SignificanceCerebrospinal fluid (CSF) rhinorrhea (leakage of brain fluid from the nose) can be difficult to identify and currently requires invasive procedures, such as intrathecal fluorescein, which requires a lumbar drain placement. Fluorescein is also known to have rare but significant side effects including seizures and death. As the number of endonasal skull base cases increases, the number of CSF leaks has also increased for which an alternative diagnostic method would be highly advantageous to patients.AimWe aim to develop an instrument to identify CSF leaks based on water absorption in the shortwave infrared (SWIR) without the need of intrathecal contrast agents. This device needed to be adapted to the anatomy of the human nasal cavity while maintaining low weight and ergonomic characteristics of current surgical instruments.ApproachAbsorption spectra of CSF and artificial CSF were obtained to characterize the absorption peaks that could be targeted with SWIR light. Different illumination systems were tested and refined prior to adapting them into a portable endoscope for testing in 3D-printed models and cadavers for feasibility.ResultsWe identified CSF to have an identical absorption profile as water. In our testing, a narrowband laser source at 1480 nm proved superior to using a broad 1450 nm LED. Using a SWIR enabling endoscope set up, we tested the ability to detect artificial CSF in a cadaver model.ConclusionsAn endoscopic system based on SWIR narrowband imaging can provide an alternative in the future to invasive methods of CSF leak detection.

SignificanceLow-frequency oscillations (LFOs) (<0.1 Hz) with respect to cerebral blood flow (CBF) have shown promise as an indicator of altered neurologic activity in the abnormal brain. Portable optical instruments have evolved to offer a noninvasive alternative for continuous CBF monitoring at the bedside compared with many large neuroimaging modalities. However, their utilization for acquiring LFOs of CBF has only been studied to a limited extent.AimWe aim to optimize an innovative speckle contrast diffuse correlation tomography (scDCT) system for the detection of LFOs within CBF variations.ApproachThe scDCT was optimized to achieve a higher sampling rate and a faster image reconstruction using a moving window 3D reconstruction algorithm with parallel computation. Power spectral density (PSD) analysis was performed to investigate altered LFOs during transient global cerebral ischemia in neonatal piglets.ResultsTransient global cerebral ischemia resulted in reductions in both CBF and PSD compared with their baseline values.ConclusionsSpontaneous LFOs, combined with CBF, provide a more comprehensive assay with the potential to clarify pathological mechanisms involved in brain injury. These results support scDCT’s inclusion and application in the growing area of LFO analysis and demonstrate its inherent advantage for neurological studies in preclinical and clinical settings, such as neonatal intensive care units.

SignificanceThe clinical use of optical methods for in vivo skin imaging is limited by skin strong scattering properties, which reduce image contrast and probing depth. The efficiency of optical methods can be improved by optical clearing (OC). However, for the use of OC agents (OCAs) in a clinical setting, compliance with acceptable non-toxic concentrations is required.AimOC of in vivo human skin, combined with physical and chemical methods to enhance skin permeability to OCAs, was performed to determine the clearing-effectiveness of biocompatible OCAs using line-field confocal optical coherence tomography (LC-OCT) imaging.ApproachNine types of OCAs mixtures were used in association with dermabrasion and sonophoresis for OC protocol on three volunteers hand skin. From 3D images obtained every 5 min for 40 min, the intensity and contrast parameters were extracted to assess their changes during the clearing process and evaluate each OCAs mixture’s clearing efficacy.ResultsThe LC-OCT images average intensity and contrast increased over the entire skin depth with all OCAs. The best image contrast and intensity improvement was observed using the polyethylene glycol, oleic acid, and propylene glycol mixture.ConclusionsComplex OCAs featuring reduced component concentrations that meet drug regulation-established biocompatibility requirements were developed and proved to induce significant skin tissues clearing. By allowing deeper observations and higher contrast, such OCAs in combination with physical and chemical permeation enhancers may improve LC-OCT diagnostic efficacy.

SignificanceFluorescently guided minimally invasive surgery is improving patient outcomes and disease-free survival, but biomarker variability hinders complete tumor resection with single molecular probes. To overcome this, we developed a bioinspired endoscopic system that images multiple tumor-targeted probes, quantifies volumetric ratios in cancer models, and detects tumors in ex vivo samples.AimWe present a new rigid endoscopic imaging system (EIS) that can capture color images while simultaneously resolving two near-infrared (NIR) probes.ApproachOur optimized EIS integrates a hexa-chromatic image sensor, a rigid endoscope optimized for NIR-color imaging, and a custom illumination fiber bundle.ResultsOur optimized EIS achieves a 60% improvement in NIR spatial resolution when compared to a leading FDA-approved endoscope. Ratio-metric imaging of two tumor-targeted probes is demonstrated in vials and animal models of breast cancer. Clinical data gathered from fluorescently tagged lung cancer samples on the operating room’s back table demonstrate a high tumor-to-background ratio and consistency with the vial experiments.ConclusionsWe investigate key engineering breakthroughs for the single-chip endoscopic system, which can capture and distinguish numerous tumor-targeting fluorophores. As the molecular imaging field shifts toward a multi-tumor targeted probe methodology, our imaging instrument can aid in assessing these concepts during surgical procedures.

