Significance: Photothermal optical coherence tomography (PT-OCT) has the promise to offer structural images coregistered with chemical composition information, which can offer a significant impact in early detection of diseases such as atherosclerosis.

Aim: We take the first step in understanding the relation between PT-OCT signals and the endogenous tissue composition by considering the interplay between the opto-thermo-physical properties of tissue as a function of its lipid composition and the ensuing effects on the PT-OCT signals.

Approach: Multiparameter theoretical estimates for PT-OCT signal as a function of composition in a two-component lipid–water model are derived and discussed. Experimental data from various concentrations of lipid in the form of droplets and injections under bovine cardiac muscle align with theoretical predictions.

Results: Theoretical and experimental results suggest that the variations of heat capacity and mass density with tissue composition significantly contribute to the amount of optical path length difference measured by OCT phase.

Conclusion: PT-OCT has the potential to offer key insights into the chemical composition of the subsurface lipid pools in tissue; however, the interpretation of results needs to be carried out by keeping the nonlinear interplay between the tissue of opto-thermo-physical properties and PT-OCT signals in mind.

Significance: A new concept of a biotissue phantom for terahertz (THz) biomedical applications is needed for reliable and long-term usage.

Aim: We aimed to develop a new type of biotissue phantom without water content and with controllable THz optical properties by applying graphite powders into a polyvinyl chloride plastisol (PVCP) matrix and to give a numerical description to the THz optical properties of the phantoms using the Bruggeman model (BM) of the effective medium theory (EMT).

Approach: The THz optical properties of graphite and the PVCP matrix were measured using THz time-domain spectroscopy, which works in the frequency range from 0.1 to 1 THz. Two phantoms with 10% and 12.5% graphite were fabricated to evaluate the feasibility of describing phantoms using the EMT. The EMT then was used to determine the concentration of graphite required to mimic the THz optical properties of human cancerous and healthy oral tissue.

Results: The phantom with 16.7% of graphite has the similar THz optical properties as human cancerous oral tissue in the frequency range of 0.2 to 0.7 THz. The THz optical properties of the phantom with 21.9% of graphite are close to those of human healthy oral tissue in the bandwidth from 0.6 to 0.8 THz. Both the refractive index and absorption coefficient of the samples increase with an increase of graphite concentration. The BM of the EMT was used as the numerical model to describe the THz optical properties of the phantoms. The relative error of the BM for the refractive index estimation and the absorption coefficient is up to 4% and 8%, respectively.

Conclusions: A water-free biotissue phantom that mimics the THz optical properties of human cancerous oral tissue was developed. With 21.9% of graphite, the phantom also mimics human healthy oral tissue in a narrow frequency range. The BM proved to be a suitable numerical model of the phantom.

Significance: Reducing the bit depth is an effective approach to lower the cost of an optical coherence tomography (OCT) imaging device and increase the transmission efficiency in data acquisition and telemedicine. However, a low bit depth will lead to the degradation of the detection sensitivity, thus reducing the signal-to-noise ratio (SNR) of OCT images.

Aim: We propose using deep learning to reconstruct high SNR OCT images from low bit-depth acquisition.

Approach: The feasibility of our approach is evaluated by applying this approach to the quantized 3- to 8-bit data from native 12-bit interference fringes. We employ a pixel-to-pixel generative adversarial network (pix2pixGAN) architecture in the low-to-high bit-depth OCT image transition.

Results: Extensively, qualitative and quantitative results show our method could significantly improve the SNR of the low bit-depth OCT images. The adopted pix2pixGAN is superior to other possible deep learning and compressed sensing solutions.

Conclusions: Our work demonstrates that the proper integration of OCT and deep learning could benefit the development of healthcare in low-resource settings.

Significance: Differential interference contrast (DIC) microscopes allow noninvasive in vivo observation of transparent microstructures in tissue without the use of fluorescent dyes or genetic modification. We show how to modify a DIC microscope to measure the sample phase distribution accurately and in real-time even deep inside sample tissue.

Aim: Our aim is to improve the DIC microscope’s phase measurement to remove the phase bias that occurs in the presence of strong scattering.

