This PDF file contains the front matter associated with SPIE Proceedings Volume 11641 including the Title Page, Copyright information, and Table of Contents.

Quick, macroscopic examination of breast tissue from surgical excisions or biopsies can guide breast cancer patient management. To this purpose, we examined dual-contrast fluorescence imaging with intravital dye Methylene Blue (MB) and disease-specific molecular marker pH low insertion peptide (pHLIP) conjugated with fluorescent Alexa532 (Alexa532-pHLIP) as contrast agents. Samples were stained with both MB and Alexa532-pHLIP and imaged with multimodal wide-field system. Co- and cross-polarized reflectance and fluorescence images were acquired. Then, specimens were processed for H and E paraffin embedded histopathology. Wide-field imaging demonstrated increased pHLIP-Alexa 532 fluorescence emission and high MB fluorescence polarization in cancerous regions.

Skin cancer is the most common human malignancy. The goal of this pilot study was to validate a novel handheld optical polarization imaging (OPI) device for preoperative detection of basal cell carcinoma (BCC) margins. Ten patients with biopsy proven basal cell carcinoma (BCC) were imaged prior to Mohs surgery at UMASS Memorial Medical Center. Preliminary results from analysis of 10 BCC lesions show a strong correlation between optical imaging and histopathology. These findings indicate OPI may be a valuable tool for optimizing surgical treatment of skin cancer.

The crucial problem of brain tumor surgery is the accurate detection the tumor border for safe and complete tumor resection. Whereas it is quite easy to identify brain tumor in preoperative magnetic resonance imaging, it is often difficult to differentiate solid tumor tissue from infiltrated white matter during surgery with conventional surgical intra-operative microscope. To address this problem we suggest exploring the optical anisotropy of healthy brain white matter which represents a highly ordered structure consisting of axons that are joined together in fiber tracts. Tumor cells grow chaotically and erase the optical anisotropy of healthy brain. Instead of detecting the tumor itself, we suggest to visualize healthy white matter by means of its fiber tracts by detecting the optical anisotropy of brain tissue. For this purpose we used a wide-field imaging Mueller polarimetric system operating in the visible wavelength range in backscattering configuration. The Mueller matrix images of the thick (~1cm) fixed human brain specimen and thick (~1cm) fresh veal brain specimen were measured at 633 nm in reflection. Lu Chipman decomposition was applied pixel-wise to the experimental Mueller matrices. The maps of azimuth of fast optical axis of linear birefringent medium showed a compelling correlation with the fiber tracts directions on histology image of thin whole mount silver-stained brain tissue section, that is gold standard for ex-vivo brain fiber tract visualization. Thus, label-free non-contact imaging Mueller polarimetry shows potential for the intra-operative visualization of brain white matter fiber tracts. Further studies are ongoing.

Quantitative cancer detection method utilizing fluorescence polarization imaging of aqueous methylene blue will be presented. It holds the potential to provide unique capability to detect cancer accurately and rapidly in vivo at the cellular level.

Here we proposed an imaging strategy to monitor more fluorescent objectives synchronously in the living animals with multicolor two-photon excited fluorescence microscopy. We used highly nonlinear photonic crystal fibers and a 100-fs Ti: Sapphire oscillator to generate 200 nm wide continuum pulses, which covers the two-photon excitation spectra of conventional fluorophores. Then, we used the phase shaping method to compensate for the dispersion and switch excitation wavelength rapidly. Next, we implemented non-negative matrix factorization (NMF) to unmix images with cross-talk. Images of 8-color HeLa cells and 4-color mice tumor under the mouse dorsal skinfold chamber were acquired.

Intracellular motion (IM) is originated from the motion of molecules and organelles in the cytoplasm of eukaryotic cells. IM is essential for the proper functioning of cells. Instead of tracking specific molecules, coherent gated methods measure the speckle variation induced by IM. Initially, holographic optical coherence imaging was used to image the IM of tumor spheroids and drugs' responses^1,2 . Later on, with optical coherence tomography (OCT), IM has been used as an endogenous contrast to reveal the cellular and subcellular structures with freshly excised tissue. The uniqueness of the coherent gated method is that it can detect IM at different depths without requiring fluorescence tagging. Therefore, the imaged objects can stay at a more natural status.

