of a preferred Linear Feedback Shift Register.

[0018] Fig. 5 is a functional block diagram of a preferred signal detector.

[0019] Fig. 6 is a functional block diagram of a preferred frame accumulator.

[0020] Fig. 7 is a functional block diagram of a preferred frame correlator. [0021] Fig. 8 is a mechanical view of an embodiment of the present invention. [0022] Fig. 9 is an experimental curve indicating the binding of biotin and streptavidin in solution obtained with the present invention.

[0023] Fig. 1OA and 1OB depict an embodiment of the present invention using a 64- element photomultiplier array.

[0024] Fig. 11 is an embodiment of the present invention using an 11x11 array of fibers to deliver light between the sources or detectors and the patient. [0025] Fig. 12 is a mechanical view of a preferred embodiment.

[0026] Fig. 13 is a functional representation of a preferred embodiment using a needle- based optical probe coupled to a source and a detector with a single optical fiber. [0027] Fig. 14 depicts a preferred needle-based probe.

Detailed Description of the Drawings

[0028] Fig. 1 depicts a functional block diagram of a preferred measurement system 100 which can be a fluorescence measurement system or a photon measurement system. In case of the fluorescene measurement system, the present system 100 is used to measure the fluorescence lifetime of a sample 5. In case of the photon measurement system, the system 100 is used to measure the interaction of photons with the sample 5. In certain applications, the sample 5 may be a chemical or combination of chemicals contained in a solution. The sample 5 may be human breast tissue or fat tissue but it could just as well be any semitransparent material. In other applications the sample may be cellular matter or other biological material or any material in which fluorescence can be excited. The measurement system 100 preferably includes Temporal Response Analysis Engine 11. The Temporal Response Analysis Engine 11 generates a digital modulation signal for driving an illumination light source that is used to interrogate the sample. The Temporal Response Engine 11 also provides a means for processing a detected optical signal from the sample 5 to extract information about the sample 5. Preferably a digital modulation signal 16 is generated in the signal generator 1 and transmitted to the transmit signal conditioner 2. The digital modulation signal 16 is the digital representation of a chosen code sequence. The code sequence is preferably chosen from the known pseudorandom binary sequences (PRJBS), Gold codes, Golay codes, Kasami codes, Walsh codes, or other codes that possess the preferred desirable property of large auto-correlation values and low cross-correlation values. The digital modulation signal 16 may represent a single code pattern or multiple repeats of the same pattern. A single complete set of code patterns is designated a modulation frame or code pattern frame. The digital modulation signal 16 is preferably transmitted to the signal detector 10 as an electronic reference signal 17. The transmit signal conditioner 2 formats the digital modulation signal 16 as necessary to drive an optical illumination source 3. If system 100 is a fluorescence measurement system, the modulated optical source 3 is a 635nm diode laser made by Sanyo Corp. If system 100 is a photon measurement system, the modulated optical source 3 is a 785nm diode laser made by Hitachi Corp. In some embodiments a 785 nm continuous wave diode laser may be preferred. Formatting of the digital modulation signal 16 in the preferred embodiment involves converting the digital modulation signal 16 to an analog voltage waveform that is coupled through a 50-ohm bias-T to the DC drive current of the optical illumination source 3. In other embodiments, the optical illumination source 3 may be a different laser diode, a light-emitting diode, or a light source used together with an external optical modulator. The optical illumination source 3 generates the modulated optical wave 20 which is preferably transmitted to the sample 5 by light delivery optics 4. The preferred light delivery optics 4 is a 3mm diameter fiber bundle located between the optical illumination source 3 and the sample 5 to deliver the modulated optical wave 20 from the optical illumination source 3 to the sample 5. In other embodiments the light delivery optics 4 comprises other arrangements of optical fibers, lenses, mirrors or other optical delivery components. In case of fluorescence measurement system, when the modulated optical wave 20 illuminates the sample 5, fluorescence optical waves 21 are generated. In case of photon measurement system, the sample 5 is treated with a fluorescent material that will fluoresce when it is illuminated by the modulated optical wave 20, and in this case optical waves 21 are scattered optical waves. In the preferred measurement system, the optical waves 21 are fluorescence generated from a fluorescent material within the sample 5. The fluorescent material is preferably an exogenous contrast agent added to or injected into the sample 5 or alternatively it is preferably some constituent component of a material that exhibits endogenous fluorescence. The detection optics 6 are situated so that a portion of the modulated optical waves 21 are detected by the detection optics 6. In the preferred measurement system 100, the detection optics 6 include an optical filter for separating the optical wave 21 from the modulated optical waves 20. The optical filter preferably transmits the higher wavelength fluorescence and blocks the lower wavelength illumination light. In applications where the portion of the modulated optical wave 20 scattered in the direction of the detection optics 6 is small compared to the fluorescence optical wave, or where the scattered optical waves 21 of interest are not fluorescing, an optical filter is not required.

[0029] In the preferred measurement system 100, the detection optics 6 preferably include a second 3mm diameter fiber bundle located between the optical filter and the optical detector 7. The optical detector 7 converts the optical waves 21 to an electronic signal. In the preferred fluorescence measurement system 100, the optical detector 7 is preferably a 0.5mm-diameter silicon avalanche photodiode (APD) manufactured by Pacific Silicon Sensor. In case where system 100 is a photon measurement system, the optical detector 7 is preferably a phtomultiplier tube, model R7400U-20 from Hamamatsu Corp. In other embodiments, the optical detector 7 may be a PIN photodiode, a photomultiplier tube, an avalanche photodiode, a charge-couple device, or other suitable photosensitive element. As previously stated, the optical detector 7 preferably converts detected optical waves 21 into an electronic signal which is communicated to the detected signal conditioner 8. The detected signal conditioner 8 preferably formats the signal so it may be converted to discrete samples by an Analog to Digital (AJO) converter 9. The AfD converter 9 outputs a detected response signal 19. The detected response signal 19 is communicated to a signal detector 10, where it is preferably correlated with the electronic reference signal 17 to extract a sample transfer characteristic.