Approach: A quarter-wave plate was added in front of the polarization camera, allowing a modified phase calculation to incorporate all four polarization orientation angles (0 deg, 45 deg, 90 deg, and 135 deg) captured simultaneously by the polarization camera, followed by deconvolution.

Results: We confirm that the proposed method reduces phase measurement error in the presence of scattering and demonstrate the method using in vivo imaging of a beating heart inside a medaka egg and the whole-body blood circulation in a young medaka fish.

Conclusions: Modifying a polarization-camera DIC microscope with a quarter-wave plate allows users to image deep inside samples without phase bias due to scattering effects.

Significance: Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We propose simultaneous multiplane DPC imaging using volume holographic microscopy (VHM).

Aim: To design and implement a new configuration of DPC-VHM for multiplane imaging.

Approach: The angularly multiplexed volume holographic gratings (AMVHGs) and the wavelength-coded volume holographic gratings (WC-VHGs) are used for this purpose. To obtain asymmetric illumination for DPC images, a dynamic illumination system is designed by modifying the regular Köhler illumination using a thin film transistor panel (TFT-panel).

Results: Multidepth DPC images of standard resolution chart and biosamples were used to compare imaging performance with the corresponding bright-field images. An average contrast enhancement of around three times is observed for target resolution chart by DPC-VHM. Imaging performance of our system is studied by modulation transfer function analysis, which suggests that DPC-VHM not only suppresses the DC component but also enhances high-frequency information.

Conclusions: Proposed DPC-VHM can acquire multidepth-resolved DPC images without axial scanning. The illumination part of the system is adjustable so that the system can be adapted to bright-field mode, phase contrast mode, and DPC mode by controlling the pattern on the TFT-panel.

Significance: Two-photon (2P) fluorescence imaging can provide background-free high-contrast images from the scattering tissues. However, obtaining a multiplane image is not straightforward. We present a two-photon volume holographic imaging (2P-VHI) system for multiplane imaging.

Aim: Our goal was to design and implement a 2P-VHI system that can provide the high-contrast optically sectioned images at multiple planes.

Approach: A 2P-VHI system is presented that incorporates angularly multiplexed volume holographic gratings and a femtosecond laser source for fluorescence excitation for multiplane imaging. A volume hologram with multiplexed gratings provides multifocal observation, whereas nonlinear excitation using a femtosecond laser helps in significantly enhancing both depth resolution and contrast of images.

Results: Standard fluorescent beads are used to demonstrate the imaging performance of the 2P-VHI system. Two-depth resolved optical-sectioning images of fluorescently labeled thick mice intestine samples were obtained. In addition, the optical sectioning capability of our system is measured and compared with that of a conventional VHI system.

Conclusions: Results demonstrated that 2P excitation in VHI systems provided the optical sectioning ability that helps in reducing background noise in the images. Integration of nonlinear fluorescence excitation in the VHI provides some unique advantages to the system and has potential to design multidepth optical sectioned spatial–spectral imaging systems.

Significance: Parallel phase-shifting digital holographic microscope (PPSDHM) is powerful for three-dimensional (3D) measurements of dynamic specimens. However, the PPSDHM reported previously was directly fixed on the optical bench and imposed difficulties case, thus it is required to modify the specification of the microscope or transport the microscope to another location.

Aim: We present a modularized PPSDHM. We construct the proposed PPSDHM and demonstrate the 3D measurement capability of the PPSDHM.

Approach: The PPSDHM was designed as an inverted microscope to record transparent objects and modularized by integrating the optical elements of the PPSDHM on an optical breadboard. To demonstrate the effectiveness of the PPSDHM, we recorded a 3D motion-picture of moving Volvoxes at 1000  frames  /  s and carried out 3D tracking of the Volvoxes.

Results: The PPSDHM was practically realized and 3D images of objects were successfully reconstructed from holograms recorded with a single-shot exposure. The 3D trajectories of Volvoxes were obtained from the reconstructed images.

Conclusions: We established a modularized PPSDHM that is capable of 3D image acquisition by integrating the optical elements of the PPSDHM on an optical breadboard. The recording capability of 3D motion-pictures of dynamic specimens was experimentally demonstrated by the PPSDHM.

Significance: A scene-based adaptive-optics (AO) system is developed and a method for investigating its imaging performance is proposed. The system enables derivation of Strehl ratios from observed images via collaboration with computer simulations. The resultant Strehl ratios are comparable with those of other current AO systems.

Aim: For versatile and noninvasive AO microscopy, a scene-based wavefront-sensing technique working on a Shack–Hartmann wavefront sensor is developed in a modal control system. The purpose of the research is to clarify the imaging performance of the AO system via the derivation of Strehl ratios from observed images toward applications in microscopy of living cells and tissues.

Approach: Two imaging metrics that can be directly measured from observed images (i.e., an energy concentration ratio and unbiased maximum ratio) are defined and related to the Strehl ratio via computer simulations. Experiments are conducted using artificial targets to measure the imaging metrics, which are then converted to Strehl ratios.

Results: The resultant Strehl ratios are >0.7 and 0.5 in the cases of defocus and higher aberrations, respectively. The half-widths at half-maximum of the AO-corrected bead images are favorably comparable to those of on-focus images under simple defocus aberration, and the AO system works both under bright-field illumination and on fluorescent bead images.

Conclusions: The proposed scene-based AO system is expected to work with a Strehl ratio of more than 0.5 when applied to high-resolution live imaging of cells and tissues under bright-field and fluorescence microscopies.

Significance: Due to patients’ respiratory movement or involuntary body movements during breast cancer radiotherapy, the mismatched adjacent fields in surface exposure regions could result in insufficient dosage or overdose in these regions, which would lead to tissue injury, excessive skin burns, and potential death. Cherenkov luminescence imaging (CLI) could be used to effectively detect the matching information of adjacent radiation fields without extra radiation or invasive imaging.

Aim: Our objective was to provide a biological experimental basis for monitoring matching of adjacent radiation fields between photon and electron fields due to introduced shifts during radiotherapy by CLI technique.

Approach: A medical accelerator was used to generate photon and electron fields. An industrial camera system was adopted to image the excited CLI signal during irradiation of chicken tissue with yellow (group A and group C experiments) or black color (group B experiment). The following introduced shifts were tested: 10, 5, 2, and 0 mm toward superior or inferior direction. A model was introduced to deal with matching error analysis of adjacent radiation fields due to introduced shifts with adapted plans used to treat neoplasms of the right breast with supraclavicular nodes or internal mammary lymph node.

Results: The matching values between photon and electron fields were consistent with the tested introduced shifts during yellow chicken irradiation. In group A, average discrepancies were 0.59  ±  0.35  mm and 0.68  ±  0.37  mm for photon fields and electron fields in anterior/posterior (AP) direction, with 87% and 75% of measurement within 1 mm, respectively. In group C, average discrepancies were 0.80  ±  0.65  mm and 1.07  ±  0.57  mm for oblique photon field with gantry angles of 330 deg and 150 deg, with 66% and 65% of measurement within 1 mm, respectively. The average discrepancies were 0.44  ±  0.30  mm for electron field in the AP direction, with 94% of measurement within 1 mm. The matching error introduced by the proposed method was less than 1.5 mm for AP fields and 2 mm for oblique incidence fields. However, the field matching could not be monitored with black chicken tissue irradiation due to a weak CLI signal that could hardly be extracted from background noise in group B.

Conclusions: CLI is demonstrated for the quantitative monitoring of the field match line on light biological tissue phantoms and has potential for monitoring of field matching in surface tissue during breast cancer radiotherapy.

Significance: Prenatal exposure to ethanol causes several morphological and neurobehavioral deficits. While there are some studies on the effects of ethanol exposure on blood flow, research focusing on acute changes in the microvasculature is limited.

Aim: The first aim of this study was to assess the dose-dependent changes in murine fetal brain microvasculature of developing fetuses in response to maternal alcohol consumption. The second aim was to quantify changes in vasculature occurring concurrently in the mother’s hindlimb and the fetus’s brain after maternal exposure to alcohol.

Approach: Correlation mapping optical coherence angiography was used to evaluate the effects of prenatal exposure to different doses of ethanol (3, 1.5, and 0.75  g  /  kg) on murine fetal brain vasculature in utero. Additionally, simultaneous imaging of maternal peripheral vessels and the fetal brain vasculature was performed to assess changes of the vasculature occurring concurrently in response to ethanol consumption.

Results: The fetal brain vessel diameters (VDs) decreased by ∼47  %  , 30%, and 14% in response to ethanol doses of 3, 1.5, and 0.75  g  /  kg, respectively. However, the mother’s hindlimb VD increased by 63% in response to ethanol at a dose of 3  g  /  kg.

Conclusions: Results showed a dose-dependent reduction in vascular blood flow in fetal brain vessels when the mother was exposed to ethanol, whereas vessels in the maternal hindlimb exhibited concurrent vasodilation.

Significance: The use of cancer-targeted contrast agents in fluorescence-guided surgery (FGS) has the potential to improve intraoperative visualization of tumors and surgical margins. However, evaluation of their translational potential is challenging.

Aim: We examined the utility of a somatostatin receptor subtype-2 (SSTR2)-targeted fluorescent agent in combination with a benchtop near-infrared fluorescence (NIRF) imaging system to visualize mouse xenografts under conditions that simulate the clinical FGS workflow for open surgical procedures.

Approach: The dual-labeled somatostatin analog, Ga67-MMC(IR800)-TOC, was injected into mice (n  =  24) implanted with SSTR2-expressing tumors and imaged with the customized OnLume NIRF imaging system (Madison, Wisconsin). In vivo and ex vivo imaging were performed under ambient light. The optimal dose (0.2, 0.5, and 2 nmol) and imaging time point (3, 24, 48, and 72 h) were determined using contrast-to-noise ratio (CNR) as the image quality parameter. Video captures of tumor resections were obtained to provide an FGS readout that is representative of clinical utility. Finally, a log-transformed linear regression model was fitted to assess congruence between fluorescence readouts and the underlying drug distribution.

Results: The drug–device combination provided high in vivo and ex vivo contrast (CNRs  >  3, except lung at 3 h) at all time points with the optimal dose of 2 nmol. The optimal imaging time point was 24-h post-injection, where CNRs  >  6.5 were achieved in tissues of interest (i.e., pancreas, small intestine, stomach, and lung). Intraoperative FGS showed excellent utility for examination of the tumor cavity pre- and post-resection. The relationship between fluorescence readouts and gamma counts was linear and strongly correlated (n  =  334, R²  =  0.71; r  =  0.84; P  <  0.0001).

Conclusion: The innovative OnLume NIRF imaging system enhanced the evaluation of Ga67-MMC(IR800)-TOC in tumor models. These components comprise a promising drug–device combination for FGS in patients with SSTR2-expressing tumors.

Significance: Evaluation of vessel patency and blood flow direction is important in various medical situations, including diagnosis and monitoring of ischemic diseases, and image-guided vascular surgeries. While optical coherence tomography angiography (OCTA) is the most widely used functional extension of optical coherence tomography that visualizes three-dimensional vasculature, inability to provide information of blood flow direction is one of its limitations.

Aim: We demonstrate two-dimensional (2D) transverse blood flow direction imaging in en face OCTA.

Approach: A series of triangular beam scans for the fast axis was implemented in the horizontal direction for the first volume scan and in the vertical direction for the following volume scan, and the inter A-line OCTA was performed for the blood flow direction imaging while the stepwise pattern was used for each slow axis scan. The decorrelation differences between the forward and the backward inter A-line OCTA were calculated for the horizontal and the vertical fast axis scans, and the ratio of the horizontal and the vertical decorrelation differences was utilized to show the 2D transverse flow direction information.

Results: OCTA flow direction imaging was verified using flow phantoms with various flow orientations and speeds, and we identified the flow speed range relative to the scan speed for reliable flow direction measurement. We demonstrated the visualization of 2D transverse blood flow orientations in mouse brain vascular networks in vivo.

Conclusions: The proposed OCTA imaging technique that provides information of 2D transverse flow direction can be utilized in various clinical applications and preclinical studies.

Significance: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles.

Aim: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell’s response to treatment.

Approach: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell’s membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Results: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells.

Conclusions: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance.

Significance: One key pathological characteristic of seronegative spondyloarthropathy (SpA) is inflammation at the insertion of tendons and ligaments into the bone (enthesitis).

Aim: We explore the potential of the emerging photoacoustic (PA) imaging in diagnosis of SpA and review its feasibility in detecting SpA-associated Achilles tendon enthesitis.

Approach: A light-emitting diode (LED)-based PA and ultrasound combined system was employed. The PA images, both along the long and the short axes of each Achilles tendon insertion region, were acquired at 850-nm wavelength, which is sensitive in depicting increased blood volume (i.e., hyperemia). To assess the hyperemia indicating enthesis inflammation, two parameters were quantified in the imaged tendons, including the average intensity and the density of the color pixels in the pseudo-color PA images. Ten SpA patients, all of which met Assessment of SpA International Society (ASAS) criteria for SpA and were found to have Achilles enthesitis by clinical exam according to a board-certified rheumatologist, were included in the study.

Results: The PA and Doppler ultrasound imaging of Achilles enthesitis resulting from these 10 SpA patients were compared to those from 10 healthy volunteers, leading to statistically significant differences (p  <  0.05) in the applied t-tests.

Conclusions: This preliminary clinical study suggests that the LED-based PA imaging holds a promise for sensitive and objective assessment of SpA enthesitis in an outpatient setting of the rheumatology clinic.

Significance: Re-excision rates for women with invasive breast cancer undergoing breast conserving surgery (or lumpectomy) have decreased in the past decade but remain substantial. This is mainly due to the inability to assess the entire surface of an excised lumpectomy specimen efficiently and accurately during surgery.

Aim: The goal of this study was to develop a deep-ultraviolet scanning fluorescence microscope (DUV-FSM) that can be used to accurately and rapidly detect cancer cells on the surface of excised breast tissue.

Approach: A DUV-FSM was used to image the surfaces of 47 (31 malignant and 16 normal/benign) fresh breast tissue samples stained in propidium iodide and eosin Y solutions. A set of fluorescence images were obtained from each sample using low magnification (4  ×  ) and fully automated scanning. The images were stitched to form a color image. Three nonmedical evaluators were trained to interpret and assess the fluorescence images. Nuclear–cytoplasm ratio (N/C) was calculated and used for tissue classification.

Results: DUV-FSM images a breast sample with subcellular resolution at a speed of 1.0  min  /  cm². Fluorescence images show excellent visual contrast in color, tissue texture, cell density, and shape between invasive carcinomas and their normal counterparts. Visual interpretation of fluorescence images by nonmedical evaluators was able to distinguish invasive carcinoma from normal samples with high sensitivity (97.62%) and specificity (92.86%). Using N/C alone was able to differentiate patch-level invasive carcinoma from normal breast tissues with reasonable sensitivity (81.5%) and specificity (78.5%).

Conclusions: DUV-FSM achieved a good balance between imaging speed and spatial resolution with excellent contrast, which allows either visual or quantitative detection of invasive cancer cells on the surfaces of a breast surgical specimen.

Significance: Processing and diagnosing a set of 12 prostate biopsies using conventional histology methods typically take at least one day. A rapid and accurate process performed while the patient is still on-site could significantly improve the patient’s quality of life.

Aim: We develop and assess the feasibility of a one-hour-to-diagnosis (1Hr2Dx) method for processing and providing a preliminary diagnosis of a set of 12 prostate biopsies.

Approach: We developed a fluorescence staining, optical clearing, and 3D open-top light-sheet microscopy workflow to enable 12 prostate needle core biopsies to be processed and diagnosed within an hour of receipt. We analyzed 44 biopsies by the 1Hr2Dx method, which does not consume tissue. The biopsies were then processed for routine, slide-based 2D histology. Three pathologists independently evaluated the 3D 1Hr2Dx and 2D slide-based datasets in a blinded, randomized fashion. Turnaround times were recorded, and the accuracy of our method was compared with gold-standard slide-based histology.

Results: The average turnaround time for tissue processing, imaging, and diagnosis was 44.5 min. The sensitivity and specificity of 1Hr2Dx in diagnosing cancer were both >90  %  .

Conclusions: The 1Hr2Dx method has the potential to improve patient care by providing an accurate preliminary diagnosis within an hour of biopsy.