Prenatal alcohol exposure (PAE) causes a spectrum of abnormalities, collectively termed as fetal alcohol spectrum disorders (FASD). The severity of the defect depends on the amount of alcohol consumed and the period of gestation during which alcohol was consumed. PAE during the second trimester is known to affect fetal brain development as this period of gestation marks the peak period for fetal neurogenesis and angiogenesis. Our previous study evaluated acute changes in fetal brain vasculature after PAE. However, not much has been done to assess concurrent changes on both the fetal and maternal side. This study uses correlation mapping optical coherence angiography (cm-OCA) to evaluate changes in vasculature in the fetal brain and the maternal hindlimb after maternal exposure to alcohol. Results showed drastic vasoconstriction in the fetal brain while vasodilation was seen in the mother, unlike results from the sham group, where there was no significant change in both the fetus and the mother. Changes seen in the fetal brain was similar to our previously published results.

Mueller matrix polarimetry has been applied to assist the diagnosis of several different types of diseases. The improvement of imaging resolution using objective with high numerical aperture (NA) is important for traditional optical microscope. However, imaging using a high NA objective entails a problem, namely, the field of view (FOV) is smaller and imaging speed is slower. Our previous work found that when using Mueller matrix microscope to obtain the structural features of tissue samples, some information of anisotropic structures, such as the density and orientation distribution of fibers can be revealed by polarization parameters images with relatively low resolution. In this study, we use objectives with different numerical aperture to measure the microscopic Mueller matrix of human healthy breast duct tissues and ductal carcinoma in situ (DCIS) tissues, which have distinct typical fibrous structures. Then a group of image texture feature parameters of Mueller matrix derived parameters images under high and low imaging resolutions are quantitatively compared. The results demonstrate that with the decline of imaging resolution, the fibers density information contained in the texture features of linear retardance δ parameter image are preserved well. While for the azimuthal orientation parameter θ which is closely related to the spatial location, the high imaging resolution to obtain quantitative structural information is still needed. The study provides an important criterion to decide which information of fibrous structures can be extracted accurately using transmission Mueller matrix microscope with low numerical aperture objectives to assist diagnose clinically such as breast ductal carcinoma.

In our work, we developed a method of THz solid immersion microscopy for continuous-wave reflection-mode imaging of soft biological tissues with a sub-wavelength spatial resolution. We have analyzed the performance of the developed THz solid immersion lens and demonstrated a 0.15λ-resolution of the proposed imaging modality at λ = 500 μm. We have applied the developed method for the THz imaging of various soft biological tissues and reconstructed refractive index distribution of the objects. The observed results justify capabilities of the proposed THz imaging modality in biophotonics.

Optical clearing (OC) increases the depth of light penetration and improves the outcomes of optical imaging measurements in situ and in vivo. In vivo OC protocols have to be biocompatible and should result in transient effects with minimal long-term damage to the tissues. The effects of various OC compositions in vivo were previously studied by using primarily optical imaging. Multimodality registration of optical and magnetic resonance imaging (MRI) signals in the same voxels of live tissue could be useful for improving accuracy of optical image reconstruction. We investigated OC effects on fluorescence intensity (FI) imaging of red fluorescent TagRFP protein marker in tumor cells and combined it with MRI. The OC effects of diamagnetic glycerol/DMSO/water and a paramagnetic magnetic resonance (MR) imaging agent (gadobutrol) and its mixtures were measured by using whole body FI, a single-photon counting FI setup and three MRI pulse sequences: 1) T2-weighted fast spin-echo; 2) diffusion-weighted and 2) 3D gradient-echo. A time-dependent increase of TagRFP FI resulted in tumor FI/skin ratio improvement at 15-30 min after OC. 0.7M solution of gadobutrol in DMSO/water was more efficient than 1.M gadobutrol (30-35% vs.15-20% increase of FI). The observed MRI signal intensity changes were most likely due to a combination of several effects, i.e. 1) longitudinal proton relaxation time shortening in subcutaneous tumor; 2) magnetic susceptibility effects of gadobutrol; 3) transient increase of T1w signal due to gadobutrol penetration through the skin and dilution in extracellular volume. The obtained results indicate that MRI can be instrumental in enabling mechanistic studies of OC effects in the skin and peripheral subcutaneous tissue.

