Hyperspectral imaging and spectral analysis quantifies fluorophore concentration during fluorescence-guided surgery1-6. However, acquisition of the multiple wavelengths required to implement these methods can be time-consuming and hinder surgical workflow. To this end, a snapshot hyperspectral imaging system capable of acquiring 64 channels of spectral data simultaneously was developed for rapid hyperspectral imaging during neurosurgery. The system uses a birefringent spectral demultiplexer to split incoming light and redirect wavelengths to different sections of a large format microscope sensor. Its configuration achieves high optical throughput, accepts unpolarized input light and exceeds channel count of prior image-replicating imaging spectrometers by 4-fold. Tissue-simulating phantoms consisting of serial dilutions of the fluorescent agent characterize system linearity and sensitivity, and comparisons to performance of a liquid crystal tunable filter based hyperspectral imaging device are favorable. The new instrument showed comparable, if not improved, sensitivity at low fluorophore concentrations; yet, acquired wide-field images at more than 70-fold increase in frame rate. Image data acquired in the operating room during human brain tumor resection confirm these findings. The new device is an important advance in achieving real-time quantitative imaging of fluorophore concentration for guiding surgery.

Background Opioid use disorder (OUD) affects more than 14 million Americans and poses a high risk of relapse, overdose, and death. Current treatments are not tailored to individual needs and do not monitor the effectiveness of the medication. We propose a novel method to measure the occupancy of mu opioid receptors (MOR), which are key targets for opioid pharmacotherapy, in peripheral tissues with high MOR density. We developed a fluorescent peptide agonist that binds to MOR and can be detected by non-invasive point-of-care techniques. We present in vitro and in vivo results that demonstrate the feasibility and potential of this method to assess MOR availability and treatment efficacy in OUD patients. Methods A new fluorescent-labeled synthetic peptide agonist [Lys7]Dermorphin-IRDye800CW, called DRM-800, was synthesized and characterized in vitro to evaluate binding and internalization. Wildtype and MOR knock-out mice were used to quantify plasma kinetics and, using a cyromacrotome, fluorescence images were acquired post-mortem on whole-body sections 150 um apart. These volumes were used to compare in vivo enhancement of MOR-rich structures. Results In vitro assays and microscope visualization of DRM-800 showed high MOR-affinity and rapid, robust internalization. Plasma half-life following intravenous injection in mice was 8-12 minutes. Specific binding by tissue structures of interest, measured by the ratio of relative fluorescent units in wild-type vs. MOR knockout mice showed high binding in dorsal root ganglia, spiral ganglia and trigeminal ganglion, as well as in the small and large intestine. Conclusions The pharmacokinetics and distribution, binding kinetics and rapid internalization suggests that MOR-specific fluorescence enhancement corresponding to opioid rich structures could serve as a potential biomarker in opioid use disorder.

Timely assessment of bone perfusion in orthopaedic trauma surgery plays an important role in successful treatment outcome. For guiding accurate debridement of bones with impaired blood supply, fluorescence-guided surgery (FGS) technique have gained increasingly popularity. Compared to other imaging modalities like computed tomography and nuclear magnetic resonance imaging that are time consuming and less practical during surgery, fluorescence imaging can be performed intraoperatively and is able to visualize the bone blood flow in real time. In order to link the blood flow fluorescence imaging to quantitative bone perfusion numbers, in this study we are using a modified fluorescent microsphere (FM) approach called microsphere quantification using imaging cryomacrotome (mQUIC). Bone perfusion is assessed by identifying the density of deposited microspheres in reconstructed imaging volumes, which are proportional to the regional blood flow. In the rabbit model presented here, cryoimaging was used to scan femurs injected with three colors of microspheres corresponding to three conditions: baseline, post-osteotomy and post-periosteal stripping. Image processing, such as top-hat transform and object-based colocalization, was used to enable accurate counting of FMs to produce their 3D-localization within the bones. FM density volumes were converted to bone perfusion units (mL/min/100g) using the reference organ technique. This study provides a groundwork for direct comparison with our DCE-FI technique for measuring bone perfusion in orthopaedic trauma surgery models.