SignificanceWide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore’s diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, θ imaging is still rarely investigated in general and in carbon dots (CDs) in particular.AimTo extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and θ, together with the steady-state images of fluorescence intensity (FI) and FA (r).ApproachThe proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates.ResultsThe FLT of fluorescein samples was found to decrease from 4.01 ± 0.01 to 3.56 ± 0.02 ns, whereas both r and θ were significantly increased from 0.053 ± 0.012 to 0.252 ± 0.003 and 0.15 ± 0.05 to 11.25 ± 1.87 ns, respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of r from 0.100 ± 0.011 to 0.150 ± 0.013 and θ from 0.98 ± 0.13 to 1.65 ± 0.20 ns for the first CDs and from 0.280 ± 0.008 to 0.310 ± 0.004 and 5.55 ± 1.08 to 7.95 ± 0.97 ns for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs.ConclusionsThrough the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, r, and θ). Nevertheless, θ was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

SignificanceThis third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery.AimNational and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed.ApproachPrincipal investigators presenting at the Perelman School of Medicine Abramson Cancer Center’s third clinical trials update on IMI were selected to discuss their clinical trials and endpoints.ResultsDyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed.ConclusionsIMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.

SignificancePhotobleaching of the photosensitizer reduces fluorescence observation time and the intensity of fluorescence emitted for tumor detection during 5-aminolevulinic acid-based photodynamic diagnosis.AimThis study aims to utilize the concept of fluorescence photoswitching, which uses the fluorescence emission from photosensitizer excitation followed by the simultaneous excitation of the photosensitizer and its photoproduct to increase the fluorescence detection intensity during PDD of deeply located tumors.ApproachThe fluorescence photobleaching of protoporphyrin IX (PpIX) and the formation of its photoproduct, photoprotoporhyrin (Ppp), caused by exposure to 505 nm light were investigated in solution, ex vivo, and in vivo, and the fluorescence photoswitching was analyzed. The fluorescence observations of PpIX and Ppp were performed with 505 and 450 or 455 nm excitation, respectively, which is the suited wavelength for the primary excitation of each fluorophore.ResultsFluorescence photoswitching was observed in all forms of PpIX investigated, and the fluorescence photoswitching time, fluorescence intensity relative to the initial PpIX and Ppp intensity, and fluorescence intensity relative to PpIX after photobleaching were obtained. The dependence of the fluorescence photoswitching time and intensity on the irradiation power density was noted. A fluorescence intensity increase between 1.6 and 3.9 times was achieved with simultaneous excitation of PpIX and Ppp after fluorescence photoswitching, compared with the excitation of PpIX alone.ConclusionsWe have demonstrated the potential of fluorescence photoswitching for the improvement of the fluorescence observation intensity for the PDD of deeply located tumors.

SignificanceEdema occurs in the course of various skin diseases. It manifests itself in changes in water concentrations in skin layers: dermis and hypodermis and their thicknesses. In medicine and cosmetology, objective tools are required to assess the skin’s physiological parameters. The dynamics of edema and the skin of healthy volunteers were studied using spatially resolved diffuse reflectance spectroscopy (DRS) in conjunction with ultrasound (US).AimIn this work, we have developed a method based on DRS with a spatial resolution (SR DRS), allowing us to simultaneously assess water content in the dermis, dermal thickness, and hypodermal thickness.ApproachAn experimental investigation of histamine included edema using SR DRS under the control of US was conducted. An approach for skin parameter determination was studied and confirmed using Monte-Carlo simulation of diffuse reflectance spectra for a three-layered system with the varying dermis and hypodermis parameters.ResultsIt was shown that an interfiber distance of 1 mm yields a minimal relative error of water content determination in the dermis equal to 9.3%. The lowest error of hypodermal thickness estimation was achieved with the interfiber distance of 10 mm. Dermal thickness for a group of volunteers (7 participants, 21 measurement sites) was determined using SR DRS technique with an 8.3% error using machine learning approaches, taking measurements at multiple interfiber distances into account. Hypodermis thickness was determined with root mean squared error of 0.56 mm for the same group.ConclusionsThis study demonstrates that measurement of the skin diffuse reflectance response at multiple distances makes it possible to determine the main parameters of the skin and will serve as the basis for the development and verification of an approach that works in a wide range of skin structure parameters.

SignificanceDiffuse correlation spectroscopy (DCS) is an indispensable tool for quantifying cerebral blood flow noninvasively by measuring the autocorrelation function (ACF) of the diffused light. Recently, a multispeckle DCS approach was proposed to scale up the sensitivity with the number of independent speckle measurements, leveraging the rapid development of single-photon avalanche diode (SPAD) cameras. However, the extremely high data rate from advanced SPAD cameras is beyond the data transfer rate commonly available and requires specialized high-performance computation to calculate large number of autocorrelators (ACs) for real-time measurements.AimWe aim to demonstrate a data compression scheme in the readout field-programmable gate array (FPGA) of a large-pixel-count SPAD camera. On-FPGA, data compression should democratize SPAD cameras and streamline system integration for multispeckle DCS.ApproachWe present a 192 × 128 SPAD array with 128 linear ACs embedded on an FPGA to calculate 12,288 ACFs in real time.ResultsWe achieved a signal-to-noise ratio (SNR) gain of 110 over a single-pixel DCS system and more than threefold increase in SNR with respect to the state-of-the-art multispeckle DCS.ConclusionsThe FPGA-embedded autocorrelation algorithm offers a scalable data compression method to large SPAD array, which can improve the sensitivity and usability of multispeckle DCS instruments.

SignificancePositive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative.AimCurrent intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients.ApproachDual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer.ResultsTargeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues.ConclusionsPrevious work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker.