Systems and methods for fiber-based laser speckle imaging

Abstract

An illumination system for laser speckle imaging includes a laser light source, two or more sections of optical fiber, a fiber-coupled acousto-optic modulator (FCAOM) coupled to the light source by a first section of the two or more sections of optical fiber, and a collimating optical system that focuses light output by the laser to illuminate an object within a field of view (FOV), the collimating optical system being coupled to the FCAOM by a second section of the two or more sections of optical fiber. In some implementations, the illumination system is incorporated into a laser speckle imaging system that includes an image capture device for capturing an image of the object within the FOV. In some implementations, the image is captured and processed using an intra-exposure modulation speckle imaging technique.

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant Nos. R01EB011556 and R01NS108484 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 478,264, filed January 3, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Monitoring cerebral blood flow (CBF) plays a critical role in countless neurosurgical and neuroscience applications. In the operating room, applications of CBF monitoring range from tumor resection, cerebral artery bypass, arteriovenous malformation (AVM) resection, and microvascular clipping of cerebral aneurysms. In neuroscience and preclinical research, CBF monitoring can play a major role in understanding the effects of stroke and stroke recovery. Many imaging techniques are available for monitoring CBF, ranging from optical techniques such as indocyanine green fluorescence angiography (ICGA) to radiographic techniques such as digital subtraction angiography (DSA). However, these techniques suffer from the need for contrast agents, interruption of the surgical procedure when used intraoperatively, and, in the case of DSA, radiation exposure.

[0004] Laser speckle contrast imaging (LSCI) has emerged as a powerful technique for continuous imaging of CBF without the use of contrast agents. LSCI has been applied in both stroke research and various surgical and neurosurgical applications. LSCI is a label-free optical technique that can provide continuous monitoring of CBF using simple instrumentation. However, LSCI suffers from several drawbacks that limit its effectiveness in quantifying blood flow. For example, although LSCI reliably detects qualitative changes in flow, it cannot accurately quantify flow changes or differences in flow between different regions or tissue types. This is largely because LSCI measurements are highly instrument-dependent, fail to account for the effects of static scatterers present in real tissue, and are unaware of noise. Due to these limitations, LSCI is typically limited to measuring relative changes in blood flow within a single subject during a single experiment. Summary of the Invention

[0005] One implementation of the present disclosure is an illumination system for laser speckle imaging, the illumination system including: a light source configured to output light having a wavelength in the range of 600 nm to 2000 nm; two or more sections of optical fiber; a fiber-coupled acousto-optic modulator (FCAOM), the FCAOM coupled to the light source by a first section of the two or more sections of optical fiber; and a collimating optical system that focuses light output by a wavelength-stable laser to illuminate an object within a field of view (FOV), the collimating optical system coupled to the FCAOM by a second section of the two or more sections of optical fiber.

[0006] Another implementation of the present disclosure is a laser speckle imaging system that includes a light source having an operating wavelength in a range of 600 nm to 2000 nm, an optical fiber, a fiber-coupled acousto-optic modulator (FCAOM), the FCAOM coupled to the light source by a first section of the optical fiber, a collimating optical system that focuses light output by the light source to illuminate a field of view (FOV), the collimating optical system coupled to the FCAOM by a second section of the optical fiber, and an image capture device for capturing an image of the FOV as the FOV is illuminated by the light source.

[0007] Yet another implementation of the present disclosure is a method of speckle imaging, including operating a light source and an acousto-optic modulator (AOM) of a laser speckle imaging system to illuminate a field of view (FOV) at a plurality of different modulation frequencies within a single exposure time of the laser speckle imaging system, capturing at least one image of the FOV at each of the plurality of different modulation frequencies, calculating a speckle contrast for each captured image to create one or more sets of speckle contrast images, and extracting a value of decorrelation time at each pixel using the one or more sets of speckle contrast images.

[0008] Yet another implementation of the present disclosure is a method of speckle imaging, including operating a light source and an acousto-optic modulator (AOM) of a laser speckle imaging system to generate, within a first exposure time of the laser speckle imaging system, a first set of pulses having a first time delay therebetween, wherein light output by the light source illuminates a field of view (FOV); operating the light source and the AOM to generate, within a second exposure time of the laser speckle imaging system, a second set of pulses having a second time delay therebetween, the second time delay being different from the first time delay; capturing a series of images of the FOV within each of the first and second exposure times; calculating a speckle contrast for each image in the series of images to create a corresponding set of speckle contrast images; and extracting a value of the anti-correlation time at each pixel using the set of speckle contrast images.

[0009] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description, as claimed, are exemplary and explanatory only and are not restrictive.

[0010] Various objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by reference to the detailed description in conjunction with the accompanying drawings, in which like reference characters identify corresponding elements throughout, and in which like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 1 is a diagram of an exemplary free-space multiple exposure speckle imaging (MESI) system, according to some implementations. [Figure 1B]FIG. 1 is a diagram of another exemplary MESI system, according to some implementations. [Figure 2] FIG. 1 is a diagram of an optical fiber coupled laser speckle imaging system, according to some implementations. [Figure 3] FIG. 1 is a diagram of an exemplary image processing pipeline, according to some implementations. [Figure 4A] 1 is a graph illustrating exemplary gating of a MESI pulse train, according to some implementations. [Figure 4B] 1 is a graph illustrating exemplary gating of a MESI pulse train, according to some implementations. [Figure 5] 1 is a graph illustrating relative microfluidics flows for exemplary microfluidics flow protocols, according to some implementations. [Figure 6A] 1B is a graph illustrating the percent deviation of precision and repeatability measurements using the traditional MESI system of FIG. 1A and the FCMESI system of FIG. 2, according to some implementations. [Figure 6B] 1B is a graph illustrating the percent deviation of precision and repeatability measurements using the traditional MESI system of FIG. 1A and the FCMESI system of FIG. 2, according to some implementations. [Figure 7] 1B is an example of in vivo imaging using the traditional MESI system of FIG. 1A and the FCMESI system of FIG. 2, according to some implementations. [Figure 8A] 3 is an exemplary in vivo image captured using the FCMESI system of FIG. 2 in a model of stroke, according to some implementations. [Figure 8B] 8B is a graph of average speckle variance based on the in vivo image of FIG. 8A according to some implementations. [Figure 9] FIG. 1 is a flow diagram of a process for intra-exposure modulated speckle imaging using frequency modulation, according to some implementations. [Figure 10]FIG. 1 is a flow diagram of a process for intra-exposure modulated speckle imaging using time-delay modulation, according to some implementations. [Figure 11] 10 is an exemplary diagram illustrating the intra-exposure modulated speckle imaging process of FIG. 9 according to some implementations. [Figure 12] 11 is an exemplary diagram illustrating the intra-exposure modulated speckle imaging process of FIG. 10 according to some implementations. [Figure 13A] 1 is a diagram of an exemplary temporal relationship between intensity modulation and camera exposure, according to some implementations. [Figure 13B] FIG. 1 illustrates an example autocorrelation function, according to some implementations. [Figure 13C] FIG. 1 illustrates a workflow for extracting correlation time of a two-pulse modulated multi-exposure image, according to some implementations. [Figure 14A] 1 is an example of an original image and a speckle contrast image acquired using a two-pulse modulation approach, according to some implementations. [Figure 14B] 14B is a graph comparing the measured flow rate for the images in FIG. 14A according to some implementations. [Figure 15A] 10A-10C illustrate test results of the two-pulse modulation approach described herein, according to some implementations. [Figure 15B] 10A-10C illustrate test results of the two-pulse modulation approach described herein, according to some implementations. [Figure 15C] 10A-10C illustrate test results of the two-pulse modulation approach described herein, according to some implementations. [Figure 16A] 10 is a graph comparing measured flow rates using two-pulse and sinusoidal modulation, according to some implementations. [Figure 16B] 10 is a graph comparing measured flow rates using two-pulse and sinusoidal modulation, according to some implementations. DETAILED DESCRIPTION OF THE INVENTION

[0012] To address certain limitations mentioned above with respect to LSCI, multiple exposure speckle imaging (MESI) was developed as an extension of LSCI. MESI requires the collection of LSCI images over a wide range of exposure times, and from this series of images, a quantitatively accurate amount of CBF can be extracted. This is possible because MESI allows the effects of instrumentation, static scattering, and noise to be separated from the actual CBF. MESI has been shown to quantify flow changes with substantially greater accuracy than LSCI, even in the presence of static scattering.

[0013] Because MESI requires varying exposure times, the intensity of the light incident on the camera is modulated for several reasons: first, to ensure sufficient signal at short exposure times; second, to prevent saturation at longer exposure times; and finally, to create a similar average intensity over the exposure time to minimize camera and shot noise variations. Traditionally, such intensity modulation is achieved using an acousto-optic modulator (AOM), which acts as a variable amplitude gate to the illumination. This additional instrumentation significantly increases the complexity of MESI compared to traditional single-exposure LSCI. A preliminary clinical study of intraoperative MESI during brain tumor resection surgery revealed improved quantitative measurements of CBF compared to single-exposure LSCI, but the study was limited to very low temporal resolution because constraints of the clinical environment prevented the use of an AOM and required manual adjustment of the light intensity.