[0030] Information about the temporal properties of the photons is preferably calculated from the sample transfer characteristic. In case of fluorescence measurement sytem, this information preferably includes such properties as the fluorescence lifetime. The estimate of fluorescence lifetime can preferably be used to estimate characteristics such as the degree of chemical binding or to infer properties of the chemical environment surrounding the fluorescing material. For photon measurement system, this information preferably includes such properties as direct measurements of photon time-of- flight and the fluorescence lifetime. The estimate of photon times-of-flight is then preferably used to estimate characteristics of the tissue such as the absorption coefficient, scattering coefficient, or location of fluorescing material.

[0031] Another embodiment of the measurement system 100 includes an optical reference generator 22. The optical reference generator 22 preferably includes an optical splitter 12A or 12B that routes a portion of the modulated optical wave 20 to a secondary optical detector 13. The position of the optical splitter 12A or 12B can be either before or after the light delivery optics. The output of the secondary optical detector 13 is preferably routed to a secondary signal conditioner 14 whose output is communicated to a secondary A/D converter 15. The secondary A/D converter 15 preferably outputs a source reference signal 18 which can be correlated with the detected response 19 to extract the sample transfer characteristic. Using the source reference signal 18 as opposed to the electronic reference signal 17 allows the filtering of the temporal properties of the signal conditioner 2 and the modulated optical source 3 from the measured transfer characteristic. [0032] The preferred hardware implementation of the A/D converter module and its interfaces to the signal detector 10 are shown in Fig. 2. An array of N A/D converters 90 preferably receives the analog signal 95 in parallel from the signal conditioner 8 or 14 . The output samples 18 or 19 from the A/D converters 90 are preferably communicated to the First-In-First-Out buffers (FIFOs) 91 where they are buffered for distribution to the internal components of the signal detector 10. In the preferred measurement system 100 the A/D converters 90 are eight MAX 108 integrated circuits made by Maxim operating at 250 Msample/sec and outputting two data samples at a time in parallel at 125MHz. The FIFOs 91 are preferably implemented within a Xilinx 4 FPGA. The acquisition synchronizer 92 preferably controls signal acquisition and digital data distribution through the conversion clock (CCIk) signals 96.

[0033] The acquisition synchronizer 92 is preferably synchronized with an externally provided synchronization clock (SCIk) 40 which is also preferably used to synchronize the signal generator 1. The signals CCIk[I .. N] are preferably generated within the acquisition synchronizer 92 and preferably have the same frequency as SCIk 40 but are offset in phase from SCIk 40 in N fixed increments of (360 ÷ N)°, with the phase of CCIk[I] set to the fixed offset of Z°. In the preferred system the internal clock generation capabilities of the Xilinx FPGA are used to implement the acquisition synchronizer 92 directly. The A/D converters 90 preferably perform their conversions in sync with the conversion clocks 96 such that they generate samples at N discrete sample times spread evenly throughout the fundamental sample interval defined by the period of SCIk 40. The effective sample rate for the array of converters is preferably N times the rate defined by SCIk 40. This process of using multiple A/D converters sampling out of phase to increase the effective sample rate is what we call parallel over-sampling. In the preferred measuring system, parallel over-sampling results in an effective sample rate of 2Gsamples/sec. The offset value Z allows the entire sample set to be offset by some phase from the synchronization clock 40. The acquisition synchronizer 92 preferably is configured such that the value of Z can be varied synchronously with the modulation frame, or with a block of frames called a frame block. This allows Z to follow a sequence of K values smaller than (360 ÷ N)⁰ such that on successive modulation frames/frame blocks the effective sampling phases (relative to the synchronization clock) take on K values intermediate to those created by the N conversion clocks in any given frame. In this case preferably the input signal at any given A/D converter 90 will be sampled at K discrete phases over K blocks. The detected response 19 is preferably assumed to be stationary with respect to the start of the code pattern block over that time interval. The preferred K discrete sampling phases correspond to K discrete sample times and the effective temporal resolution of the sampling process is preferably increased by a factor of K. This process is referred to as temporal over-sampling.

[0034] In the preferred measuring system the value of Z is always zero and temporal over-sampling is achieved by adjusting the phase of the modulation as described below rather than by adjusting the phase of the A/D converter sampling. Preferably the FIFOs latch input data to the A/D converters 90 synchronously with the corresponding conversion clock 96. The FIFO 91 output data is preferably provided to the internal components of the signal detector 10 synchronously with the synchronization clock 40 such that all further processing is synchronized with the synchronization clock 40.

[0035] The preferred implementation of the Temporal Response Analysis Engine 11 are shown in Figs. 3 through 7; the preferred signal generator 1 is shown in Figs. 3 and 4, while the preferred signal detector 10 is shown in Figs. 5, 6, and 7. In the preferred system the Temporal Response Analysis Engine 11 is implemented as logic blocks within a Xilinx 4 FPGA.