We present an ultrafast laser surgery probe for bone tissue microsurgery. A custom miniaturized CaF₂ mitigated the strong multiphoton absorption observed in our previous ZnS-based design while providing tighter beam focusing over a larger ablation field-of-view. The objective produced a beam waist radius of 1.71 μm covering a 130×130 μm² scan area, delivering fluences >8 J/cm² at the tissue surface at 53% transmission efficiency. The entire opto-mechanical system, enclosed within a 14 mm diameter metal housing with a 2.6 mm probe tip, exhibited material removal rates >0.1 mm³/min in bovine cortical bone. We performed simulations when using a high-power fiber laser and found that material removal rates >40 mm³/min could be achieved through selection of optimal laser surgery parameters. The device can serve as a clinically viable solution for minimally invasive spinal surgery applications.

Complex decorrelation-based OCT angiography (OCTA) has the potential for quantitively monitoring hemodynamic activities. To improve the dynamic range and uncertainty for quantification, an adaptive spatial-temporal (ST) kernel was proposed. The ensemble size in decorrelation computation was enlarged by collecting samples in the spatial/ temporal dimensions. The spatial sub-kernel size was adaptively changed to suppress the bulk motion influence by solving a maximum entropy model. The improvement of dynamic range and uncertainty were validated by theoretical analyzation, numerical simulation, and in vitro/ in vivo experiments. Furthermore, proved by the in vivo experiments, the adaptive ST-kernel can also improve the separability between different stimuli and allow a reliable temporal analysis of the hemodynamic response.

The ability of diffuse correlation spectroscopy (DCS) to measure tissue perfusion paves the way for monitoring cerebral blood flow (CBF) non-invasively. However, during measurements on human forehead, the measured blood flow index (BFi) is susceptible to contamination due to the blood flow in extracerebral tissue. Time domain DCS addresses this problem by selecting photons based on their travel time to obtain BFi at various depths. We have determined the gate start time(s) and width(s) that can lead to optimal sensitivity of BFi to CBF during actual measurements on human subjects through simulations. The simulated parameters were compared with measurement data.

Photosynthetic single-celled diatom algae, due to their unique structure and properties, represent promising candidates for various applications in technology and biomedicine. These nanostructured objects, enveloped within a silica cell wall called a frustule, play a significant role in Earth’s ecology. In this study, we proposed new techniques for monitoring the growth of diatoms—in situ fluorescence measurements using the IVIS imaging system and photoacoustic measurements with a raster scanning optoacoustic mesoscopy (RSOM) setup. Two different diatom cultures, Achnanthidium sibiricum and Encyonema silesiacum, were cultivated under the optimal conditions in the incubator and monitored over the period of 70 days. Our results showed that the total radiant efficiency increases with increasing incubation time for E. silesiacum. Simultaneously, for A. sibiricum it slightly decreases after 56 days, indicating that diatoms were at the end of their exponential growth phase. The photoacoustic signal from E. silesiacum was lower than from A. sibiricum, which is in good agreement with spectroscopic characterization results. The IVIS imaging system made it possible to assess the growth and viability of diatom cells without compromising cell integrity. In contrast, photoacoustic imaging has proved to be suitable for the rapid detection and thorough in situ assessment of the density of diatom colonies due to the presence of light-absorbing chromophores. These methods can be used to monitor the growth of diatoms and facilitate the harvesting of bioactive substances derived from diatoms for pharmaceutical and biomedical purposes.

Photoacoustic imaging offers “x-ray vision” to see beyond tool tips and underneath tissue during surgical procedures, yet no ionizing x-rays are required. Instead, optical fibers and acoustic receivers enable photoacoustic sensing of major structures – like blood vessels and nerves – that are otherwise hidden from view. The process is initiated by delivering laser pulses through optical fibers to illuminate regions of interest, causing an acoustic response that is detectable with ultrasound transducers. Beamforming is then implemented to create a photoacoustic image. In this talk, I will highlight novel light delivery systems, new spatial coherence beamforming theory, deep learning alternatives to beamforming, and robotic integration methods, each pioneered by the Photoacoustic and Ultrasonic Systems Engineering (PULSE) Lab to enable an exciting new frontier of photoacoustic-guided surgery. This new paradigm has the potential to eliminate the occurrence of major complications (e.g., excessive bleeding, paralysis, accidental patient death) during a wide range of delicate surgeries and procedures, including neurosurgery, cardiac catheter-based interventions, liver surgery, spinal fusion surgery, hysterectomies, biopsies, and teleoperative robotic surgeries.