[0014] To further address these and other limitations of traditional MESI systems, an optical fiber coupled MESI (FCMESI) illumination system using a fiber coupled laser and a fiber coupled AOM (FCAOM) is described herein in several implementations. This system is smaller and much simpler than other MESI systems and, unlike other systems, utilizes an FCAOM. The FCMESI system described herein is generally based on the principles of traditional free-space MESI systems but alleviates many of the instrumentation issues of traditional systems through the use of fiber-based components. As discussed in more detail below, the FCMESI system described herein performs as well as or better than traditional MESI systems in both microfluidics and in vivo experiments. Furthermore, the illumination arm of the FCMESI system described herein can be used with many other types of speckle imaging and is not limited to MESI applications only.

[0015] In addition to the FCMESI system described above, a method of LSCI called "intra-exposure modulated speckle imaging" or intensity-modulated imaging is also described herein. Traditional illumination methods involve maintaining a constant intensity of laser light throughout the camera exposure time. The exposure time can be varied to increase sensitivity up to a certain flow rate range. However, achieving reproducible blood flow values ​​with this traditional method can be difficult. Currently, the only way to improve LSCI sensitivity to large flow rates is to reduce the camera exposure time to very short values ​​(e.g., microseconds). These very short camera exposure times require high illumination power to detect enough light to capture the speckle pattern. Such high power is often difficult to achieve due to laser diode limitations and / or safety restrictions. Notably, modulated intensity imaging is much more sensitive to large flow rates without the need to reduce the camera exposure time to prohibitively short values, thereby enabling imaging of large flow rates at lower average power.

[0016] Intra-exposure modulated speckle imaging involves varying the intensity of laser illumination within the exposure time of an imaging device (e.g., a camera). The speckle contrast of the image from the modulated illumination can then be related to the underlying flow dynamics in a more quantitative manner. The intensity modulation within the camera exposure can be pulsed, sinusoidal, or any other function. Because the laser illumination is coherent, the speckle contrast of the modulated illumination integrated over the camera exposure time will vary depending on the temporal characteristics of the laser illumination. Therefore, the sensitivity of the blood flow image can be adjusted to different flow levels by changing the nature of the intensity modulation. Further details are provided below.

[0017] overview In LSCI, decorrelation of the speckle pattern by dynamic scattering events causes blurring over the camera exposure time, which is quantified by the speckle contrast K, defined as:

number

number

[0018] MESI is based on a more rigorous model that considers static scattering events and non-ideal conditions, resulting in the MESI equation:

number

[0019] Multiple Exposure Speckle Imaging (MESI) Referring first to FIG. 1A, a diagram of an exemplary free-space multiple exposure speckle imaging (MESI) system 100 is shown, according to some implementations. System 100 is generally an example of a “traditional” MESI system. As shown, system 100 includes a light source 102 that emits light for imaging. In some implementations, light source 102 is a laser or laser diode, e.g., emitting light in the 600 nm to 2000 nm range. System 100 includes an isolator 104, followed by a section of optical fiber 106 and an acousto-optic modulator (AOM) 108. In some implementations, optical fiber 106 is configured for optical correction of the anomalous beam shape provided by light source 102. Specifically, optical fiber 106 is terminated with a collimating lens to generate a circular beam. The collimated output can then pass through AOM 108, and an iris 110 can be used to select the first diffraction order of AOM 108. Generally, the AOM 108 is configured to use acoustic waves to diffract and / or shift the frequency of light, or in other words, can be used to control the power / intensity of the light emitted by the light source 102.

[0020] As shown, in some implementations, a series of mirrors and / or lenses may be used to direct the light emitted by the light source 102, although it should be understood that the number and / or arrangement of mirrors and / or lenses may vary based on the particular implementation of the system 100. In this example, multiple mirrors direct light toward a flow phantom 112 through which a fluid sample passes for testing. However, in use, the light may also be directed toward a blood vessel to measure blood flow. More generally, the light emitted from the light source 102 is directed toward a field of view (FOV) of the image capture system 114. In this case, the FOV of the image capture system 114 encompasses at least a portion of the flow phantom 112. In some implementations, the image capture system 114 includes a camera 116 or other suitable device or sensor for capturing images. In some such implementations, the camera 116 is a black-and-white camera. In some implementations, the image capture system 114 includes one or more lenses for expanding the FOV.

[0021] In some implementations, system 100 includes a radio frequency (RF) driver 120 for controlling the light throughput of AOM 108. Specifically, in some such implementations, RF driver 120 may output electrical signals at a controlled frequency that excite piezoelectric transducers or other similar components of AOM 108 to modulate or adjust the light output by AOM 108. A data acquisition device (DAQ) 122 may also be included for synchronizing image acquisition via image capture system 114 with the modulation of AOM 108. DAQ 122 may generally be configured to provide command signals to RF driver 120 to control the modulation of AOM 108 and may also receive captured image data from image capture system 114. In some implementations, system 100 further includes a computing device 124 that may interface with DAQ 122 to receive and further process image data and / or to otherwise control RF driver 120 and DAQ 122. In some such implementations, computing device 124 may be a desktop computer, a laptop computer, a server, or any other suitable computing device.

[0022] 1B is a diagram of another exemplary MESI system 150, according to some implementations. Similar to system 100, as previously described, system 150 includes a light source 102 and an isolator 104, followed by a section of optical fiber 106 and an AOM 108. In some implementations, MESI system 150 may include one or more mirrors and / or lenses positioned between the isolator 104 and the optical fiber 106 to direct the light emitted by the light source 102. For example, in the illustrated configuration, system 150 includes two mirrors (labeled "M") followed by a first lens (L1) before the optical fiber 106. Similarly, a second lens (L2) and respective mirror are positioned after the optical fiber 106. However, it should be understood that this particular configuration is not intended to be limiting; rather, the number, arrangement, and / or inclusion of mirrors and / or lenses may vary based on the application, layout, etc.

[0023] In the illustrated implementation, two illumination light paths are established, for example, after passing through the AOM 108, including a wide-angle path shown in solid lines and a focused path shown in dashed lines. While the MESI system 150 is shown in some implementations to include a flip mirror (labeled "FM") to switch the light between the two paths, it should be understood that the light is generally modulated by the same pulse train. After contacting the target 160 (e.g., a sample, flow phantom 112, etc.), the diffusely reflected light may be collected, for example, by an objective lens (L5) and then separated by a beam splitter 152. A first portion of the light is passed toward a camera 156, while a second portion of the light is reflected toward an avalanche photodiode (APD) 154 for use, for example, in pulse train control. In some implementations, beam splitter 152 is a 50 / 50 beam splitter, for example, where the first and second portions of light are approximately equal, although this disclosure is not intended to be limiting in this regard.

[0024] As shown, the camera 156 collects a first portion of the reflected light and forwards the image data to the computing device 124, e.g., for further processing and / or display. A second portion of the light is shown passing through a lens (L6) and a fiber coupler (FC) to a second optical fiber 158. In some implementations, the second optical fiber 158 is a single-mode fiber (SMF). The light exiting the second optical fiber 158 passes through one or more lenses (L7, L8) before reaching the APD 154. As noted above, the APD 154 generates an electrical signal in response to the received light, which is provided to the DAQ 122 to facilitate pulse train control. In some implementations, the electrical signal output from the APD 154 passes through a low-pass filter (LFP) or other suitable filter.

[0025] Optical fiber coupled laser speckle imaging system Referring now to FIG. 2 , a diagram of an optical fiber-coupled laser speckle imaging system 200 is shown, according to some implementations. Generally, the operating principles of system 200 are similar to those of system 100 described above. For example, system 200 includes an illumination arm 202 having a light source 204 that illuminates an FOV (e.g., including, in this example, flow phantom 112) for speckle imaging. However, illumination arm 202 of system 200 is generally constructed from fiber-coupled components, significantly reducing the system's complexity and size. For example, system 200 generally does not require numerous mirrors to focus and steer light emitted from the light source, as does system 100. As shown, system 200 also does not include isolator 104 or iris 110. Thus, system 200 may generally be easier to set up, operate, and use than system 100, and has fewer components and therefore fewer potential drawbacks. In some examples, system 200 may even be less expensive to construct than system 100.

[0026] Additionally, the use of fiber-based components and mating sleeves eliminates the need for careful alignment and realignment of optical components and minimizes the number of parts that can collect dust, which is especially important in clinical settings and non-optical laboratories. While LSCI applications are expanding, MESI adoption has been slow, so system 200 can remove barriers to MESI adoption for new applications, both intraoperatively and in emerging research settings. Given the benefits of MESI, the adoption of FCMESI in settings where LSCI is currently used could enable accurate monitoring of CBF in many applications ranging from neurosurgery to neuroscience.