[0036] The functional blocks of the preferred signal generator 1 are shown in Fig. 3. The top 41 and bottom 42 signal paths are two preferred variants for generating different code patterns for the modulation signal 16. In the top path 41 a Linear Feedback Shift Register (LFSR) 30 is preferably used to create a PRBS code. The specific code pattern is preferably determined by the number of state bits within the LFSR 30 and the gain code 36 input to the LFSR 30. In one preferred implementation the gain code 36 is stored in a gain memory 31, which is preferably configured to allow the code pattern 16 to be changed during operation either by selecting one of several gain codes from a read-only memory or by setting a new gain code into a writable memory. In other embodiments the gain code 36 may be hard-wired into the LFSR 30, or a code-specific state-machine designed to generate a desired code through a series of state transformations may be used in place of the LFSR 30. In the bottom path 42 the entire code pattern is preferably stored as a bit sequence in a pattern memory 32. The sequence in which pattern bits are presented is preferably determined by an address sequencer 33 which preferably provides the cell addresses 37 for the memory. The address sequencer 33 is preferably configured to allow changing the code pattern 16 during operation either by selecting one of several patterns stored in a read-only memory or by inputting a new pattern into a writable memory.

[0037] The modulation signal 16 for both the LFSR 30 or pattern memory implementation is preferably buffered by an output buffer 35 to make the signals 16 more robust when driving external components. Timing for presentation of the code pattern bits is preferably controlled by a generation synchronizer 34 which preferably generates the master clock (MCIk) 38 for the LFSR 30 and the address sequencer 33. The master clock 38 is preferably synchronized to a system synchronization clock (SCIk) 40 which preferably controls both code pattern generation and response signal acquisition. MCIk 38 preferably operates at the same frequency as SCIk 40 but is preferably offset in phase by an amount specified by the phase input 39, which is preferably an externally programmable parameter. This phase offset allows the relative phase between the modulation signal 16 and the detected response 19 to be adjusted. If the phase is adjusted by some increment, (360÷K)°, at the end of each code pattern block or set of blocks the detected response resulting from the modulation signal will preferably be sampled at K discrete phases over K blocks. In this embodiment of the measuring system as with the preferred embodiment, the detected response 19 is assumed to be stationary with respect to the start of the code pattern block over that time interval so that the K discrete sampling phases correspond to K discrete sample times and the effective temporal resolution of the sampling process is increased by a factor of K. [0038] This temporal over-sampling is functionally equivalent to the technique described for temporal over-sampling in the A/D converter embodiment. In other embodiments the external phase specification may represent the phase increment rather than the absolute phase, and the generation synchronizer 34 may increment the phase internally.

[0039] The preferred implementation of the LFSR 30 is shown in Fig. 4. The LFSR 30 is preferably a state-machine comprising M standard LFSR cells 48 which hold and transform the state. The LFSR cells 48 are preferably linked in a numbered sequence, and the output from the LFSR 30 is the current state of cell number zero. Each cell preferably comprises a state latch 45 which holds a single bit of state information, a gain element 46 to control the feedback gain for the cell based on the externally provided gain code 36, and an accumulator 47. The accumulator 47 preferably adds the feedback from the cell to the cumulative feedback from all previous cells. At each clock increment the state for a cell is updated to match the previous state from the next higher cell in the chain; the state of the last cell in the chain is updated with the accumulated feedback from all the previous cells. The accumulator 47 for the last cell in the chain may be omitted if desired. The pattern generated by the LFSR 30 is preferably determined by the number of cells in the chain and by the gain code. In a preferred embodiment the gain code is provided from an external source to allow the code pattern to be modified. In other embodiments the gain code may be a fixed value. In embodiments in which the gain code is fixed, the implementation of the gain elements and accumulators for each cell may be optimized for the specific gain code for that cell rather than implemented in the generalized fashion shown. The clock for the LFSR 30 and for all its internal latches is preferably the signal generator master clock 38.

[0040] The preferred functional blocks for the signal detector 10 are shown in Fig. 5. The detected response 19 and either the electronic reference signal 17 or the source reference signal 18 are received at two frame accumulators 50 and 51, where the samples for each discrete sample time are accumulated with samples from identical sample times from different modulation frames to form the aggregated detected response 58 and the aggregated reference signal 59. As a result of this aggregation, the effective data rate at which samples are preferably processed in following blocks is reduced by a factor equal to the number of frames aggregated into each sample point. The frame accumulators 50 and 51 are preferably replicated N times to handle the N channels of the A/D converter independently. The internal details of the frame accumulators 50 and 51 for the detected response and the reference signal may differ, depending on the digital format of the two signals. For example, if the reference signal used for analysis is the electronic reference signal 17 rather than the source reference signal 18 its value for each sample time is known a priori to be identical for every frame and to take on only two possible binary values, 0 or 1. In that case preferably the frame accumulator 51 for the reference signal 17 need only store one bit per sample time, equal to the value of the modulation signal for that sample time. At some point between the output of the frame accumulators and final output of the sample transfer characteristic 57 the N acquisition/accumulation channels are preferably re-interleaved into a single data stream. In one preferred embodiment two multiplexers 52 and 53 perform this reintegration at the output of the frame accumulators 50 and 51. In other embodiments this re-integration may take place at any other desired point in the signal processing chain. With or without re-integration the aggregated detected response 58 and the aggregated reference signal 59 are routed to the frame correlator 55 where the two signals 58 and 59 are preferably combined by a cross- correlation algorithm to produce the correlated signal 61 which preferably comprises a single value for each complete aggregated frame of samples. The correlated signal 61 represents the degree to which the aggregated response signal 58 contains components matching the aggregated reference signal 59. If the aggregated reference signal 59 is delayed by a time τ before presentation to the correlator 55 then the correlated signal 61 represents the degree to which the aggregated response signal 58 contains components of the delayed version of the aggregated reference signal 60. The sample transfer characteristic 57 comprises a set of correlated signals calculated for a range of J such delay times. Phase delay blocks 54 generate the delayed versions of the aggregated reference signal 60. For simplicity the J phase delay blocks 54 are illustrated as discrete components operating in parallel and each providing the complete delay required for one correlated signal. In one preferred embodiment they comprise a cascade of J phase delay blocks each providing the time increment between one correlated signal and the next. The phase delays for the correlated signals are preferably discrete and correspond to integral multiples of the synchronization clock 40 period. The phase delay blocks 54 are preferably implemented as shift registers or FIFOs of the appropriate depth and clocked by the synchronization clock 40. In other embodiments the time delay may be implemented using other methods. In one preferred embodiment each phase delay is processed by a corresponding frame correlator 55. In other embodiments a single frame correlator 55 may be used to calculate the correlated signal 61 for multiple phase delays by presenting the detected response data to its input multiple times, using a different phase delayed version of the reference signal 60 for each iteration. In this case fewer frame correlators 55 are required.