In orthopedic trauma surgery, timely assessment of bone tissue perfusion plays a vital role in the successful treatment outcome. Fluorescence-guidance is gaining increased surgical interest, especially with respect to hemodynamic assessment of bone. Intraoperative dynamic contrast-enhanced fluorescence imaging (DCE-FI) not only enables visualization of the perfused areas of the injured bone, but with subsequent analysis using kinetic models, may also provide a valuable quantitative bone blood flow information to a surgeon. In this study, we are validating this quantitative approach with a modified fluorescent microsphere (FM) technique using a custom-built four-channel imaging cryomacrotome. We demonstrate that FMs of four different colors can be accurately detected in controlled phantoms and evaluate their detection accuracy in real blood samples. In a rabbit model of orthopaedic trauma, we show that blood flow measurements using the DCE-FI technique can be compared with the FM technique. This feasibility pilot study provides the groundwork for investigation of the correlation between bone perfusion measurements using DCE-FI and using fluorescent microspheres, in units of ml/min/100g.

Glioblastoma remains the deadliest type of brain tumor: half of patients do not live more than 16 months, even when treated with surgery, chemotherapy, and radiation. Tracking systems can help neurosurgeons precisely identify tumor on MRI images. Nevertheless, the tumor often regrows a few centimeters from where the original tumor was. This is because at the time of the first surgery, the actual tumor cells are already invading the healthy tissue around the tumor. These ‘invaders’ are difficult to cut out because even when looking through the surgical microscope, the tumor margins and normal brain tissue look very similar. The emerging utilization of fluorescing biomarkers (e.g., 5-ALA) sensitive to genetic downregulation present in cancer cells improves the detectability of marginal glioma, albeit requiring to switch to the surgical microscope excitation (blue light) mode and dim the operation room lights, imposing difficulties for neurosurgeons and staff. Here, we present a portable fluorescence-guided surgery optical imaging system integrated into the conventional surgical microscope to give neurosurgeons a better tool to predict which tissue is normal and which contains the start of tumor invasion without the need to switch to the excitation mode. The system operates under the microscope’s white light illumination using pulsed fluorophore excitation with gated acquisition and provides helpful tumor tissue fluorescing contrast. Tissue-mimicking phantom imaging confirmed protoporphyrin IX detection down to 0.1μg/mL concentration. Brain tissue imaging ex-vivo and pre-clinical intracranial tumor resection demonstrated the system’s capability to provide a typical operating environment with auxiliary or augmented visualization of PpIX possible.

-BACKGROUND: Tumors that take up and metabolize 5aminolevulinic acid emit bright pink fluorescence when illuminated with blue light, aiding surgeons in identifying the margin of resection. The adoption of this method is hindered by the blue light illumination, which is too dim to safely operate under and therefore necessitates switching back and forth from white-light mode. The aim of this study was to examine the addition of an optimized secondary illuminant adapter to improve usability of blue-light mode without degrading tumor contrast. -METHODS: Color science methods were used to evaluate the color of the secondary illuminant and its impact on color rendering index as well as the tumor-to-background color contrast in data collected from 7 patients with highgrade gliomas (World Health Organization grade III and IV). A secondary illuminant adapter was built to provide 475e600 nm light the intensity of which can be controlled by the surgeon and was evaluated in 2 additional patients. -RESULTS: Secondary illuminant color had opposing effects on color rendering index and tumor-to-background color contrast; providing surgeon control of intensity allows this trade-off to be balanced in real time. Demonstration in 2 high-grade glioma cases confirms this, showing that additional visibility adds value when intensity can be controlled by the surgeon. -CONCLUSIONS: Addition of a secondary illuminant may mitigate surgeon complaints that the operative field is too dark under the blue light illumination required for 5aminolevulinic acid fluorescence guidance by providing improved color rendering index without completely sacrificing tumor-to-background color contrast.

Fluorescence-guided surgery (FGS) enhances intraoperative visualization of tumors to maximize safe resection, and quantitative fluorescence imaging (qFI) of protoporphyrin IX (PpIX) has provided additional information for guidance during intracranial tumor surgery. Previous developments in fluorescence quantification have demonstrated that the depth of fluorescence signals can be estimated given known optical properties in a lab setting, and now with the work described here that these optical properties can be determined in vivo in human brain tissue in the operating room (OR) during tumor resection procedures. More specifically, we report the first depth estimation of subsurface tumor intraoperatively, achieved with the combination of spatial frequency domain imaging (SFDI) for optical property measurement and red-light excitation of PpIX. We modified a commercial surgical microscope (Zeiss) with a digital light processing module (DLI Austin, TX) to modulate light from a xenon arc lamp to illuminate the field. White-light excitation and a liquid crystal tunable filter (LCTF Verispec) were used to measure diffuse reflectance at discrete wavelengths of 670 nm and 710 nm on a sCMOS camera. An illumination-side filter wheel allowed excitation of PpIX fluorescence at 405 nm and 635 nm, and the LCTF measured fluorescence emissions at 670 nm and 710 nm. Data acquisition and processing generated wide-field images of the depth of PpIX fluorescence within 1 minute in the OR. The ability of the clinical microscope to perform optical property mapping with SFDI and convert these wide-field estimates into images of the depth of fluorescence was tested in tissue simulating phantoms and in vivo during a craniotomy for brain tumor resection. Results indicate that wide-field optical property estimates with SFDI can be combined with depth sensing algorithms to produce maps of the depth of PpIX when exposed to red-light in the OR.