[0027] It should also be understood that system 200 is not limited to MESI applications. For example, system 200 can be used for multiple-exposure speckle imaging, although illumination arm 202 and its components make system 200 suitable for other forms of laser speckle imaging. In some implementations, system 200 can be used to perform an intra-exposure modulation method of speckle imaging, as described in more detail with respect to FIGS. 9-12 . Intra-exposure modulation is a technique for speckle imaging that involves modulating the intensity of light applied to an object within a FOV. Intra-exposure modulation may also be referred to as intensity-modulated speckle imaging. Accordingly, the present disclosure contemplates system 200 as being suitable for various modulated speckle imaging techniques, including MESI and intra-exposure modulation.

[0028] As shown, the illumination arm 202 of the system 200 further includes a fiber-coupled AOM (FCAOM) 208 coupled to the light source 204 via a first section of an optical fiber 206. In some implementations, the light source 204 is a volume holographic grating (VHG) stabilized laser diode that outputs light having a primary wavelength of 785 nm. It should be understood that a VHG stabilized laser is provided by way of example only. The present disclosure contemplates the use of other laser light sources, including, for example, wavelengthless stabilized lasers. Additionally, it should be understood that 785 nm is provided by way of example only for the primary wavelength. The present disclosure contemplates the use of light sources having primary wavelengths greater than or less than 785 nm. For example, the light source 204 may operate at a wavelength within the range of 600 nm to 2000 nm.

[0029] In some implementations, the FCAOM 208 has a rise time of 50 ns, although the FCAOM 208 may be configured for other rise times as contemplated herein. In some implementations, the optical fiber 206 (or at least a portion of the optical fiber 206) is part of or fixedly coupled to the light source 204. Similarly, in some implementations, a portion of the optical fiber 206 or the entire optical fiber 206 may be part of or fixedly coupled to the FCAOM 208. For example, a first portion of the optical fiber 206 may extend from an output side of the light source 204, and a second portion of the optical fiber 206 may extend from an input side of the FCAOM 208. In some such implementations, the portions of the optical fiber 206 may be coupled by a mating sleeve. However, it should be understood that the particular configuration of the system 200 is not limited to the present description. For example, in other implementations, the optical fiber 206 may be a separate component from the light source 204 and / or the FCAOM 208 and, therefore, may be removably coupled to both components.

[0030] In some implementations, the illumination arm 202 includes a second section of optical fiber 210 that couples the FCAOM 208 to the collimating optic 212. The collimating optic 212 is optionally an adjustable focal length collimating optic. In some such implementations, the adjustable focal length collimating optic 212 can be used to adjust the illumination of the FOV (e.g., generally encompassing a portion of the flow phantom 112 in the illustrated example). Like the optical fiber 206, in some examples, the optical fiber 210 or a portion thereof can be part of the FCAOM 208 (e.g., can be fixedly coupled to the FCAOM 208). For example, the optical fiber 210 can extend from an output of the FCAOM 208. In other implementations, the optical fiber 210 is a separate component from the FCAOM 208 and, therefore, can be removably coupled to the FCAOM 208 and / or the adjustable focal length collimating optic 212. Generally, one or both of the optical fibers 206, 210 are single-mode optical fibers. It should be understood that the adjustable focal length collimating optic is provided by way of example only, and the present disclosure contemplates the use of other collimating optics.

[0031] Like system 100, system 200 is shown to include an image capture system 214 that includes one or more lenses and an image capture device 216. In some implementations, image capture device 216 is any suitable camera or image sensor, such as a black-and-white camera (e.g., a 155 μm camera). In the illustrated example, image capture system 214 includes two lenses. In some implementations, at least one of the lenses is configured to expand the FOV for image capture device 216. In some implementations, image capture system 214 includes a long-pass filter to filter out visible light. For example, the long-pass filter may be one of the lenses shown in FIG. 2. Image capture device 216 is coupled to a DAQ 222 that may receive and optionally process image data captured by image capture device 216. In some implementations, DAQ 222 is further configured to trigger image capture device 216 (e.g., cause image capture device 216 to capture an image). Optionally, the DAQ 222 may be communicatively coupled to an RF driver 220, which modulates the light throughput of the FCAOM 208 by applying an electrical signal to the FCAOM 208. In particular, the DAQ 222 may communicate with the RF driver 220 to synchronize the light output or modulation with the trigger of the image capture device 216.

[0032] In some implementations, system 200 includes a controller 230 that communicates with one or both of RF driver 220 and DAQ 222. Generally, controller 230 is configured to receive, process, and / or store image data from DAQ 222. In some implementations, controller 230 performs all of the functions of DAQ 222, such that DAQ 222 may not be included. In some implementations, controller 230 provides control signals to RF driver 220 (e.g., as opposed to DAQ 222 providing control signals), thereby coordinating the operation of components of illumination arm 202 and image capture system 214. It will be understood that any such arrangements and implementations of the components of system 200 are contemplated herein.

[0033] As shown, the controller 230 generally includes a processor 232 and a memory 234. Accordingly, the controller 230 may be any suitable computing device (e.g., a laptop computer, a server, etc.). The processor 232 may be a general-purpose processor, an application-specific integrated circuit (ASIC), one or more field-programmable gate arrays (FPGAs), a group of processing components, or other suitable electronic processing structure. In some embodiments, the processor 232 is configured to execute program code stored in the memory 234 to cause the controller 230 to perform one or more operations, as described in more detail below. In some implementations, the controller 230 may be part of another computing device (e.g., the DAQ 222 or another computer), such that the components of the controller 230 may be shared with or the same as the host device.

[0034] Memory 234 may include one or more devices (e.g., memory units, memory devices, storage devices, etc.) for storing data and / or computer code for completing and / or facilitating various processes described in this disclosure. In some embodiments, memory 234 includes a tangible (e.g., non-transitory) computer-readable medium that stores code or instructions executable by processor 232. Tangible computer-readable medium refers to any physical medium that can provide data that causes controller 230 to operate in a specific manner. Examples of tangible computer-readable media may include, but are not limited to, volatile, non-volatile, removable, and non-removable media implemented in any method or technology for storing information such as computer-readable instructions, data structures, program modules, or other data. Thus, memory 234 may include RAM, ROM, hard drive storage, temporary storage, non-volatile memory, flash memory, optical memory, or other suitable memory for storing any software objects and / or computer instructions. Memory 234 may include database components, object code components, script components, or any other type of information structure for supporting the various activities and information structures described in this disclosure. Memory 234 may be communicatively coupled to processor 232 and may include computer code for executing (e.g., by processor 232) one or more processes described herein.

[0035] Although depicted as individual components, it will be understood that processor 232 and / or memory 234 can be implemented using a variety of different types and amounts of processors and memory. For example, processor 232 may represent a single processing device or multiple processing devices. Similarly, memory 234 may represent a single memory device or multiple memory devices. Additionally, in some embodiments, controller 230 may be implemented within a single computing device (e.g., a server, a chassis, etc.). In other embodiments, controller 230 may be distributed across multiple devices (e.g., which may be in distributed locations). For example, controller 230 may include multiple distributed computing devices (e.g., multiple processors and / or memory devices) in communication with each other that cooperate to perform operations.

[0036] Experimental setup and results The ability to gate laser illumination for both the AOM108 and FCAOM208 was tested using appropriate photodiodes. Pulse trains covering the first 10 exposure times of the MESI pulse train were delivered to each of the AOM108 and FCAOM208 (e.g., via their respective light sources 102 and 204), and the light output was measured. A microfluidic flow phantom (e.g., flow phantom 112) was used to test the system's ability to quantify flow changes. For these tests, the flow phantom 112 was constructed from polydimethylsiloxane (PDMS) with the addition of titanium dioxide to mimic the scattering properties of tissue. A 300 x 300 μm square channel was embedded within the flow phantom, with a glass coverslip bonded on top, and plastic tubing was connected to create the channel's inlet and outlet.

[0037] A solution of 1.1 μm diameter polystyrene microspheres in deionized water was used to create a solution to mimic the scattering properties of blood at 785 nm (e.g., the wavelength of light source 204). Specifically, Mie theory was used to calculate the scattering coefficient of the microspheres, and then the reduced scattering coefficient of blood (1.3 mm -1 The solution consisted of 4.8% by volume of microsphere solution, 0.1% Tween® 20 (P1379-100ML, Sigma-Aldrich) to prevent microsphere aggregation, and the remainder was deionized water.

[0038] Flow within the system was regulated by an external flow control system (not shown). Briefly, the polystyrene solution was contained in a reservoir connected to a pressure regulator, and the reservoir outlet was connected to plastic tubing connecting the reservoir to the microfluidic channel inlet. The microfluidic channel outlet was placed in a separate collection reservoir. Flow was monitored at two different locations using two separate flow sensors (one before and one after the channel). Both flow sensors were connected to a control hub that interfaced with a control computer (e.g., controller 230).

[0039] The flow rate was set as a step function, ranging from 1 to 10 mm / s, in 1 mm / s increments. Each step was held for 90 seconds, and the entire protocol was followed by a 30-second period during which the flow rate was set to 0 mm / s. The total experimental time was 15.5 minutes. This protocol was performed both during imaging with the free-space MESI system, System 100, and during imaging with the FCMESI system, System 200. MESI images were acquired sequentially throughout the protocol for a total of 1,950 image sequences, with each sequence consisting of 15 images collected at 15 different exposure times. For System 200, the protocol was performed three times, with the start of each test separated by approximately 20 minutes. Because free-space MESI systems such as System 100 have previously been thoroughly tested by others in the field, the microfluidics protocol using it was performed only once.

[0040] For the study, mice were anesthetized with isoflurane, and body temperature was maintained with a heating pad throughout the procedure. Craniotomies were performed on two mice, and a portion of the skull was removed and replaced with a glass coverslip fixed in place with dental cement. One mouse underwent photothrombotic stroke by injecting rose bengal dye, followed by illuminating an area of ​​the cortical surface with 532 nm light. One mouse underwent photothrombotic stroke by retroorbital injection of rose bengal dye (15 mg / kg), followed by focusing 532 nm light on the perforating arterioles of the motor cortex. Three weeks after stroke, MESI was performed, and each mouse was imaged using both the free-space and FCMESI systems (System 100 and System 200, respectively). For each imaging session, 56 trains of 15 frames at 15 different exposure times were acquired.

[0041] Processing was then performed on the collected images based on the exemplary image processing pipeline 300 shown in FIG. 3. For each collected raw image, shown as raw image 302, the speckle contrast was calculated over a sliding window of 7x7 pixels to generate speckle contrast image 304. For the microfluidics experiments, a 40x40 pixel region of interest (ROI) was selected to correspond to the width of the microfluidics channel in the FCMESI (e.g., system 200) image. The arithmetic mean of the speckle contrast images was calculated within this ROI to generate a single speckle contrast value at each exposure time, shown in graph 306. This mean value was used to calculate the measured K 2ICT was found by fitting (T) to Equation 3 above. Given the issue of numerical stability of the resulting fit in Equation 3, β was chosen to be a constant value during the fitting process to remove one of the four variables from the fitting process. The value of β was chosen by finding the median value of K per exposure time for frames in 1 mm / s steps in the microfluidics step function, fitting this data to Equation 3, and selecting the resulting β value as its true constant value.

[0042] To eliminate the effect of transition times between flow rates in the flow protocol, 25 frames of data were removed on either side of the midpoint of the transition time between rates. This data trimming and subsequent fitting of the ICT generated a series of ICT values ​​corresponding to 10 steps in the step function. All ICT values ​​were normalized to the average ICT value for the lowest flow rate, generating the rICT time course shown in graph 308. The relative microfluidics flow (rM) was found by taking the average flow value of the two flow sensors, then taking the average at each flow rate and normalizing to the first step. rICT was then plotted against the relative microfluidics flow for each flow rate, as shown in graph 310.

[0043] For each step in the step function, the arithmetic mean of ICT was found for each test. Furthermore, using the rICT and relative microfluidics (rM) values ​​at each step, the mean percent deviation of precision at each step (Δ ACC ) and the mean percent deviation of reproducibility (Δ REP ) can be calculated according to the following formula:

number

[0044] These metrics allow for the change in flow (Δ ACC ) and the stability of their measurements (Δ REPThree separate tests were performed on the system 200, allowing the ability to accurately determine Δ ACC and Δ REP The mean and standard deviation of were found.

[0045] For in vivo imaging, speckle contrast was calculated for each image. All images captured at the same exposure were averaged together to generate a single dataset consisting of 15 averaged images for each of 15 different exposure times. ICT was then found across the relevant FOV by fitting the data at each pixel to the MESI equation. For stroke model imaging using the FCMESI system, three ROIs corresponding to vessels, parenchyma, and infarct were selected, and the fit to the MESI equation was determined.

[0046] Referring now to FIGS. 4A and 4B, graphs illustrating exemplary gating of MESI pulse trains according to some implementations are shown. As shown, both the free-space AOM (e.g., AOM108) and the FCAOM208 demonstrated similar capabilities for gating optical signals for MESI trains of different exposure times. FIG. 4A shows the signal intensity for both AOM108 and FCAOM208 over, for example, 10 exposure times. For the 10 different exposure times, each AOM modulates the optical throughput, decreasing the instantaneous optical output as the exposure time increases. Because AOM108 and FCAOM208 have unique calibration curves, each produces pulse trains with comparable shapes, although the absolute values ​​of the optical output differ between each pulse train. Furthermore, each individual pulse in the train had similar shape, morphology, and rise time, despite the different absolute measurements. FIG. 4B illustrates these characteristics through a close-up view of the second pulse shown in FIG. 4A. These results indicate that there is no substantial difference between the two systems in their ability to generate MESI pulse trains.

[0047] FIG. 5 shows an exemplary graph of rICT plotted against rM for each of 10 speeds in a step function for four test runs of a microfluidics flow protocol (e.g., three using System 200 and one using System 100, as described above). rICT and rM are not equal across all steps, but are similar throughout the step function. Significantly, rICT from System 100 was almost always within the range of values ​​from tests of System 200, demonstrating that the performance of System 200 in measuring flow changes in microfluidics channels is comparable to that of more traditional free-space MESIs (e.g., System 100). The average Δ for System 200 ACC is lower than that of system 100 at all speeds, but Δ ACC is large enough at lower velocities (eg, below 4 mm / s) that the performance of system 100 falls within the range of expected performance of system 200, as shown in FIG. 6A.

[0048] 6A and 6B show, inter alia, the mean percent deviation of the precision and repeatability measurements, with error bars indicating the range of standard deviations from the mean. The performance of system 200 is shown in blue bars, while the performance of system 100 is shown in orange. In FIG. 6A, the percent deviation of precision (Δ ACC ) versus flow velocity. The mean error of precision for system 200 is lower than that of older systems (e.g., system 100) at all flow rates, but the error bars for steps between 2 and 4 mm / s are larger. Figure 6B shows the percent deviation of repeatability (Δ REP ) shows that the repeatability did not show a consistent trend across all flow rates, but the upper bound of the error at each rate was less than 6%, with no substantial difference between the two systems. In summary, this data clearly shows that system 200 had comparable precision and repeatability compared to system 100, despite the hardware changes.

[0049] Because flow velocities in the aforementioned microfluidics systems have been shown to be very stable, the accuracy and reproducibility issues of rICT are generally caused by MESI imaging and fitting to the MESI equation. From an accuracy perspective, MESI appears to systematically underestimate flow in almost all cases, especially for velocities between 3 and 5 mm / s. This issue is likely caused by the stability of the numerical calculations, particularly the fact that β was considered a constant, potentially introducing a systematic bias. This could be addressed by using a different way of fitting β, a different fitting algorithm, or even by utilizing different models or several models depending on the flow conditions. Despite these potential numerical deficiencies, FCMESI (e.g., System 200) was able to quantify flow changes with the same level of accuracy and reproducibility as previous MESI systems (e.g., System 100), and has been shown to have significant advantages in quantifying flow changes compared to single-exposure LSCI.

[0050] Referring now to FIG. 7, exemplary in vivo images from the aforementioned mouse experiment are shown according to some implementations. Specifically, in FIG. 7, the upper left corner shows an image of a control mouse collected with system 100; the upper right corner shows an image of a stroke model collected with system 100 (infarcts are boxed); the lower left corner shows an image of a control mouse collected with system 200; and the lower right corner shows an image of a stroke model collected with system 200 (infarcts are boxed). In imaging the mice, both systems 100 and 200 were able to detect infarcts in the case of stroke mice and map the vasculature of healthy mice. Due to the different magnifications for both systems, the vascular networks are not necessarily equivalent; system 100 has a higher resolution due to the higher magnification. However, all major features on the cortical surface are clearly visible in both sets of images, indicating that FCMESI (e.g., system 200) can image the cerebral vascular network.