[0041] The details of the preferred frame accumulator 50 or 51 are shown in Fig. 6. Samples from the signal 17, 18, or 19 are preferably accumulated in the adder 70 by summing them with values taken from the memory 71 ; the resulting aggregated signal 58 or 59 is routed to the output of the accumulator and stored back into the memory at the same location from which the original data was taken. Each discrete sample time for the channel is represented by a single addressed cell within the memory. The size of the memory is preferably determined by two parameters, K and R, which preferably encode the sampling scheme. K represents the number of discrete phases at which samples are preferably taken in various frames during temporal over-sampling. R is the ratio of the number of samples in a modulation frame to the number of sampling channels provided in the A/D converter 90 for parallel over-sampling and signifies the number of samples that must be accommodated by each channel within a single frame. A preferred sample enable gate 72 is provided to restart the accumulation process at the beginning of each set of frames by clearing the cells in the memory. The address sequencer 73 selects the cell of the memory to be addressed for each sample point. The frame accumulators 50 or 51 preferably run synchronously with the synchronization clock 40 (although out of phase), so only a single address sequencer is required to address all the frame accumulators. [0042] The details of the preferred frame correlator 55 is shown in Fig. 7. The ideal method for correlating the signals is to take the integral of the detected response 19 weighted by the reference signal 17 or 18. Because the preferred embodiment is a sampled system the integration is approximated by summation over all the samples within a frame set using the adder 81 to generate the correlation signal 61. The weighting of the aggregated detected response 58 by the aggregated reference signal 59 is preferably performed by a multiplier 80. Other embodiments may employ other weighting and integration schemes, including scaling and integration in the analog domain directly on the detected signals. A sample enable gate 82 is preferably provided to restart the accumulation process at the beginning of each set of frames by clearing the correlator .

[0043] The geometric relationship shown in Fig. 1 between the light delivery optics 4, the sample 5, and the detection optics 6 is schematic and not intended to reflect actual physical geometry. In practice, the delivery optics and the detection optics can be placed on the same side of the sample, on opposite sides of the sample, or at arbitrary positions with respect to the sample. Fig. 8 depicts a mechanical view of one embodiment of the fluorescence measurement system. An electronics unit 85 includes the modulated optical source, optical detector, temporal response analysis engine, and associated electronics. The illumination light is delivered to the sample contained in a sample container 88 using the delivery fiber bundle 86. The response optical signal is delivered from the sample to the electronic units using the detection fiber bundle 87. The fiber bundles are flexible and easily repositioned with respect to the sample. An optical filter 89 that transmits the fluorescence light while rejecting excitation light is placed between the sample container 88 and the detection fiber bundle 87.

[0044] A method for using the present invention for examining chemical binding is as follows. A substance that exhibits fluorescence is placed in a sample holder. The substance may naturally exhibit fluorescence or it may be a material that has been modified by the addition of a fluorescent label. The fluorescence from the sample is then measured as described above to obtain a temporal transfer characteristic. One or more additional substances is then added to the sample holder and allowed to interact with the first substance. The fluorescence is measured again. By comparing the temporal transfer characteristic obtained before the second material was added to that obtained after the material is added, one can estimate the change in the system caused by adding the second material. If the two materials interact, the width and/or shape of the measured temporal transfer characteristic typically will change. A binding curve is generated by measuring the change as a function of the relative concentrations of the two materials.