Diabetic foot is a well-known problem among patients suffering from peripheral arterial diseases (PAD). An optical sensor was built for a contactless measurement of the anatomical site, composing of a diode-laser and a high-speed camera. The laser illuminates the inspected tissue and the back reflected light forms time changing speckle patterns. We used second order autocorrelation function (ACF) decay time as merit for blood flow estimation. Clinical study with 15 subjects was conducted. An occlusion test was introduced to provoke a statistical parameter to distinguish between low perfused and a healthy foot.

Time-domain diffuse optics exploits near infrared light pulses diffused in turbid samples to retrieve their optical properties e.g., absorption and reduced scattering coefficients. Typically, interference effect are discarded, but speckle effects are exploited in other techniques e.g., diffuse correlation spectroscopy (DCS) to retrieve information regarding the tissue dynamics. Here, using a highly coherent Ti:Sapphire mode-locked laser and a single-mode detection fiber, we report the direct observation of temporal fluctuations in the measured distribution of time-of-flights (DTOF) curve. We study the dependence of these fluctuations on the sample dynamical properties (moving from fluid to rigid tissue-mimicking phantoms) and on the area of the detection fiber, which is directly linked to the number of collected coherence areas. Our observation agree with a time-resolved speckle pattern, and may enable the simultaneous monitoring of the tissue optical and dynamical properties.

Dynamic laser speckle imaging (DLSI) is an optical technique that directly measures the temporal speckle intensity fluctuations with sufficient temporal sampling to correctly model and predict the underlying blood flow changes. The speckle measurements are limited to superficial tissues and it lacks three-dimensional imaging of blood flow. To perform depth-resolved measurement, we developed Interferometric Dynamic Laser Speckle Imaging (iDLSI) capable of three-dimensional volumetric measurements of the dynamics. Here, we present the analytical expression for g²,iDLSI(τ) dependent on particle dynamics, coherence properties, sample, and reference intensity. The numerical validations are performed in a homogeneous and in spatially varying dynamic regions.

Cerebral blood flow is an important biomarker of brain health and function, as it regulates the delivery of oxygen and substrates to tissue and the removal of metabolic waste products. Diffuse Correlation Spectroscopy (DCS) is a promising noninvasive optical technique for monitoring cerebral blood flow and for measuring cortex functional activation tasks. However, the current state-of-the-art DCS adoption is hindered by a trade-off between sensitivity to the cortex and signal-to-noise ratio (SNR). Here we report on a multi-speckle DCS (mDCS) system based on a 1024-pixel single-photon avalanche diode (SPAD) camera that removes this trade-off and demonstrated a 32-fold increase in SNR with respect to traditional single-speckle DCS.

Speckle contrast optical spectroscopy/tomography (SCOS/SCOT) is a low-cost, non-invasive, and real-time optical imaging modality for measuring cerebral blood flow with increased signal-to-noise ratio relative to diffuse correlation spectroscopy. However, the recent camera-based detector system is not ideal for imaging a large area of the human brain because of the limited area of focus over the contour of the head and hair occluding the field of view. Here we demonstrated the feasibility of using inexpensive multi-mode fiber bundles to build a SCOS system for mapping the flow of fluids, and we showed a statistical method for distinguishing noise and speckle signals.

Laser Speckle Contrast Imaging (LSCI) measurements provide sufficient information on the changes in the tissue dynamics using spatial contrast measurements over an integration time. To allow the adoption of LSCI in humans, we propose a fiber-based LSCI system that has the potential to overcome free-space imaging of Speckle Contrast Optical Tomography (SCOT) while maintaining the high-speed imaging of LSCI. Here, we propose Dynamic Speckle Model (DSM) to develop the noise model for fiber-based LSCI (fb-LSCI) taking into account all the noise sources. We have identified operating parameter space i.e. small speckle to pixel ratio and long exposure time to minimise the impact of noise sources on the contrast measured. The performance of fb-LSCI is compared with other methods that measure changes in tissue dynamics such as DCS.

Diffuse correlation spectroscopy (DCS) is an established diffuse optical technique that uses the analysis of temporal speckle intensity fluctuations to measure blood flow in tissue. Recent advances in the field have seen the introduction of iDWS/iDCS, which have allowed for the use of conventional photodetectors to replace the single photon counting detectors required to measure the traditional, homodyne DCS signal. Here we detail a high framerate, highly parallel iDCS system at 1064 nm which allows for improved signal to noise ratio at extended source detector separations.

Optical coherence tomography (OCT) is a well-established modality for structural and functional imaging of the biological samples. Conventional scanning OCT combines the low temporal coherence with confocal gating to reject multiply scattered light. However, OCT uses a spatially coherent light source, and thus, is susceptible to speckle noise, which reduces the transverse resolution. We use dynamic light scattering to improve the transverse resolution. The dynamic scattering particles induce speckles, that change over time due to particle displacement. By incoherently averaging OCT images acquired under different particle distributions, we effectively suppress the spatial coherence and improve transverse image resolution.

We utilized time-domain diffuse correlation spectroscopy (TD-DCS) to quantify depth-resolved blood flow changes for in vivo experiments on arm and forehead adult humans. We illustrated that conventional TD-DCS processing is incapable of estimating blood flow changes at short source-detector separations, as expected. To tackle this problem, we introduced a novel model. We recovered the relative blood flow index of the forearm muscle during the cuff occlusion challenge and human forehead under variable pressure accurately.

We summarize and discuss multifunctional medical instruments based on sapphire shaped crystals. Such instruments are able to combine several modalities, such as interstitial exposure to laser radiation, fluorescent diagnosis, as well as tissue resection, aspiration and cryodestruction. Sapphire instruments enable biocomopatability, withstand extremely low and high temperatures, along with operation in harsh environment. Sapphire fibers and waveguides also allows imaging of biological objects in THz range. In visible and infrared ranges, these instruments can be combined with optical fibers via small internal channels, which can be produced during sapphire crystal growth by means of the edge-defined film-fed growth (EFG) technique. This talk covers the resent developments and experimental investigations of sapphire needles, cryoapplicators, scalpels, and fibers.

Laser-assisted relaxation of internal stresses can be used to stabilize the new shape of cartilaginous implants. The ability of cartilage tissue to experience irreversible deformation under certain conditions, as well as to restore its original configuration after external mechanical stress, is studied in near real time using a new method of optical coherent elastography (OCE), developed to visualize slow deformation. The OCE technique allows one to monitor the efficiency of laser-induced stress relaxation at 1–2 minute intervals using 2D subsurface strain mapping. It has been shown that the redistribution of the interstitial fluid due to the applied load is an important factor for the mechanism of "shape memory" of the cartilage. Deviations in the behavior of cartilage from the usual elastic properties of homogeneous materials, such as subsurface dilatation opposing the applied load, are identified and analyzed.

We developed a high-resolution multimodal system for mouse embryonic imaging that combines Optical Coherence Tomography (OCT) and Light Sheet Fluorescence Microscopy (LSFM). LSFM Illumination is restricted to fluorophores in the focal volume, and collecting the light using a microscope objective increases the signal from that plane and reduces the noise coming from outside of the plane. Colinearly aligning this modality with the OCT beam allows one to acquire the structural information from the same plane that is illuminated by the LSFM beam. A 3D image of 9.5 day mouse embryo was captured using this multimodal system.

The luminescence spectra of upconversion NaYF₄:Yb, Er nanoparticles located under layers of biological tissue of different thickness were experimentally obtained. Luminescence of the particles was excited by laser radiation with a wavelength of 980 nm through a layer of biological tissue. The radiation power was 0.5 W. Luminescence was collected using a collimator with an aperture of 0.15 located at a distance of 20 cm from the sample. The spectra were recorded using a QE 65000 spectrometer (Ocean Optics, US). As the tissue samples, layers of rat skin, muscle and adipose tissue were used. The thicknesses of the samples were varied in the range of 1-2 mm. Distortions of the luminescence spectra resulting from its attenuation in an absorbing-scattering sample are shown. The dependences of distortions on the type and thickness of the sample, as well as on the luminescence wavelength, are obtained. The results obtained are important in determining the temperature of nanoparticles from the luminescence spectra, since the distortions of the spectra lead to an error in determining the temperature.

This work was aimed at determining the optical characteristics of biological samples at different temperatures. The work investigated the change in the intensity and shape of the absorption spectra of various biological tissues in vitro, depending on the sample temperature.

In this study, polyethylene glycol (PEG) with a molecular weight of 6000 Daltons was used for optical clearing of human skin in vivo. This choice supposes skin optical clearing based on the tissue dehydration since penetration of large molecules into the skin is impossible. OCT tomograms were recorded using Spectral Radar OCT System OCP930SR 022 at the 930 nm before and during application of PEG solution on the skin surface of volunteers. The obtained data were used to calculate the time-dependence of the light attenuation coefficient in the skin. Then the characteristic time, degree, efficiency and rate of skin optical clearing were estimated. Also the time-dependence of skin probing depth was obtained.