[0051] To further demonstrate the wide-field mouse imaging capabilities of FCMESI, different ROIs were selected within the FCMESI image of the stroke model. Each of these three ROIs corresponds to a different key feature: blood vessels, parenchyma, and infarct, as highlighted in Figure 8A. The speckle contrast was averaged over each ROI and fitted to Equation 3, and the fit was plotted against the measured data, as shown in Figure 8B. Calculating τ c The trend of was as expected, with the highest in infarction (τ c = 938 μs), and slightly lower in soft tissues (τ c =349 μs), and is much lower in blood vessels (τ c =75.4μs), the result is τ c The fit is consistent with an inverse correlation between FCMESI and CBF. The fit, as determined by mean square error (MSE), was best for vessels (MSE=0.0054) and worst for infarcts (MSE=0.0171), with intermediate fit quality for parenchymal tissues (MSE=0.0109), suggesting a correlation between increased flow and increased fit to the MESI equation. Collectively, these fit measures clearly demonstrate that FCMESI can distinguish between different flow rates in different tissue structures of the mouse brain, providing further evidence that system 200 can be used in clinical settings with complex anatomy.

[0052] Intra-exposure modulated speckle imaging As described above, intra-exposure modulation speckle imaging generally involves varying the intensity of laser illumination within the exposure time of an imaging device (e.g., a camera). In some implementations, varying the intensity of the laser (e.g., light source) is achieved by modulating the light throughput of an AOM (e.g., AOM 108, FCAOM 208). The modulation function of the AOM is denoted m(t), the raw speckle signal is denoted I(t), and the modulated speckle signal is denoted I(t). m (t) can be defined as: I m (t)=I(t)m(t)

[0053] Then the intensity of pixel i of an image capture device (e.g., a camera sensor) (within the intensity-modulated exposure time T) is:

number

[0054] The intensity modulation can then be defined as:

number

number

[0055] For square wave modulation, the speckle contrast equation can be defined as:

number

number

number

[0056] By substituting the assumed g2(τ) into this equation, we can establish the following relationship between speckle contrast and correlation time for different g2(τ) models:

number

number

number

[0057] Referring now to FIG. 9 , a flow diagram of a process 900 for intra-exposure modulated speckle imaging using frequency modulation is shown, according to some implementations. In some implementations, the process 900 is performed by the system 200, as described above. For example, the process 900 may be performed, at least in part, by the controller 230. Additionally or alternatively, the process 900 may be performed, at least in part, by the RF driver 220 and / or the DAQ 222. However, it should be understood that the process 900 may also be performed by the system 100 or the system 150 (e.g., by the computing device 124) or other suitable laser speckle imaging system. In some examples, certain steps of the process 900 may be optional, and the process 900 may be performed using fewer than all of the steps. It should also be understood that the order of the steps shown in FIG. 9 is not intended to be limiting.

[0058] It should also be noted that one or more steps of process 900, as described below, may be performed within a single exposure time (defined by time T) of an image capture device (e.g., camera 156, image capture device 216), hence the term "intra-exposure modulation" speckle imaging. In some implementations, at least steps 902 and 904 are performed within the exposure time (T), while steps 906 and / or 908 may also be performed within the exposure time. Throughout the following description of FIG. 9, reference may be made to various equations discussed above.

[0059] In step 902, the intensity of light applied to the field of view (FOV) of the MESI system is varied over an exposure time (T). In some implementations, the light intensity is varied sinusoidally, although other waveforms are contemplated herein. In this regard, the FOV is illuminated at a plurality of different modulation frequencies. In some implementations, the light intensity is modulated by controlling a light source, such as the isolator 104 or the light source 204. For example, the controller 230 may control the light source 204 by sending a control signal and / or modulation power to the light source 204. In some implementations, the light intensity is modulated by controlling an AOM, such as the AOM 108 or the FCAOM 208. In some such implementations, the controller 230 may cause the RF driver 220 to modulate the light throughput of the FCAOM 208 to illuminate the FOV at various modulation frequencies. Alternatively, the RF driver 220 may control the FCAOM 208 based on data provided by the DAQ 222. Similarly, in some implementations, the DAQ 122 and / or the computing device 124 may control the AOM 108 to modulate the light throughput of the AOM 108.

[0060] However, it should be understood that controlling the light source and / or AOM of a laser speckle imaging system (e.g., system 150) is not the only way to modulate / vary the intensity of light over time. Accordingly, the present disclosure contemplates various other methods of achieving modulation of light intensity. For example, the intensity of light can be modulated using (e.g., controlled by) one or more of an electro-optic modulator (EOM), direct modulation (e.g., electrical modulation) of laser diode current, or mechanical modulation (e.g., chopper wheel) in, for example, any of systems 100, 150, or 200. These and other techniques for modulating light intensity, e.g., as in step 902, are contemplated herein.

[0061] An example of modulation of light illuminating the FOV is shown in FIG. 11 , which illustrates five different modulation frequencies (ω) within an exposure time T of an image capture device (e.g., camera 156, image capture device 216). In some implementations, the light illuminating the FOV may be modulated at each modulation frequency within a single exposure time T. For example, the light throughput of FCAOM 208 may be projected (e.g., onto the FOV) at each modulation frequency within a single exposure time such that the modulation frequency of the light varies throughout the exposure.

[0062] In step 904, at least one image of the FOV is captured at each modulation frequency. In some implementations, the DAQ 222 and / or the controller 230 may store images over an exposure time T of the image capture device 216 to generate a series of images at different modulation frequencies. Similarly, the computing device 124 may store images over an exposure time T of the camera 156. Then, in step 906, the speckle contrast (K) is calculated for each captured image. Generally, the speckle contrast varies as a function of the modulation frequency. In FIG. 11, for example, five different modulation frequencies are used to create a set of speckle contrast images: K(ω1), K(ω2), K(ω3), K(ω4), and K(ω5).

[0063] In step 908, the value of the anti-correlation time (τ c ) is calculated using the speckle contrast images. In some implementations, each set of speckle contrast images (e.g., K(ω1), K(ω2), K(ω3), K(ω4), and K(ω5)) is used to calculate the value of the decorrelation time (τ c ) is extracted. c ) is described above. Optionally, in step 910, blood flow (g2) is determined based on the value of the decorrelation time at each pixel, again using the formula described above.

[0064] Referring now to FIG. 10 , a flow diagram of a process 1000 for intra-exposure modulated speckle imaging using time-delay modulation is shown, according to some implementations. In some implementations, the process 1000 is performed by the system 200, as described above. For example, the process 1000 may be performed, at least in part, by the controller 230. Additionally or alternatively, the process 1000 may be performed, at least in part, by the RF driver 220 and / or the DAQ 222. However, it should be understood that the process 1000 may also be performed by the system 100 or the system 150 (e.g., by the computing device 124) or other suitable laser speckle imaging system. In some examples, certain steps of the process 1000 may be optional, and the process 1000 may be performed using fewer than all of the steps. It should also be understood that the order of the steps shown in FIG. 10 is not intended to be limiting. Reference may be made to various equations described above throughout the following description of FIG. 10 .

[0065] In step 1002, the intensity of light projected onto the field of view (FOV) of the MESI system is varied over an exposure time (T) and a time delay (t d) apart. In some implementations, the intensity of the light is modulated by controlling a light source, such as the isolator 104 or the light source 204. For example, the controller 230 may control the light source 204 by sending a control signal and / or modulation power to the light source 204. In some implementations, the intensity of the light is modulated by controlling an AOM, such as the AOM 108 or the FCAOM 208. In some such implementations, the controller 230 may cause the RF driver 220 to modulate the light throughput of the FCAOM 208 to illuminate the FOV at various modulation frequencies. Alternatively, the RF driver 220 may control the FCAOM 208 based on data provided by the DAQ 222. Similarly, in some implementations, the DAQ 122 and / or the computing device 124 may control the AOM 108 to modulate the light throughput of the AOM 108.

[0066] However, it should be understood that controlling the light source and / or AOM of a laser speckle imaging system (e.g., system 150) is not the only way to modulate / vary the intensity of light over time. Accordingly, the present disclosure contemplates various other methods of achieving modulation of light intensity. For example, the intensity of light can be modulated using (e.g., controlling) one or more of an EOM, direct modulation of laser diode current (e.g., electrical modulation), or mechanical modulation (e.g., chopper wheel) in, for example, any of systems 100, 150, or 200. These and other techniques for modulating light intensity, e.g., as in step 1002, are contemplated herein.

[0067] An example of modulation of the light illuminating the FOV is shown in Figure 12. In this example, three different sets of pulses are shown, each with a different time delay (t) between the pulses. d) In some implementations, each set of pulses is emitted in a separate exposure. For example, in FIG. 12, three different exposure times may be required to capture each set of pulses. However, each set of pulses is generally performed within a single exposure time.

[0068] In step 1004, at least one image of the FOV is captured for each set of pulses, or in other words, for each time delay. For example, in some implementations, the DAQ 222 and / or the controller 230 capture a set of images to generate a series of images at different delay times. Then, in step 1006, the speckle contrast (K) is calculated for each captured image. Generally, the speckle contrast is a function of the delay time (t d ) as a function of time. In FIG. 12, for example, a set of speckle contrast images, K(t d1 ), K(t d2 ), and K(t d3 )

[0069] In step 1008, the value of the anti-correlation time (τ c ) is calculated using the speckle contrast images. In some implementations, for each set of speckle contrast images (e.g., K(t d1 ), K(t d2 ), and K(t d3 )) to calculate the value of the anti-correlation time (τ c ) is extracted. c ) is described above. Optionally, in step 1010, blood flow (g2) is determined based on the value of the decorrelation time at each pixel, again using the formula described above.