[0045] The present invention can also be used for monitoring the kinetics of chemical interactions. In this case, the output of the fluorescence measurement system is monitored continuously or at multiple discrete time intervals after the second substance is added to the sample. The kinetics of the interaction is determined by measuring the change in the temporal transfer characteristic as a function of time. [0046] In one embodiment, the present invention was utilized to investigate the binding of biotin, a colorless crystalline vitamin of the vitamin B complex, to streptavidin, a protein that has a high affinity to biotin. The streptavidin was labeled with Cy 5, a fluorescing dye that can be excited with wavelengths around 635nm. The starting solution consisted of 1 μM concentration of streptavidin in a buffer solution. The light source was a Sanyo DL5038-21 635nm diode laser. The photodetector was a 0.5mm- diameter APD, part number AD500-1.3G-TO5 from Pacific Silicon Sensor. The Temporal Response Analysis Engine was implemented with a 2.5Gsample/sec data acquisition card from Z-Tec for signal detection and a Tektronix DG2040 digital pattern generator for signal generation. Correlation calculations were carried out in software on a personal computer. The result of the correlation is the sample transfer characteristic. A change in the width of the sample transfer characteristic is a direct measure of the change in fluorescence lifetime. The laser was modulated at a bit rate of 125Mb/sec with a 31-bit PRBS code. Measurements of the sample transfer characteristic width were made for different concentrations of biotin added to the solution containing the labeled streptavidin. As the concentration of biotin increased, the fluorescence lifetime of the Cy5 dye changed due to the binding of biotin molecules to the streptavidin molecules. This change in lifetime was reflected as a change in the width of the sample transfer characteristic. For each measurement, the code sequence was repeated 20 times, with the data averaged over the 20 cycles. Correlation was performed on the averaged data. A plot of change in transfer characteristic width as a function of biotin concentration is shown in Fig. 9. [0047] When the measurement system 100 is a photon measurement system, it can be useful for interrogating a section of tissue located generally between the light delivery optics and the detection optics. In order to interrogate a larger tissue volume, it is useful to have a system where the photon measurement system is replicated so that separate tissue sections can be interrogated with separate source-detector pairs. One embodiment of such a system is shown in Fig. 1OA and 1OB. Eight fiber bundles 105 are used to deliver light from eight different sources to the tissue. The fiber bundles are shown encircled by the dotted line in Fig. 1OA. The detectors are a 64-element photomultiplier array 106 manufactured by Hamamatsu with the individual elements in an 8x8 arrangement. Fluorescent light from the tissue passes through an optical filter 108 that blocks light at the excitation wavelength. The fluorescent light is coupled to the detector array by a 2.5:1 tapered imaging fiber bundle 107 made by Schott Corp. An exploded view of the detector array 106, filter 108, and imaging fiber bundle 107 is given in Fig. 1OB. Each source-detector pair can be coupled to electronics as shown functionally in Fig. 1 to form an individual photon measurement system. Each source-detector pair yields information about photon time of flight through a somewhat different section of tissue than any other pair. Each source can be turned on sequentially, while all the detectors can be sampled simultaneously while a given source is on. Alternatively, each source can be driven with a different code such that any code is orthogonal to the others. In this case, the sources can be driven simultaneously and the low cross-correlation of the respective reference signals allows separation of the signals. The sequential case will exhibit improved signal-to-noise ratio compared to the simultaneously on case due to the non-ideal cross-correlations obtained in practice. Another embodiment of the present invention is shown in Fig. 11. In this case, the imaging instrument 1 10 includes an 11x1 1 array 1 1 1 of multimode fibers for coupling light from the sources and detectors in an electronics module 112 to the tissue. Each fiber can be coupled to either a source or a detector. The fibers are spaced at lcm intervals on the imaging head 1 15. The image reconstructed from the measured data is displayed on the monitor 113. The imaging head 115 can easily be manipulated to image various parts of the patient 114. The present invention is not limited to the particular geometries described here. The use of the photon measurement system 100 is possible with various combinations of sources and detectors and various positions of the sources and detectors. In the examples described, the geometry is a reflection geometry with the sources and detectors effectively on the same side of the tissue. In other embodiments, the detection optics can be placed on the opposite side of the tissue from the light delivery optics. The particular number of sources and detectors can also be varied depending on the resolution and fϊeld-of-view required for a particular application. In the present embodiments, the instrument is intended to cover an area of approximately 10cm x 10cm area. Imaging a larger area can be accomplished by moving the instrument head across the area. The embodiments described utilize a photomultiplier array as the optical detectors. In other embodiments, it is possible to use PIN photodiodes, avalanche photodiodes, individual photomultiplier tubes, detector arrays, charge-coupled device arrays, or other photosensitive elements.

[0048] The present invention is utilized for sentinel lymph node mapping as presently described. A patient is injected near the site of a malignancy with a dye that fluoresces when exposed to near-infrared light. In particular, indocyanine green (ICG) can be excited at wavelengths around 785 nm and fluoresces at wavelengths around 830nm. The dye serves both as a visual guide for the surgeon and as a contrast agent for the optical imaging system. ICG has the advantage that it is already approved for use in medical procedures such as angiography; however, several alternative contrast agents are also available. Imaging proceeds as follows. Assuming the imaging is performed reasonably soon after injection of the dye, the dye will be relatively well-localized in the sentinel node or nodes. If the dye is ICG, this amount of time is one the order of minutes. The imaging head is placed in contact or in close proximity to the tissue suspected of containing the sentinel node. The correlator output, or characteristic transfer function, is measured for each source-detector pair. For any given source and detector position, it is possible to calculate a priori the expected characteristic transfer function for a given location of fluorescence dye. In practice, because the tissue is so highly scattering, neighboring source-detector pairs can have somewhat overlapping interrogation regions. The image reconstruction problem consists of estimating the most likely distribution of dye given all the measurements of characteristic transfer functions from all the source- detector combinations. Various techniques are known for performing such an inversion problem, including such methods as singular-value decomposition and the Algebraic Reconstruction Technique, also known as the Gauss-Seidel method. The result of the inversion is a volumetric map of the location of dye within the tissue. Because the dye collects predominantly in the sentinel node(s), this map is effectively a map of the sentinel node location. This map is displayed in the form of an image or images on a monitor attached to the instrument. The surgeon uses this image to plan his surgical incisions. The estimated positions of the sentinel node with respect to the instrument are also displayed on the monitor, allowing the surgeon or other operator to mark the body before the surgery begins.

