Significance: Common-path interferometers have the advantage of producing ultrastable interferometric fringes compared with conventional interferometers, such as Michelson or Mach–Zehnder that are sensitive to environmental instabilities. Isolating interferometric measurements from mechanical disturbances is important in biodynamic imaging because Doppler spectroscopy of intracellular dynamics requires extreme stability for phase-sensitive interferometric detection to capture fluctuation frequencies down to 10 mHz.

Aim: The aim of this study was to demonstrate that Doppler spectra produced from a common-path interferometer using a grating and a spatial filter (SF) are comparable to, and more stable than, spectra from conventional biodynamic imaging.

Approach: A common-path interferometer using a holographic diffraction grating and an SF was employed with a low-coherence source. Simulations evaluated the spatial resolution. DLD-1 (human colon adenocarcinoma) spheroids were used as living target tissue samples. Power spectra under external vibrations and drug-response spectrograms were compared between common-path and Fourier-domain holographic systems.

Results: The common-path holography configuration shows enhanced interferometric stability against mechanical vibrations through common-mode rejection while maintaining sensitivity to Doppler frequency fluctuations caused by intracellular motions.

Conclusions: A common-path interferometer using a grating and an SF can provide enhanced interferometric stability in tissue-dynamics spectroscopy for drug screening assays.

Significance: The modern use of fluorescence in surgery came iteratively through new devices and pre-existing imaging agents, with indications that were paved via regulatory approvals and device clearances. These events led to a growing set of surgery subspecialty uses.

Aim: This article outlines the key milestones that initiated commercially marketed systems and agents by highlighting temporal sequences and strategic decisions between them, with the goal of helping to inform future successes.

Approach: A review of successful regulatory approvals and the sequences between them was completed for companies that achieved US Food and Drug Administration (FDA) premarket approval or new drug approvals (NDAs) or device clearances in the fields of fluorescent imaging agents, open surgery imaging devices, and their approved medical indications.

Results: Angiography agents, indocyanine green and fluorescein, were approved for human use as absorbing dyes, and this use in retinal imaging was the precursor to lateral translation into tissue perfusion imaging in the last two decades with a growing number of devices. Many FDA cleared devices for open fluorescence-guided surgery used the predicate created by the SPY SP2000 system. This first system was 510(k) cleared for angiography imaging with a unique split predicate from x-ray imaging of vasculature and retinal fluorescence angiography. Since that time, the lateral spread of open surgery devices being cleared for new indications has been occurring with a growth of adoption in surgical subspecialties. Growth into new surgical subspecialties has been achieved by leveraging different NDAs and clearances between indications, such that medical uses have broadened over time.

Conclusions: Key decisions made by developers to advance specific device clearances and NDAs have been based upon existing optical fluorescent agents. The historical lessons and regulatory trends in newer indications and contrast agents can help the field evolve via successful investment in new systems and applications.

Guest editors Toyohiko Yatagai, Osamu Matoba, Yoshihisa Aizu, Yasuhiro Awatsuji, and Yuan Luo introduce the articles in the Special Series on Biomedical Imaging and Sensing.

Significance: Untreated methemoglobinemia may cause severe hypoxemia and even death when methemoglobin levels in the blood stream exceed 70%. Although CO-oximetry can be used to monitor the response to treatment for methemoglobinemia, it is costly and requires an invasive procedure for collecting blood samples from patients. A pulse CO-oximeter with a contact probe can be used to continuously and non-invasively measure the percentage of methemoglobin, as well as the percutaneous oxygen saturation. In terms of the prevention of infectious diseases, however, it is desirable to monitor methemoglobin and oxygen saturation levels in a non-contact manner. Diffuse reflectance spectral imaging is promising as a non-contact, non-invasive, and cost-effective clinical diagnostic tool for methemoglobinemia.

Aim: To demonstrate the feasibility of visible spectral diffuse reflectance for in vivo monitoring of hemoglobin derivatives and evaluating methemoglobin production and reduction as well as hypoxemia during methemoglobinemia in rats.

Approach: A new imaging approach based on the multiple regression analysis aided by Monte Carlo simulations for light transport was developed to quantify methemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin using a hyperspectral imaging system. An in vivo experiment with rats exposed to sodium nitrite (NaNO₂) at different doses was performed to confirm the feasibility of the method for evaluating the dynamics of methemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin during methemoglobinemia. Systemic physiological parameters, including the percutaneous arterial oxygen saturation, heart rate (HR), and pulse distention, were measured by a commercially available pulse oximeter, and the results were compared to those obtained by the proposed method.