[0070] Additional experimental results 13A-16B, further details and associated experimental results are provided generally regarding the disclosed intra-exposure modulation techniques. While the results described herein were obtained using an experimental setup similar to the configuration shown in FIG. 1B (e.g., system 150), it should be understood that these results more generally represent the feasibility of the disclosed intra-exposure modulation techniques for various MESI systems, including, in some examples, system 100 and / or system 200.

[0071] FIG. 13A illustrates an exemplary temporal relationship between intensity modulation and camera exposure. In this diagram, the x-axis is time. The AOM line represents the voltage signal of an AOM (e.g., AOM 108) or other method for modulating the intensity of the laser. As shown, the target is illuminated only when the AOM modulation voltage is high. Thus, I t For τ, only the signal when the AOM is high is recorded and integrated onto the raw camera image. Figure 13B illustrates the autocorrelation function of a two-pulse modulated waveform. The intensity modulated waveform m(t) can be defined as m(t)∈[0,1]. The autocorrelation of m(t), defined as M(τ), consists of two pulses, denoted M0 and M1 in this figure. T m As approaches zero, M(τ) becomes the sum of two delta functions. Figure 13C illustrates the workflow for extracting correlation time from a two-pulse modulated multi-exposure original image. First, the two-pulse modulated speckle contrast K 2 2P is calculated from the modulated speckle raw image, and then its trace along the third dimension T is fitted with different electric field autocorrelation g1(τ) models (n=2, 1 or 0.5). The best g1(τ) model is selected based on the coefficient of determination R 2 is determined by maximizing

[0072] 14A and 14B generally show normalized K 2 2PFigure 14A shows an experimental verification of the consistency between g(τ) and g2(τ). Figure 14A includes an image acquired in the two-pulse modulation approach (left) and a speckle contrast image calculated from the two-pulse modulation original image (right). Figure 14B shows the normalized measured K under flow rates ranging from 0 to 100 μL / min with a step of 10 μL / min. 2 2P 2(τ) (shown as a solid line) with the measured g2(τ) (shown as a dotted line).

[0073] Figures 15A-15C generally show normalized K 2 2P Figure 15 illustrates experimental verification of the consistency between the in vivo g2(τ) and the normalized measured K2(τ). Figure 15A is a speckle contrast image calculated from the two-pulse modulated raw image. Figure 15B is a normalized measured K2(τ) at three different spatial locations, denoted P1, P2, and P3, similar to Figure 15A. 2 2P Figure 15C shows graphs comparing g(τ) (shown as dots) and g(τ) (shown as a solid line). The tilde above the sign in the legend indicates the normalized quantity. 2 2P Figure 10 is a graph comparing anticorrelation time (ICT) values ​​extracted from and in vivo g(τ), clearly demonstrating the excellent agreement between the two measurement types. These example images show 28 points from four mice.

[0074] 16A and 16B generally illustrate the experimental validation of sinusoidal modulation within an exposure in a flow phantom. Figure 16A shows the normalized K measured with two-pulse modulation for flow rates ranging from 0 to 80 μL / min. 2 2P (T) and g2(τ). 2 c11 is a graph of the normalized power spectral density (PSD) (shown as a solid line) extracted from single-point intensity measurements for flow rates ranging from 0 to 80 μL / min. In this example, ω represents the angular modulation frequency of the light intensity within the camera exposure time T (FIG. 11). 2 c The (ω) values ​​coincide with the PSD values ​​when the modulation frequency ω is changed.

[0075] Configuration of a specific implementation The configurations and arrangements of the systems and methods shown in various implementations are merely exemplary. While only a few implementations have been described in detail in this disclosure, many modifications are possible (e.g., changes in the size, dimensions, structure, shape, and proportions of various elements, parameter values, mounting arrangements, material use, color, orientation, etc.). For example, the positions of elements may be reversed or otherwise changed, and the nature or number of individual elements or positions may be changed or varied. Accordingly, all such modifications are intended to be included within the scope of this disclosure. The order or sequence of any process or method steps may be changed or rearranged according to alternative implementations. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of implementations without departing from the scope of this disclosure.

[0076] The present disclosure contemplates methods, systems, and program products on any machine-readable media for accomplishing various operations. Implementations of the present disclosure may be implemented using existing computer processors, by dedicated computer processors for suitable systems incorporated for this or other purposes, or by hardwired systems. Implementations within the scope of the present disclosure include program products that include machine-readable media for carrying or storing machine-executable instructions or data structures. Such machine-readable media can be any available medium that can be accessed by a general-purpose or special-purpose computer or other machine with a processor. By way of example, such machine-readable media include RAM, ROM, EPROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or machine-executable instructions or data structures that can be accessed by a general-purpose or special-purpose computer or other machine with a processor, containing desired program code.

[0077] When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless) to a machine, the machine properly views the connection as a machine-readable medium. Thus, any such connection is properly termed a machine-readable medium. Combinations of the above are also included within the scope of machine-readable media. Machine-executable instructions comprise, for example, instructions and data that cause a general-purpose computer, special-purpose computer, or special-purpose processing machines to perform a certain function or group of functions.

[0078] While the figures show a specific order of method steps, the order of the steps may differ from that depicted. Also, two or more steps may be performed concurrently or with partial concurrence. Such variations depend on the software and hardware systems selected and the designer's choice. All such variations are within the scope of this disclosure. Similarly, software implementations may be achieved using standard programming techniques with rule-based logic and other logic to accomplish the various connecting, processing, comparing, and determining steps.

[0079] It is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only, and is not intended to be limiting.

[0080] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another implementation includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another implementation. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0081] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0082] Throughout the description and claims of this specification, the word "comprise" and variations of words such as "comprising" and "comprises" mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers, or steps. "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal implementation. "Such as" is used for descriptive purposes, not limiting.

[0083] Components that can be used to implement the disclosed methods and systems are disclosed. These and other components are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these components are disclosed, it is understood that specific reference to each of the various individual and collective combinations and permutations thereof may not be expressly disclosed, and that for all methods and systems, each is specifically contemplated and described herein. This applies to all aspects of the present application, including, but not limited to, the steps of the disclosed methods. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed by any specific implementation or combination of implementations of the disclosed methods.

[0084] Exemplary Implementations Clause 1. An illumination system for laser speckle imaging, the illumination system including: a light source configured to output light having a wavelength in the range of 600 nm to 2000 nm; two or more sections of optical fiber; a fiber-coupled acousto-optic modulator (FCAOM), the FCAOM coupled to the light source by a first section of the two or more sections of optical fiber; and a collimating optical system that focuses light output by the wavelength-stable laser to illuminate an object within a field of view (FOV), the collimating optical system being coupled to the FCAOM by a second section of the two or more sections of optical fiber.

[0085] Clause 2. The illumination system of clause 1, wherein the light source is a laser or a laser diode.

[0086] Clause 3. The illumination system of clause 2, wherein the wavelength-stable laser is a volume holographic grating (VHG)-stable laser diode.

[0087] Clause 4. An illumination system according to any one of clauses 1 to 3, wherein the collimating optics has an adjustable focal length.

[0088] Clause 5. An illumination system as described in any one of clauses 1 to 4, wherein the first section of the optical fiber comprises a first portion integrated with the light source and a second portion integrated with the FCAOM.

[0089] Clause 6. The lighting system of clause 5, wherein the first part and the second part are joined by a mating sleeve.

[0090] Clause 7. An illumination system described in any one of clauses 1 to 6, wherein the image capture device is configured to capture an image of an object within the FOV when the FOV is illuminated by the light source.

[0091] Clause 8. The illumination system of clause 7, wherein the image capture device comprises at least one magnifying lens that expands the FOV.

[0092] Clause 9. The lighting system of clause 7, wherein the image capture device comprises a black and white camera.

[0093] Clause 10. The illumination system of clause 9, wherein the image capture device comprises a long-pass filter positioned between the FOV and the monochrome camera.

[0094] Clause 11. The illumination system of any one of clauses 1 to 10, wherein the optical fiber is a single-mode optical fiber.

[0095] Clause 12. An illumination system according to any one of clauses 1 to 11, wherein the collimating optics has an adjustable focal length.

[0096] Clause 13. The lighting system of any one of clauses 1 to 12, further comprising a radio frequency (RF) driver configured to modulate the output of the FCAOM.

[0097] Clause 14. The lighting system of clause 13, further comprising a controller configured to control the RF driver, wherein the controller synchronizes the output of the FCAOM with the operation of an image capture system that captures an image of a target within the FOV.