[0049] A preferred imaging method for locating the sentinel lymph node or nodes is as follows. The patient is injected with fluorescent material near the site of a malignancy. Imaging begins after an amount of time sufficient for the fluorescent material to reach the sentinel lymph node or nodes. The instrument head is placed over the patient at a position that represents an initial estimate for the location of the sentinel node. With the instrument head in position, the first optical source is turned on for an amount of time corresponding to the desired number of repeats of the code sequence. Scattered optical waves are measured at each corresponding detector. The output of each detector is correlated with the reference signal as described above to produce a temporal transfer characteristic corresponding to the source-detector combination. The temporal transfer characteristics for each source-detector combination are stored in memory. The process is repeated for each subsequent optical source until temporal transfer characteristics are collected for all desired source-detector pairings. The acquired temporal transfer characteristics are then used to reconstruct an image of the underlying tissue volume using an algorithm implemented in software. The algorithm is based on the ability to estimate a priori the temporal transfer characteristic that will be obtained for any source- detector pairing for any particular location of fluorescent dye. The algorithm generates a most likely estimate of the fluorescent material locations based on the a priori models given the measured temporal transfer characteristics. This estimate of fluorescent material locations is displayed in the form of a volumetric image on a monitor connected to the instrument. The user of the instrument can conclude based on the image whether or not the underlying tissue contains a sentinel node. Generally, the node will be imaged as a subset of the volume with a high estimated concentration of fluorescent material. If the user judges that the sentinel node has been located, he may physically mark the body where the instrument head had been placed with a pen to indicate the area in which to cut. Alternatively, he may save the image on the screen or on a printout so that it may be referred to during surgery. If the user concludes that the sentinel lymph node has not been located, he moves the instrument to a different location and the process is repeated.

[0050] As in case of fluorescent measurement system, for photon measurement system, the geometric relationship between the light delivery optics 4, the sample 5, and the detection optics 6 of the preferred photon measurement system 100 depicted in Fig. 1 is schematic and not intended to limit the possible actual physical geometry of the system. For example, the delivery optics and the detection optics may be placed on the same side of the sample 5, on opposite sides of the sample 5, or at arbitrary positions with respect to the sample 5 so long as the scattered optical wave 21 is detectable. Fig. 12 depicts a mechanical view of a preferred embodiment of the photon measurement system 100. An electronics unit 125 preferably includes the modulated optical source 3, optical detector 7, temporal response analysis engine 11, and associated electronics. Preferably the modulated optical wave 20 is delivered to the sample 5 using the delivery fiber bundle 126. The scattered optical wave 21 is delivered from the sample to the electronic unit 125 using the detection fiber bundle 127. The fiber bundles are flexible and easily repositioned with respect to the sample. [0051] Fig. 13 depicts a functional representation of another preferred embodiment in which a single optical fiber serves as part of both the light delivery optics and the detection optics. The light 131 output by the modulated optical source 130 is reflected by an optical filter 137 toward coupling optics 132. The coupling optics focuses the light into an optical fiber 133 which delivers the light to an optical probe 134 with a removable needle 135. The removable needle includes a section of optical fiber allowing the light to be transmitted from the probe to the end of the needle. The needle can be inserted into a tissue sample 136 so that greater depth can be interrogated than in the case of a surface- based probe. A portion of light scattered by the tissue is coupled back into the fiber and delivered back to the coupling optics 132. A portion of this return light 138 is transmitted by the optical filter to an optical detector 139. The modulated optical source 130 and the optical detector 139 are connected to the temporal response analysis engine and associated electronics as described previously. A mechanical view of the needle-based probe is given in Fig. 14. The needle assembly 135 connects to the probe 134 with a standard SMA connector 141. Ih addition to the embodiments described here, other geometric arrangements of light delivery optics, detection optics, and sample are possible.

Claims

1. A fluorescence measurement system comprising a continuous- wave light source modulated with a digital waveform for interrogating a sample containing a fluorescing material; a photo-sensitive detector for measuring the fluorescence light from the sample; and digital electronics for sampling the detector output and performing a correlation of the output signal with the modulation waveform; the electronics configured to calculate parameters of the fluorescence lifetime distribution from the measured correlation.

2. A method for examining chemical binding comprising inserting a fluorescing material to be examined in a sample; measuring fluorescence emanating from the sample using a continuous-wave light source modulated with a digital waveform photon measuring system; a photo-sensitive detector for measuring the fluorescence light from the sample, and digital electronics for sampling the detector output and performing a correlation of the output signal with the modulation waveform to obtain a first temporal transfer characteristic; adding at least one substance to the sample; measuring the fluorescence using the continuous-wave light source modulated with the digital waveform photon measuring system; the photo-sensitive detector for measuring the fluorescence light from the sample, and the digital electronics for sampling the detector output and performing the correlation of the output signal with the modulation waveform to obtain a second temporal transfer characteristic after the fluorescing material and the added substance has had time to interact; comparing the first and the second temporal transfer characteristics to determine whether the fluorescing material and the added substance interacted.

3. The method of claim 2 wherein the width or shape of the first and the second measured temporal transfer characteristics are compared to determine if there has been an interaction.

4. The method of claim 2 wherein a binding curve is generated by measuring and comparing the temporal characteristics as a function of relative concentrations of the fluorescing material and the added substance.

5. The method of claim 2 wherein neither of the materials to be examined for chemical binding naturally exhibit fluorescence comprising modifying the first material by addition of a fluorescent label.