Whole-animal fluorescence cryo-imaging is an established technique that enables visualization of the biodistribution of labeled drugs, contrast agents, functional reporters and cells in detail. However, many tissues produce endogenous autofluorescence, which can confound interpretation of the cryo-imaging volumes. We describe a multi-channel, hyperspectral cryo-imaging system that acquires densely-sampled spectra at each pixel in the 3-dimensional stack. This information enables the use of spectral unmixing to isolate the fluorophore-of-interest from autofluorescence and/or other fluorescent reporters. In phantoms and a glioma xenograft model, we show that the approach improves detection limits, increases tumor contrast, and can dramatically alter image interpretation.

The ability to directly measure whole-body fluorescence can enable tracking of labeled cells, metastatic spread, and drug bio-distribution. We describe the development of a new hyperspectral imaging whole body cryo-macrotome designed to acquire 3-D fluorescence volumes in large specimens (whole animals) at high resolution. The use of hyperspectral acquisition provides full spectra at every voxel, enabling spectral decoupling of multiple fluorohpores and autofluorescence. We present examples of tissue spectra and spectral fitting in a rodent glioma xenograft.

This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2 mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. Ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R-2 = 0.92) but not well with total tumor volume changes (R-2 = 0.34). In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.

BACKGROUND:Three patients enrolled in a clinical trial of 5-aminolevulinic-acid (5-ALA)-induced fluorescence-guidance, which has been demonstrated to facilitate intracranial tumor resection, were found on neuropathological examination to have focal cortical dysplasia (FCD). OBJECTIVE:To evaluate in this case series visible fluorescence and quantitative levels of protoporphyrin IX (PpIX) during surgery and correlate these findings with preoperative magnetic resonance imaging (MRI) and histopathology. METHODS:Patients were administered 5-ALA (20 mg/kg) approximately 3 h prior to surgery and underwent image-guided, microsurgical resection of their MRI- and electrophysiologically identified lesions. Intraoperative visible fluorescence was evaluated using an operating microscope adapted with a commercially available blue light module. Quantitative PpIX levels were assessed using a handheld fiber-optic probe and a wide-field imaging spectrometer. Sites of fluorescence measurements were co-registered with both preoperative MRI and histopathological analysis. RESULTS:Three patients with a pathologically confirmed diagnosis of FCD (Types 1b, 2a, and 2b) underwent surgery. All patients demonstrated some degree of visible fluorescence (faint or moderate), and all patients had quantitatively elevated concentrations of PpIX. No evidence of neoplasia was identified on histopathology, and in 1 patient, the highest concentrations of PpIX were found at a tissue site with marked gliosis but no typical histological features of FCD. CONCLUSION:FCD has been found to be associated with intraoperative 5-ALA-induced visible fluorescence and quantitatively confirmed elevated concentrations of the fluorophore PpIX in 3 patients. This finding suggests that there may be a role for fluorescence-guidance during surgical intervention for epilepsy-associated FCD.

The potential to image subsurface fluorescent contrast agents at high spatial resolution has facilitated growing interest in short-wave infrared (SWIR) imaging for biomedical applications. The early but growing literature showing improvements in resolution in small animal models suggests this is indeed the case, yet to date, images from larger animal models that more closely recapitulate humans have not been reported. We report the first imaging of SWIR fluorescence in a large animal model. Specifically, we imaged the vascular kinetics of an indocyanine green (ICG) bolus injection during open craniotomy of a mini-pig using a custom SWIR imaging instrument and a clinical-grade surgical microscope that images ICG in the near-infrared-I (NIR-I) window. Fluorescence images in the SWIR were observed to have higher spatial and contrast resolutions throughout the dynamic sequence, particularly in the smallest vessels. Additionally, vessels beneath a surface pool of blood were readily visualized in the SWIR images yet were obscured in the NIR-I channel. These first-in-large-animal observations represent an important translational step and suggest that SWIR imaging may provide higher spatial and contrast resolution images that are robust to the influence of blood.