Results: Both the methemoglobin concentration and methemoglobin saturation rapidly increased with a half-maximum time of <20  min. They reached their maximal values nearly 60 min after the administration of NaNO₂. Tissue oxygen saturation dramatically dropped to a minimum of 33.7  %    ±  0.4  %  , 23.1  %    ±  5.6  %  , 8.8  %    ±  1.7  %  , and 9.7  %    ±  5.1  %   on average for NaNO₂ doses of 25, 37.5, 50, and 75 mg/kg, respectively. Changes in methemoglobin concentration and tissue oxygen saturation are indicative of the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Profound increases in the HR and pulse distention implied an elevated cardiac output caused by tachycardia and the resultant increase in peripheral blood volume to compensate for the hypoxia and hypoxemia during methemoglobinemia. This was in agreement with the time course of the peripheral hemoglobin volume concentration obtained by the proposed method.

Conclusions: The proposed method is capable of the in vivo non-contact simultaneous evaluation of methemoglobin levels and hypoxemia during methemoglobinemia, and that it has potential as a tool for the diagnosis and management of methemoglobinemia.

Significance: Management of skin cancer worldwide is often a challenge of scale, in that the number of potential cases presented outweighs the resources available to detect and treat skin cancer.

Aim: This project aims to develop a polarimetry probe to create an accessible skin cancer detection tool.

Approach: An optical probe was developed to perform bulk tissue Stokes polarimetry, a technique in which a laser of known polarization illuminates a target, and the altered polarization state of the backscattered light is measured. Typically, measuring a polarization state requires four sequential measurements with different orientations of polarization filters; however, this probe contains four spatially separated detectors to take four measurements in one shot. The probe was designed to perform at a lower cost and higher speed than conventional polarimetry methods. The probe uses photodiodes and linear and circular film polarizing filters as detectors, and a low-coherence laser diode as its illumination source. The probe design takes advantage of the statistical uniformity of the polarization speckle field formed at the detection area.

Results: Tests of each probe component, and the complete system put together, were performed to evaluate error and confirm the probe’s performance despite its low-cost components. This probe’s potential is demonstrated in a pilot clinical study on 71 skin lesions. The degree of polarization was found to be a factor by which malignant melanoma could be separated from other types of skin lesions.

Significance: Digital holographic microscopy (DHM) is a promising technique for the study of semitransparent biological specimen such as red blood cells (RBCs). It is important and meaningful to detect and count biological cells at the single cell level in biomedical images for biomarker discovery and disease diagnostics. However, the biological cell analysis based on phase information of images is inefficient due to the complexity of numerical phase reconstruction algorithm applied to raw hologram images. New cell study methods based on diffraction pattern directly are desirable.

Aim: Deep fully convolutional networks (FCNs) were developed on raw hologram images directly for high-throughput label-free cell detection and counting to assist the biological cell analysis in the future.

Approach: The raw diffraction patterns of RBCs were recorded by use of DHM. Ground-truth mask images were labeled based on phase images reconstructed from RBC holograms using numerical reconstruction algorithm. A deep FCN, which is UNet, was trained on the diffraction pattern images to achieve the label-free cell detection and counting.

Results: The implemented deep FCNs provide a promising way to high-throughput and label-free counting of RBCs with a counting accuracy of 99% at a throughput rate of greater than 288 cells per second and 200  μm  ×  200  μm field of view at the single cell level. Compared to convolutional neural networks, the FCNs can get much better results in terms of accuracy and throughput rate.

Conclusions: High-throughput label-free cell detection and counting were successfully achieved from diffraction patterns with deep FCNs. It is a promising approach for biological specimen analysis based on raw hologram directly.

Significance: Tumor detection and margin delineation are essential for successful tumor resection. However, postsurgical positive margin rates remain high for many cancers. Raman spectroscopy has shown promise as a highly accurate clinical spectroscopic diagnostic modality, but its margin delineation capabilities are severely limited by the need for pointwise application.

Aim: We aim to extend Raman spectroscopic diagnostics and develop a multimodal computer vision-based diagnostic system capable of both the detection and identification of suspicious lesions and the precise delineation of disease margins.

Approach: We first apply visual tracking of a Raman spectroscopic probe to achieve real-time tumor margin delineation. We then combine this system with protoporphyrin IX fluorescence imaging to achieve fluorescence-guided Raman spectroscopic margin delineation.

Results: Our system enables real-time Raman spectroscopic tumor margin delineation for both ex vivo human tumor biopsies and an in vivo tumor xenograft mouse model. We then further demonstrate that the addition of protoporphyrin IX fluorescence imaging enables fluorescence-guided Raman spectroscopic margin delineation in a tissue phantom model.

Conclusions: Our image-guided Raman spectroscopic probe-tracking system enables tumor margin delineation and is compatible with both white light and fluorescence image guidance, demonstrating the potential for our system to be developed toward clinical tumor resection surgeries.

Significance: Recovering accurate oxygenation estimations in the breast with quantitative photoacoustic tomography (QPAT) is not straightforward. Accurate light fluence models are required, but the unknown ground truth of the breast makes it difficult to validate them. Phantoms are often used for the validation, but most reported phantoms have a simple architecture. Fluence models developed in these simplistic objects are not accurate for application on the complex tissues of the breast.