6. A method for monitoring kinetics of chemical interactions comprising placing a fluorescing material to be examined in a sample receiver; measuring fluorescence of the fluorescing material using a continuous-wave light source modulated with a digital waveform photon measuring system, a photo-sensitive detector for measuring the fluorescence light from the sample, and digital electronics for sampling the detector output and performing a correlation of the output signal with the modulation waveform to obtain a temporal transfer characteristic; adding at least one substance to the sample; measuring the fluorescence of the sample using the continuous- wave light source modulated with the digital waveform photon measuring system, the photosensitive detector for measuring the fluorescence light from the sample, and digital electronics for sampling the detector output and performing the correlation of the output signal with the modulation waveform to obtain the temporal transfer characteristic; monitoring the output signal after the second substance is added to the sample receiver; determining the kinetics of an interaction of the fluorescing material and the added substance by measuring change in the temporal transfer characteristic as a function of time.

7. The method of claim 6 wherein the monitoring is done continuously

8. The method of claim 6 wherein the monitoring is done at multiple discrete time intervals.

9. The method of claim 6 wherein neither of the materials to be examined for chemical binding naturally exhibit fluorescence comprising modifying the first material by addition of a fluorescent label.

10. The method of claim 6 wherein the sample receiver is of a translucent material.

11. An imaging method for locating nodes in a mammal comprising: injecting the mammal with fluorescent material near the site of a malignancy obtaining a temporal transfer characteristic for each of a plurality of source- detector combinations by a method comprising causing a continuous wave optical source from the plurality of source- detector combinations to be modulated with a digital waveform for a predetermined amount of time to generate a modulated optical wave; directing the modulated optical wave at a position that represents an initial estimate for the location of a node causing the injected fluorescent material to fluoresce, detecting the fluorescence light using a photo-sensitive detector of the source-detector combination that generated the modulated optical wave; using digital electronics for sampling the detector output and performing a correlation of the detector output signal with the digital modulation waveform to produce the temporal transfer characteristic corresponding to the source-detector combination; storing the temporal transfer characteristics obtained from each of the plurality of the source-detector combinations in accessible memory; reconstructing an image of the underlying tissue volume based in part on the stored temporal transfer characteristics.

12. The method of claim 1 1 wherein the mammal is a human.

13. The method of claim 11 wherein method for reconstructing the image comprises ability to estimate a priori the temporal transfer characteristic that will be obtained for any source-detector pairing for any particular location of fluorescent dye.

14. The method of claim 13 wherein the method for reconstructing comprises generating a most likely estimate of the fluorescent material locations based on the a priori models given the measured temporal transfer characteristics.

15. The method of claim 14 wherein the estimate of fluorescent material locations is displayed in the form of a volumetric image on a monitor.

16. A node imaging system comprising plurality of optical source-detector combinations; the optical sources of the optical source-detector combinations are continuous- wave light sources modulated with a digital modulation waveforms to generate modulated optical waves; the detectors of the optical source-detector combinations are photo-sensitive detectors capable of detecting fluorescence light emitted from a fluorescing material injected into a mammal whose nodes are to be imaged; and digital detector electronics for sampling the detector output from each optical source-detector combination and performing a correlation of the output signal of each detector with the digital modulation waveform modulating the continuous wave light source that generated the output signal to generate a temporal transfer correlation signal; and an image reconstruction engine that is configured to reconstruct an image of the underlying tissue volume based in part on the temporal transfer correlation signal.

17. The node imaging system of claim 16 wherein there are eight optical-source detector combinations.

18. The node imaging system of claim 16 wherein the optical source-detector combinations each comprise a single optical source and a plurality of detectors.

19. The node imaging system of claim 18 wherein the same plurality of detectors is part of each source-detector combination.

20. The node imaging system of claim 19 wherein the optical sources are configured to generate the modulated optical waves sequentially while the plurality of detectors are sampled simultaneously.

21. The node imaging system of claim 16 wherein the optical source-detector combinations each comprise a single optical source and a dedicated detector.

22. The node imaging system of claim 21 wherein the dedicated detectors comprise an array of detectors.

23. The node imaging system of claim 21 wherein each optical source is modulated by a different digital modulation wave.

24. The node imaging system of claim 23 wherein the digital modulation waves are orthogonal to each other.

25. The node imaging system of claim 23 wherein the optical sources are configured to simultaneously generate the modulated optical waves.

26. The node imaging system of claim 16 including directing optics configured to direct the modulated optical wave to the estimated location of the node to be imaged and to direct the fluorescence light emitted from the fluorescing material to the detectors.

27. A photon measurement system comprising a continuous wave photon source and a corresponding detector ; a modulator comprising a modulation code, the modulator coupled to the continuous wave photon source, the continuous wave photon source generating optical wave in accordance with the modulation code; delivery optics coupled to the continuous wave photon source to direct the modulated optical wave at a desired material; the corresponding detector arranged to detect scattered optical wave from the material; the corresponding detector having as an output a signal indicative of energy in the detected scattered optical wave; the detector output signal communicated to a correlator, the correlator coupled to the modulator; the correlator configured to generate and output a flight time signal indicative of the time it takes to detect a photon after the photon is generated by the source based in part on the modulation code.

28. The photon measurement system of claim 27 further comprising a memory module, the memory module including a correlator input; the correlator input coupled to the correlator to communicate the flight time signal to the memory module.