The rate of complete resection of glioma has improved with the introduction of 5‐aminolevulinic acid‐induced protoporphyrin IX (PpIX) fluorescence image guidance. Surgical outcomes are further enhanced when the fluorescence signal is decoupled from the intrinsic tissue optical absorption and scattering obtained from diffuse reflectance measurements, yielding the absolute PpIX concentration, [PpIX]. Spatial frequency domain imaging was used previously to measure [PpIX] in near‐surface tumors under blue fluorescence excitation. Here, we extend this to subsurface [PpIX] fluorescence under red‐light excitation. The decay rate of the modulation amplitude of the fluorescence signal was used to calculate the PpIX depth, which was then applied in a forward diffusion model to estimate [PpIX] at depth. For brain‐like optical properties in phantoms with PpIX fluorescent inclusions, the depth can be recovered up to depths of 9.5 mm ± 0.4 mm, with [PpIX] ranging from 5 to 15 μg/mL within an average deviation of 15% from the true [PpIX] value.

The goal of fluorescence-guided surgery (FGR) is to provide real-time enhancement of tumors to maximize safe resection. The optical property mapping ability of spatial frequency domain imaging (SFDI) has enabled quantitative fluorescence imaging (qFI) of protoporphyrin IX (PpIX) in gliomas in the pre-clinical setting. The goal of this study was to evaluate the feasibility of using SFDI to allow for qFI to enhance FGR. Specifically, we modified a benchtop SFDI system to mount directly to a commercial surgical microscope(Zeiss). A commercially available digital light processing module (DLI Austin, TX) was used to modulate light from a xenon arc lamp to illuminate the field. White light excitation and a liquid crystal tunable filter (LCTF Verispec) was used to measure diffuse reflectance at discreet wavelengths from 420 nm to 720 nm on a CMOS camera. An illumination side filter wheel allowed for excitation of PpIX fluorescence at 405 nm and 635 nm and the LCTF measured fluorescence emission at 670 nm and 710 nm. The ability of the clinical microscope to perform optical mapping and qFI was tested with tissue simulating phantoms and live mouse models. The results of these tests showed that SFDI can be implemented in a clinical microscope and the optical mapping and qFI abilities of SFDI may be used to enhance FGR.

Intra-operative fluorescence-guided resection (FGR) enables maximum safe resection of glioma by providing real-time tumor contrast. In its most widely used form, FGR is mediated by the preferential overproduction of the fluorophore protoporphyrinIX (PpIX) in malignant tissue after an oral dose of its precursor 5-Aminolevulinic Acid (ALA)1. ALA-PpIX-FGR has been shown to significantly increase completeness of tumor resection. However, the subjective visual assessment and the variable intrinsic optical attenuation of tissue limit this technique to delineating only high-grade tumors that display strong fluorescence residing at the tissue surface. We have shown that wide-field quantitative assessment by extracting 2D maps of PpIX concentration in the tissue, [PpIX], significantly improves the accuracy in detecting diffuse tumors, thereby potentially extending FGR to patients with low-grade tumors. In this approach, hyperspectral fluorescence imaging is coupled to a custom-built spatial frequency domain imaging (SFDI) system. SFDI enables the recovery of tissue optical properties maps, μ_a and μ_s^'. These are used to correct the fluorescence images. The corrected hyperspectral fluorescence images are then spectrally unmixed to separate true PpIX fluorescence from that of its photoproducts and from autofluorescence. Quantitative fluorescence imaging was validated against the clinically used spectroscopic probe by comparing the recovered optical properties and [PpIX] in vivo of a rat brain tumor model. This quantitative approach was also applied to a near infrared fluorophore ZW-800 on tissue-simulating phantoms. ALA-PpIX-FGR, as it is currently implemented, is inaccessible to infiltrative residuals lying beyond the resection cavity because of the limited penetration depth of the blue excitation light used. This is problematic as these infiltrative tumors are the main cause of reccurence. Enhanced sub-surface tumor detection was shown feasible by exciting PpIX’s secondary absorption peak of 635 nm intra-operatively on patients with various intra-cranial pathologies. However, resolving strong fluorescence of a deep-seated tumor from weak fluorescence of a shallow tumor was not possible. That is because the detected fluorescence intensity is heavily dependent on fluorophore concentration, depth, and fluorophore distribution, while also being convolved with tissue turbidity. The aim of this work, therefore, is to extend quantitative ALA-PpIX-FGR to identify sub-surface tumors by resolving tumor depth from fluorophore concentration. This should assist the surgeon in making an informed decision as for whether to further resect or not. A new quantitative depth imaging method was developed by exploiting SFDI’s depth-encoding capabilities in fluorescence mode. The result is a series of spatially modulated fluorescence images, where the modulation amplitude decays with increasing spatial frequency at a rate dependent on fluorophore depth. After recovering depth, a diffusion-based fluorescent light transport model is applied to extract fluorophore concentration. The algorithm was validated using tissue-simulating phantoms and an ex vivo tissue model indicating that the maximum depth recovered is highly dependent on fluorophore concentration as well as on tissue turbidity. For the [PpIX] and optical property maps relevant for glioma tissue, our quantitative depth fluorescence technique can predict depths up to 9 mm ± 0.4 mm, while recovering [PpIX] with an accuracy of 15% for concentrations as low as 2.5 µg/ml.