[0098] Clause 15. A laser speckle imaging system comprising: a light source having an operating wavelength in the range of 600 nm to 2000 nm; an optical fiber; a fiber-coupled acousto-optic modulator (FCAOM), the FCAOM coupled to the light source by a first section of the optical fiber; collimating optics for focusing light output by the light source to illuminate a field of view (FOV), the collimating optics coupled to the FCAOM by a second section of the optical fiber; and an image capture device for capturing an image of the FOV as the FOV is illuminated by the light source.

[0099] Clause 16. The laser speckle imaging system of clause 15, wherein the light source is a laser or a laser diode.

[0100] Clause 17. The laser speckle imaging system of clause 16, wherein the wavelength-stable laser is a volume holographic grating (VHG)-stable laser diode.

[0101] Clause 18. A laser speckle imaging system according to any one of clauses 15 to 17, wherein the collimating optical system has an adjustable focal length.

[0102] Clause 19. A laser speckle imaging system as described in any one of clauses 15 to 18, wherein the first section of the optical fiber comprises a first portion integrated with the light source and a second portion integrated with the FCAOM.

[0103] Clause 20. The laser speckle imaging system of clause 19, wherein the first portion and the second portion are joined by a mating sleeve.

[0104] Clause 21. A laser speckle imaging system described in any one of clauses 15 to 20, wherein the image capture device comprises at least one magnifying lens that expands the FOV.

[0105] Clause 22. A laser speckle imaging system according to any one of clauses 15 to 21, wherein the image capture device comprises a black and white camera.

[0106] Clause 23. The laser speckle imaging system of clause 22, wherein the image capture device comprises a long-pass filter positioned between the FOV and the monochrome camera.

[0107] Clause 24. A laser speckle imaging system according to any one of clauses 15 to 23, wherein the optical fiber is a single-mode optical fiber.

[0108] Clause 25. A laser speckle imaging system according to any one of clauses 15 to 24, further comprising a radio frequency (RF) driver configured to modulate the output of the FCAOM.

[0109] Clause 26. The laser speckle imaging system of clause 25, further comprising a controller configured to control the RF driver and the image capture device, wherein the controller synchronizes the capture of images by the image capture device with the output of the FCAOM.

[0110] Clause 27. The laser speckle imaging system of clause 26, wherein the controller is further configured to control the FCAOM to illuminate the FOV at a plurality of different modulation frequencies within a single exposure time of the image capture device, capture at least one image of the FOV at each of the plurality of different modulation frequencies, calculate speckle contrast for each captured image to create one or more sets of speckle contrast images, and extract a value of the anti-correlation time at each pixel using the one or more sets of speckle contrast images.

[0111] Clause 28. The laser speckle imaging system of clause 26, further configured wherein the controller controls the FCAOM to generate, within a first exposure time of the image capture device, a first set of light pulses having a first time delay therebetween, and controls the FCAOM to generate, within a second exposure time of the image capture device, a second set of light pulses having a second time delay therebetween that is different from the first time delay; capture a series of images of the FOV within each of the first and second exposure times; calculate speckle contrast for each image in the series of images to create a corresponding set of speckle contrast images; and extract a value of the anti-correlation time at each pixel using the set of speckle contrast images.

[0112] Clause 29. A method of speckle imaging, comprising: operating a light source and an acousto-optic modulator (AOM) of a laser speckle imaging system to illuminate a field of view (FOV) at a plurality of different modulation frequencies within a single exposure time of the laser speckle imaging system; capturing at least one image of the FOV at each of the plurality of different modulation frequencies; calculating speckle contrast for each captured image to create one or more sets of speckle contrast images; and extracting a value of decorrelation time at each pixel using the one or more sets of speckle contrast images.

[0113] Clause 30. The method of clause 29, wherein operating the light source and the AOM to illuminate the FOV at a plurality of different modulation frequencies includes controlling the AOM to adjust the modulation frequency of the light directed to the FOV in accordance with a plurality of different modulation frequencies.

[0114] Clause 31. The method of clause 29, wherein operating the light source and AOM to illuminate the FOV at a plurality of different modulation frequencies includes controlling the light source to adjust the modulation frequency of the light directed to the FOV in accordance with a plurality of different modulation frequencies.

[0115] Clause 32. The method of any one of clauses 29 to 31, wherein the AOM is a fiber-coupled AOM.

[0116] Clause 33. The method of any one of clauses 29 to 32, further comprising determining blood flow from the value of the anti-correlation time at each pixel.

[0117] Clause 34. A method according to any one of clauses 29 to 33, wherein the light source of the laser speckle imaging system is a laser or a laser diode.

[0118] Clause 35. The method of clause 34, wherein the light source has an operating wavelength in the range of 600 nm to 2000 nm.

[0119] Clause 36. The method of clause 34, wherein the wavelength-stable laser is a fiber-coupled volume holographic grating (VHG)-stable laser diode.

[0120] Clause 37. A method according to any one of clauses 29 to 36, wherein the light source is coupled to the AOM via a section of optical fibre.

[0121] Clause 38. A method according to any one of clauses 29 to 37, wherein the AOM is coupled via a section of optical fibre to an adjustable focal length collimating optic, which focuses the light output by the light source to illuminate the FOV.

[0122] Clause 39. A method according to any one of clauses 29 to 38, wherein the laser speckle imaging system comprises an image capture device for capturing at least one image of the FOV, the image capture device comprising a black and white camera and at least one magnifying lens.

[0123] Clause 40. The method of clause 39, wherein the image capture device further comprises a long-pass filter positioned between the FOV and the monochrome camera.

[0124] Clause 41. A method according to any one of clauses 29 to 40, wherein operating the AOM includes sending a command to a radio frequency (RF) driver of the laser speckle imaging system, the RF driver being coupled to the AOM.

[0125] Clause 42. A method of speckle imaging, the method comprising: operating a light source and an acousto-optic modulator (AOM) of a laser speckle imaging system to generate, within a first exposure time of the laser speckle imaging system, a first set of pulses having a first time delay therebetween, wherein light output by the light source illuminates a field of view (FOV); operating the light source and AOM to generate, within a second exposure time of the laser speckle imaging system, a second set of pulses having a second time delay therebetween, the second time delay being different from the first time delay; capturing a series of images of the FOV within each of the first and second exposure times; calculating a speckle contrast for each image in the series of images to create a corresponding set of speckle contrast images; and extracting a value of the anti-correlation time at each pixel using the set of speckle contrast images.

[0126] Clause 43. The method of clause 42, further comprising determining blood flow from the value of the decorrelation time at each pixel.

[0127] Clause 44. The method of clause 42 or 43, wherein the light source of the laser speckle imaging system is a laser or a laser diode.

[0128] Clause 45. The method of clause 44, wherein the light source has an operating wavelength in the range of 600 nm to 2000 nm.

[0129] Clause 46. The method of clause 44, wherein the wavelength-stable laser is a fiber-coupled volume holographic grating (VHG)-stable laser diode.

[0130] Clause 47. A method according to any one of clauses 42 to 46, wherein the light source is coupled to the AOM via a section of optical fibre.

[0131] Clause 48. A method according to any one of clauses 42 to 47, wherein the AOM is coupled via a section of optical fibre to an adjustable focal length collimating optic, which focuses the light output by the light source to illuminate the FOV.

[0132] Clause 49. A method according to any one of clauses 42 to 48, wherein the laser speckle imaging system comprises an image capture device for capturing a series of images of the FOV, the image capture device comprising a black and white camera and at least one magnifying lens.

[0133] Clause 50. The method of clause 49, wherein the image capture device further comprises a long-pass filter positioned between the FOV and the monochrome camera.

[0134] Clause 51. A method according to any one of clauses 42 to 50, wherein controlling the AOM includes sending a command to a radio frequency (RF) driver of the laser speckle imaging system, the RF driver being coupled to the AOM.

[0135] Clause 52. The method of any one of clauses 42 to 51, wherein the AOM is a fiber-coupled AOM.

Claims

1. 1. An illumination system for laser speckle imaging, the illumination system comprising: a light source configured to output light having a wavelength in the range of 600 nm to 2000 nm; two or more sections of optical fiber; a fiber coupled acousto-optic modulator (FCAOM) coupled to the light source by a first section of the two or more sections of optical fiber; and a collimating optical system that focuses light output by a wavelength-stable laser to illuminate an object within a field of view (FOV), the collimating optical system being coupled to the FCAOM by a second section of the two or more sections of the optical fiber.

2. 10. The lighting system of claim 1, wherein the light source is a laser or a laser diode.

3. 3. The illumination system of claim 2, wherein the wavelength-stable laser is a volume holographic grating (VHG) stabilized laser diode.

4. 4. The illumination system according to claim 1, wherein the collimating optics has an adjustable focal length.