9. A system comprising a continuous wave light source and a corresponding detector; a signal generator coupled to the continuous wave light source such that output of the continuous wave light source is a modulated optical wave modulated in accordance with output from the signal generator; delivery optics coupled to the continuous wave light source to direct the modulated optical wave at a translucent material; an optical splitter coupled to the delivery optics between the delivery optics and the translucent material; the optical splitter coupled to an optical detector; the optical detector configured to generate an optical reference signal; the optical detector coupled to a signal detector such that when the optical reference signal is generated it is communicated to the signal detector; the corresponding detector arranged to detect scattered optical waves from the translucent material; the corresponding detector having as an output a signal indicative of a characteristic of the detected scattered optical wave; the corresponding detector output signal communicated to the signal detector; the signal detector configured to generate and provide an output indicative of a characteristic of the translucent material, based in part on the optical reference signal.

30. The system of claim 29 wherein the signal detector output is used as an input to an image reconstruction engine.

31. The system of claim 29 wherein the signal detector output is used to infer properties of the translucent material.

32. The system of claim 29 wherein the optical splitter is positioned between the continuous wave light source and the delivery optics.

33. The system of claim 29 wherein a first analog to digital converter is positioned between the optical detector and the signal detector to convert the optical reference signal into a digital signal; a second analog to digital converter is positioned between the corresponding detector and the signal detector to convert the corresponding detector output signal into a digital detector signal; and the signal detector configured to use digital processing techniques to generate the output indicative of the characteristic of the translucent material, based in part on the digitized optical reference signal.

34. The system of claim 33 wherein the signal detector comprises a detected response frame accumulator and a reference signal frame accumulator; the detected response frame accumulator having the digital detector signal as an input and the reference signal frame accumulator having the digitized optical reference signal as an input; the detected response frame accumulator configured to accumulate the digital detector signals over multiple sample times and generate an aggregated detected response signal; the reference signal frame accumulator configured to accumulate the digitized optical reference signals over multiple sample times and generate an aggregated reference signal; the reference signal frame accumulator is coupled to a plurality of programmed phase shifters such that the aggregated reference signals output from the reference signal frame accumulator form an input to the phase shifters; each phase shifter is configured to delay the aggregated reference signal a preset amount and output a delayed reference signal; the plurality of phase shifters are coupled to a plurality of frame correlators, number of phase shifters being equal to number of frame correlators, such that the delayed reference signal output from the phase shifter forms an input to a corresponding frame correlator; the detected response frame accumulator is coupled to each of the plurality of frame correlators such that the aggregated detected response signal output from the detected response frame accumulator forms an input to each of the frame correlators; wherein the frame correlators output a correlation signal.

35. The system of claim 29 wherein the signal detector is configured to employ temporal over-sampling to generate and provide the output indicative of the characteristic of the translucent material.

36. A system comprising a continuous wave optical source and optical detector pair and a temporal response analysis engine; the temporal response analysis engine comprising a signal generator and a signal detector; the signal generator having as an output a modulated signal; the optical source and the signal detector have as one of their inputs the modulated signal; the optical source, in response to the modulated signal outputs a modulated optical wave; the detector is configured to convert incident light waves into electrical signals indicative of characteristics of the incident light waves arid outputs the electrical signals which are communicated to an input of the signal detector; the signal detector based in part on the modulated signal and the electrical signals determines temporal delay between when the optical source generates the modulated optical wave and when the optical detector detects the modulated optical wave.

37. The system of claim 36 further comprising an optical probe and a needle, the optical probe is optically coupled at a first end to the continuous wave optical source and the detector; the needle comprising a delivery end and a connection end; the connection end is optically connected to a second end of the optical probe; the needle comprises optics to communicate light from the connection end to the delivery end and from the delivery end to the connection end.

38. A method for determining time of flight for a photon comprising: generating a modulated optical wave containing photons using a single low power continuous light source; directing the modulated optical wave to illuminate a material to generate scattered modulated optical waves; detecting the scattered modulated optical waves with a detector; converting the detected scattered modulated optical wave to an electrical signal; using digital processing techniques to determine the time it took the photons to be detected by the detector.

39. The method of claim 38 wherein the modulated optical wave is directed to a material by passing the modulated optical wave through a needle inserted in the material and passing the scattered modulated optical wave back through the needle to the detector.

40. A method of interrogating a translucent material comprising generating a light wave of a selected wavelength modulated with a digital waveform; directing the light wave at the translucent material to cause the light wave to be scattered from the material; receiving the scattered light waves at a photo-sensitive detector for measuring the light wave scattered from the material; sampling the detector output; and performing a correlation of the detector output with the modulated waveform.

41. The method of claim 40 wherein the correlation is performed utilizing temporal over- sampling.

42. A system comprising a continuous wave light source and a corresponding detector; a signal generator configured to generate a digital modulation signal; the signal generator is coupled to the continuous wave light source and a signal detector such that the digital modulation signal is communicated to the continuous wave light source and the signal detector; the continuous wave light source is configured to output a modulated optical wave modulated in accordance with the digital modulation signal delivery optics coupled to the continuous wave light source to direct the modulated optical wave at a translucent material the corresponding detector arranged to detect scattered optical waves from the translucent material; the corresponding detector having as an output a signal indicative of a characteristic of the detected scattered optical wave; the corresponding detector output signal communicated to the signal detector; the signal detector configured to generate and provide an output indicative of a characteristic of the translucent material, based in part on the digital modulation signal.

43. The system of claim 42 wherein an analog to digital converter is positioned between the corresponding detector and the signal detector to convert the corresponding detector output signal into a digital signal ; and the signal detector configured to use digital processing techniques to generate the output indicative of a characteristic of the translucent material, based in part on the digital modulation signal.

44. The system of claim 42 wherein the signal detector is configured to employ temporal over-sampling to generate and provide the output indicative of the characteristic of the translucent material.