Dynamics and Fluctuations in Biomedical Photonics XVIII

Introduction to SPIE Photonics West BiOS Conference 11641: Dynamics and Fluctuations in Biomedical Photonics XVIII

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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&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.

Cancer progression is closely related to changes in the structure and mechanical properties of the tumor microenvironment-TME. In many solid tumors the interplay among TME components leads to a desmoplastic reaction due to collagen overproduction. In this study, pancreatic tumor models were developed and tissue biopsies were obtained at different stages of cancer progression. Polarized microscopy was used in order to assess collagen-based optical signatures, while Atomic Force Microscopy was applied for the nano-mechanical characterization of the samples. The results demonstrated that pancreatic cancer presents unique collagen-based signatures that could potentially be studied as novel biomarkers for response/prognosis.

Tissue polarimetry could be identified as a complementary optical and non-invasive technique to assist the gold standard histopathology analysis of tissue. In this manuscript, both healthy and malignant tissue zones of a thick formalin-fixed colon specimen were used for Mueller matrix measurements. Additionally, two more Mueller matrices from Monte Carlo simulation and tissue mimicking phantom were also evaluated, in order to assess polarimetric characterization and modeling of turbid media. Symmetric decomposition algorithm of Mueller matrices developed in house was adopted to extract both polarization and depolarization properties, encoded in the Mueller matrix elements. The decomposition products allowed to reveal important information about the internal tissue structure and morphology.

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.

Bladder urothelium is a seven-cell-layered lining of the bladder wall and serves as a barrier between urine and the underlying bladder tissue. Under OCT, the visibility of the urothelium in the healthy bladder relies on significant stretching of bladder tissue. We report the intracellular motion observed in porcine urothelium under OCT. Using speckle variance, we tracked the intracellular motion from OCT images of stretched and non-stretched tissue. We demonstrate that the boundary of the urothelium can be clearly delineated even the urothelium boundary is not visible in normal OCT images. This technology could be used for staging bladder cancer.

While prenatal exposure to alcohol is known to be detrimental to fetal development, there have been studies done to assess these changes in different aspects of fetal development. However, concurrent changes in both the fetus and the mother due to alcohol exposure have never been studied before. This study uses correlation mapping optical coherence angiography (cm-OCA) to simultaneously evaluate changes in both the fetal brain and the hindlimb of the mother after maternal exposure to alcohol. While a dramatic vasoconstriction was observed in the fetal brain, vasodilation was observed in the mother.

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.

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 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. The obtained results indicate that MRI is important modality 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 μm^2 scan area, delivering fluences >8 J/cm^2 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 mm^3/min in bovine cortical bone. We performed simulations when using a high-power fiber laser and found that material removal rates >40 mm^3/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.

Diatom algae, due to their unique structure and properties, represent promising candidates for many applications in technology and biomedicine. An IVIS fluorescence imaging system and raster scanning optoacoustic mesoscopy were used to monitor the growth of two diatom cultures in an incubator. The total radiant efficiency increases with increasing incubation time for E. sileciacum, while for A. sibiricum it slightly decreases after 56 days. The highest photoacoustic signal from A. sibiricum is reached after 30 days. These techniques expand the possibilities of diatom cultivation and allow complex experiments to be planned without compromising cell integrity.

Diabetic foot is a well-known problem among patients suffering from peripheral arterial diseases (PAD). An optical sensor was built for 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 a merit for a blood flow estimation. A 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 light scattering 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 Light Scattering 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.

Moderate laser-produced heating avoiding thermal damage of the tissue is a perspective technology for fabrication of cartilaginous auto-implants. The so-fabricated implants can be used for a variety of applications in otolaryngology and reconstructive surgery. For the implant shape stability control, monitoring and quantitative estimation of internal residual stresses in the tissue is of upmost importance. We realized non-contact monitoring of slow strains in cartilage using the recently developed phase-resolved OCT-elastography. 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 cartilage. Deviations of cartilage behavior from the elastic properties of homogeneous materials, such as subsurface dilatation opposing the applied load, are identified and analyzed.

The mammalian oviduct hosts the start of a new life, where a series of reproductive events take place for a successful pregnancy. Our understanding of such dynamic activities has been largely assumed based on in vitro and ex vivo studies, and little is known about the in vivo reproductive process inside the oviduct. We present in vivo imaging of the dynamic events in the mouse oviduct with optical coherence tomography. This imaging approach in the mouse model opens an exciting opportunity to study the normal and disordered reproductive dynamics in the native environment, promising for new findings in mammalian reproduction.

Sperm dynamics within female reproductive tract are largely a mystery due to restricted imaging access. Specifically, hyperactivation producing a change in sperm motility is not studied in its native state. Toward this problem, we established an innovative volumetric imaging approach, which allows tracking of individual sperm trajectories within mouse oviduct. The method is based on optical coherence tomography (OCT) imaging through intravital imaging window for access to female reproductive organs. We revealed a potential quantitative approach for differentiating sperm hyperactivation status in vitro and built a method which could be implemented to study the regulation of hyperactivation in vivo.

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 up-conversion NaYF4:Yb, Er nanoparticles located under layers of biological tissue of different thicknesses were experimentally obtained. As the tissue samples, layers of rat skin, muscle and adipose tissue were used. The distortion of the luminescence spectra resulting from its attenuation in an absorbing-scattering sample is 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 distortion of the spectra leads 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.

Polyethylene glycol (PEG) with a molecular weight of 6000 Daltons was used for optical clearing of human skin in vivo. 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.