5. 5. The illumination system of claim 1, wherein the first section of the optical fiber comprises a first portion that is integrated with the light source and a second portion that is integrated with the FCAOM.

6. 6. The lighting system of claim 5, wherein the first portion and the second portion are joined by a mating sleeve.

7. 7. The illumination system of claim 1, wherein an image capture device is configured to capture an image of the object within the FOV when the FOV is illuminated by the light source.

8. The illumination system of claim 7 , wherein the image capture device comprises at least one magnifying lens that magnifies the FOV.

9. The lighting system of claim 7 , wherein the image capture device comprises a black and white camera.

10. 10. The illumination system of claim 9, wherein the image capture device comprises a long-pass filter positioned between the FOV and the monochrome camera.

11. 11. The illumination system according to any one of claims 1 to 10, wherein the optical fiber is a single-mode optical fiber.

12. 12. The illumination system according to any one of the preceding claims, wherein the collimating optics has an adjustable focal length.

13. 13. The lighting system of any preceding claim, further comprising a radio frequency (RF) driver configured to modulate the output of the FCAOM.

14. 14. The lighting system of claim 13, further comprising a controller configured to control the RF driver, the controller synchronizing the output of the FCAOM with operation of an image capture system that captures images of the object within the FOV.

15. 1. A laser speckle imaging system, comprising: a light source having an operating wavelength in the range of 600 nm to 2000 nm; An optical fiber; a fiber coupled acousto-optic modulator (FCAOM) coupled to the light source by a first section of the optical fiber; and a collimating optic that focuses the light output by the light source to illuminate a field of view (FOV), the collimating optic being coupled to the FCAOM by a second section of the optical fiber; an image capture device for capturing an image of the FOV when the FOV is illuminated by the light source.

16. The laser speckle imaging system of claim 15 , wherein the light source is a laser or a laser diode.

17. 17. The laser speckle imaging system of claim 16, wherein the wavelength-stable laser is a volume holographic grating (VHG)-stable laser diode.

18. The laser speckle imaging system of any one of claims 15 to 17, wherein the collimating optics has an adjustable focal length.

19. 19. The laser speckle imaging system of claim 15, wherein the first section of the optical fiber comprises a first portion that is integrated with the light source and a second portion that is integrated with the FCAOM.

20. 20. The laser speckle imaging system of claim 19, wherein the first portion and the second portion are joined by a mating sleeve.

21. The laser speckle imaging system of any one of claims 15 to 20, wherein the image capturing device comprises at least one magnifying lens for magnifying the FOV.

22. The laser speckle imaging system of any one of claims 15 to 21, wherein the image capture device comprises a black and white camera.

23. 23. The laser speckle imaging system of claim 22, wherein the image capture device comprises a long-pass filter positioned between the FOV and the monochrome camera.

24. The laser speckle imaging system according to any one of claims 15 to 23, wherein the optical fiber is a single-mode optical fiber.

25. The laser speckle imaging system of any one of claims 15 to 24, further comprising a radio frequency (RF) driver configured to modulate the output of the FCAOM.

26. 26. The laser speckle imaging system of claim 25, further comprising a controller configured to control the RF driver and the image capture device, wherein the controller synchronizes the capturing of an image by the image capture device with the output of the FCAOM.

27. The controller: Within a single exposure time of the image capture device, controlling the FCAOM to illuminate the FOV with a plurality of different modulation frequencies; capturing at least one image of the FOV at each of the plurality of different modulation frequencies; calculating speckle contrast for each captured image to generate one or more sets of speckle contrast images; 27. The laser speckle imaging system of claim 26, further configured to extract a value of decorrelation time at each pixel using one or more sets of the speckle contrast images.

28. The controller: controlling the FCAOM to generate a first set of light pulses having a first time delay therebetween within a first exposure time of the image capture device; controlling the FCAOM to generate a second set of light pulses within a second exposure time of the image capture device, the second set having a second time delay therebetween that is different from the first time delay; capturing a series of images of the FOV within each of the first and second exposure times; calculating speckle contrast for each image in the series of images to produce a corresponding set of speckle contrast images; 27. The laser speckle imaging system of claim 26, further configured to use the set of speckle contrast images to extract a value of decorrelation time at each pixel.

29. 1. A method of speckle imaging, comprising: within a single exposure time of a laser speckle imaging system: operating the laser speckle imaging system to illuminate a field of view (FOV) at a plurality of different modulation frequencies; capturing at least one image of the FOV at each of the plurality of different modulation frequencies; calculating speckle contrast for each captured image to generate one or more sets of speckle contrast images; and extracting a value of decorrelation time at each pixel using one or more sets of said speckle contrast images.

30. 30. The method of claim 29, wherein the laser speckle imaging system comprises a light source and an acousto-optic modulator (AOM), and wherein operating the laser speckle imaging system to illuminate the FOV at the plurality of different modulation frequencies comprises controlling at least one of the light source or the AOM.

31. 31. The method of claim 30, wherein operating the light source and the AOM to illuminate the FOV at the plurality of different modulation frequencies comprises controlling the AOM to adjust a modulation frequency of light directed to the FOV in accordance with the plurality of different modulation frequencies.

32. 31. The method of claim 30, wherein operating the light source and the AOM to illuminate the FOV at the plurality of different modulation frequencies comprises controlling the light source to adjust a modulation frequency of light directed to the FOV in accordance with the plurality of different modulation frequencies.

33. The method of any one of claims 30 to 32, wherein the AOM is a fiber-coupled AOM.

34. The method of any one of claims 30 to 33, further comprising determining blood flow from the value of the decorrelation time at each pixel.

35. The method of any one of claims 30 to 34, wherein the light source of the laser speckle imaging system is a laser or a laser diode.

36. 36. The method of claim 35, wherein the light source has an operating wavelength in the range of 600 nm to 2000 nm.

37. 36. The method of claim 35, wherein the wavelength-stable laser is a fiber-coupled volume holographic grating (VHG)-stable laser diode.

38. A method according to any one of claims 30 to 37, wherein the light source is coupled to the AOM via a section of optical fibre.

39. 39. The method of any one of claims 30 to 38, wherein the AOM is coupled via a section of optical fiber to an adjustable focal length collimating optic, the adjustable focal length collimating optic focusing the light output by the light source to illuminate the FOV.

40. 39. The method of any one of claims 29 to 38, wherein the laser speckle imaging system comprises an image capture device for capturing the at least one image of the FOV, the image capture device comprising a black and white camera and at least one magnifying lens.

41. 40. The method of claim 39, wherein the image capture device further comprises a long-pass filter positioned between the FOV and the monochrome camera.

42. 42. The method of claim 30, wherein operating the AOM comprises sending a command to a radio frequency (RF) driver of the laser speckle imaging system, the RF driver being coupled to the AOM.

43. 1. A method of speckle imaging, comprising: operating a laser speckle imaging system to generate a first set of pulses having a first time delay therebetween within a first exposure time of the laser speckle imaging system, wherein light output by a light source illuminates a field of view (FOV); operating the laser speckle imaging system to generate a second set of pulses having a second time delay therebetween that is different from the first time delay during a second exposure time of the laser speckle imaging system; capturing a series of images of the FOV within each of the first and second exposure times; calculating speckle contrast for each image in the series of images to generate a corresponding set of speckle contrast images; and extracting a value of decorrelation time at each pixel using the set of speckle contrast images.

44. 43. The method of claim 42, further comprising determining blood flow from the value of the decorrelation time at each pixel.

45. 45. The method of claim 43 or 44, wherein the laser speckle imaging system comprises a light source and an acousto-optic modulator (AOM).

46. 46. ​​The method of claim 45, wherein the light source of the laser speckle imaging system is a laser or a laser diode.

47. 47. The method of claim 46, wherein the light source has an operating wavelength in the range of 600 nm to 2000 nm.

48. 47. The method of claim 46, wherein the wavelength-stable laser is a fiber-coupled volume holographic grating (VHG)-stable laser diode.

49. A method according to any one of claims 45 to 48, wherein the light source is coupled to the AOM via a section of optical fibre.

50. 50. The method of any one of claims 45 to 49, wherein the AOM is coupled via a section of optical fiber to an adjustable focal length collimating optic, the adjustable focal length collimating optic focusing the light output by the light source to illuminate the FOV.

51. 51. The method of any one of claims 45 to 50, wherein the laser speckle imaging system comprises an image capture device for capturing the series of images of the FOV, the image capture device comprising a black and white camera and at least one magnifying lens.

52. 52. The method of claim 51 , wherein the image capture device further comprises a long-pass filter positioned between the FOV and the monochrome camera.

53. 53. The method of any one of claims 45 to 52, wherein controlling the AOM comprises sending commands to a radio frequency (RF) driver of the laser speckle imaging system, the RF driver being coupled to the AOM.

54. The method of any one of claims 45 to 53, wherein the AOM is a fiber-coupled AOM.

Images