Mapping the optical absorption and scattering properties of tissues using spatial frequency-domain imaging (SFDI) enhances quantitative fluorescence imaging of protoporphyrin IX (PpIX) in gliomas in the preclinical setting. The feasibility of using SFDI in the operating room was investigated here. A benchtop SFDI system was modified to mount directly to a commercial operating microscope. A digital light processing module imposed a selectable spatial light pattern from a broad-band xenon arc lamp to illuminate the surgical field. White light excitation and a liquid crystal-tunable filter allowed the diffuse reflectance images to be recorded at discrete wavelengths from 450 to 720 nm on a sCMOS camera. The performance was first tested in tissue-simulating phantoms, and data were then acquired intraoperatively during brain tumor resection surgery. The optical absorption and transport scattering coefficients could be estimated with average errors of 3.2% and 4.5% for the benchtop and clinical systems, respectively, with spatial resolution of better than 0.7 mm. These findings suggest that SFDI can be implemented in a clinically relevant configuration to achieve accurate mapping of the optical properties in the surgical field that can then be applied to achieve quantitative imaging of the fluorophore. (C) 2018 Society of Photo-Optical Instrumentation Engineers (SPIE)

Studies have shown that fluorescent agents demarcate tumor from surrounding brain tissue and offer intraoperative guidance during resection. However, visualization of fluorescence signal from tumor below the surgical surface or through the appearance of blood in the surgical field is challenging. We have previously described red light imaging techniques for estimating fluorescent depths in turbid media. In this study, we evaluate these methods over a broader range of fluorophore concentrations, and investigate the ability to resolve multiple fluorescent emissions in the same plane or at different depths along the axis of imaging. A tungsten halogen lamp is used as a broadband white light source for reflectance imaging. Fluorescence from Alexa Fluor 647 is excited with a 635 nm diode laser. Reflectance and fluorescence spectral data are gathered between 670 and 720 nm with the use of a liquid crystal tunable filter and recorded on a sCMOS camera. Results show that two fluorescent emissions can be resolved within 2 mm if they are in the same plane or within 3 mm if they are at different depths along the axis of imaging up to 6 mm below the surface.

Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml(-1) aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

Contrast agents have shown to be useful in the detection of cancers. The goal of this study was to compare enhancement of brain cancer contrast using reflectance and fluorescence confocal imaging of two fluorophores, methylene blue (MB) and demeclocycline (DMN). MB absorbs light in the red spectral range and fluoresces in the near-infrared. It is safe for in vivo staining of human skin and breast tissue. However, its safety for staining human brain is questionable. Thus, DMN, which absorbs light in the violet spectral range and fluoresces between 470 and 570 nm, could provide a safer alternative to MB. Fresh human gliomas, obtained from surgeries, were cut in half and stained with aqueous solutions of MB and DMN, respectively. Stained tissues were imaged using multimodal confocal microscopy. Resulting reflectance and fluorescence optical images were compared with hematoxylin and eosin histopathology, processed from each imaged tissue. Results indicate that images of tissues stained with either stain exhibit comparable contrast and resolution of morphological detail. Further studies are required to establish the safety and efficacy of these contrast agents for use in human brain.