Aim: We present a sophisticated breast phantom platform for photoacoustic (PA) and ultrasound (US) imaging in general, and specifically for QPAT. The breast phantom is semi-anthropomorphic in distribution of optical and acoustic properties and contains wall-less channels with blood.

Approach: 3D printing approaches are used to develop the solid 3D breast phantom from custom polyvinyl chloride plastisol formulations and additives for replicating the tissue optical and acoustic properties. A flow circuit was developed to flush the channels with bovine blood with a controlled oxygen saturation level. To showcase the phantom’s functionality, PA measurements were performed on the phantom with two oxygenation levels. Image reconstructions with and without fluence compensation from Monte Carlo simulations were analyzed for the accuracy of oxygen saturation estimations.

Results: We present design aspects of the phantom, demonstrate how it is developed, and present its breast-like appearance in PA and US imaging. The oxygen saturations were estimated in two regions of interest with and without using the fluence models. The fluence compensation positively influenced the SO₂ estimations in all cases and confirmed that highly accurate fluence models are required to minimize estimation errors.

Conclusions: This phantom allows studies to be performed in PA in carefully controlled laboratory settings to validate approaches to recover both qualitative and quantitative features sought after in in-vivo studies. We believe that testing with phantoms of this complexity can streamline the transition of new PA technologies from the laboratory to studies in the clinic.

Significance: The ability to detect and localize specific molecules through tissue is important for elucidating the molecular basis of disease and treatment. Unfortunately, most current molecular imaging tools in tissue either lack high spatial resolution (e.g., diffuse optical fluorescence tomography or positron emission tomography) or lack molecular sensitivity (e.g., micro-computed tomography, μCT). X-ray luminescence imaging emerged about 10 years ago to address this issue by combining the molecular sensitivity of optical probes with the high spatial resolution of x-ray imaging through tissue. In particular, x-ray luminescence computed tomography (XLCT) has been demonstrated as a powerful technique for the high-resolution imaging of deeply embedded contrast agents in three dimensions (3D) for small-animal imaging.

Aim: To facilitate the translation of XLCT for small-animal imaging, we have designed and built a small-animal dedicated focused x-ray luminescence tomography (FXLT) scanner with a μCT scanner, synthesized bright and biocompatible nanophosphors as contrast agents, and have developed a deep-learning-based reconstruction algorithm.

Approach: The proposed FXLT imaging system was designed using computer-aided design software and built according to specifications. NaGdF₄ nanophosphors doped with europium or terbium were synthesized with a silica shell for increased biocompatibility and functionalized with biotin. A deep-learning-based XLCT image reconstruction was also developed based on the residual neural network as a data synthesis method of projection views from few-view data to enhance the reconstructed image quality.

Results: We have built the FXLT scanner for small-animal imaging based on a rotational gantry. With all major imaging components mounted, the motor controlling the gantry can be used to rotate the system with a high accuracy. The synthesized nanophosphors displayed distinct x-ray luminescence emission, which enables multi-color imaging, and has successfully been bound to streptavidin-coated substrates. Lastly, numerical simulations using the proposed deep-learning-based reconstruction algorithm has demonstrated a clear enhancement in the reconstructed image quality.

Conclusions: The designed FXLT scanner, synthesized nanophosphors, and deep-learning-based reconstruction algorithm show great potential for the high-resolution molecular imaging of small animals.

Significance: Accessible tools are needed for rapid, non-destructive imaging of patient-derived cancer organoid (PCO) treatment response to accelerate drug discovery and streamline treatment planning for individual patients.

Aim: To segment and track individual PCOs with wide-field one-photon redox imaging to extract morphological and metabolic variables of treatment response.

Approach: Redox imaging of the endogenous fluorophores, nicotinamide dinucleotide (NADH), nicotinamide dinucleotide phosphate (NADPH), and flavin adenine dinucleotide (FAD), was used to monitor the metabolic state and morphology of PCOs. Redox imaging was performed on a wide-field one-photon epifluorescence microscope to evaluate drug response in two colorectal PCO lines. An automated image analysis framework was developed to track PCOs across multiple time points over 48 h. Variables quantified for each PCO captured metabolic and morphological response to drug treatment, including the optical redox ratio (ORR) and organoid area.

Results: The ORR (NAD(P)H/(FAD + NAD(P)H)) was independent of PCO morphology pretreatment. Drugs that induced cell death decreased the ORR and growth rate compared to control. Multivariate analysis of redox and morphology variables identified distinct PCO subpopulations. Single-organoid tracking improved sensitivity to drug treatment compared to pooled organoid analysis.

Conclusions: Wide-field one-photon redox imaging can monitor metabolic and morphological changes on a single organoid-level, providing an accessible, non-destructive tool to screen drugs in patient-matched samples.

Significance: We demonstrate the potential of probing the sO₂ change under blood flow in vivo using photoacoustic (PA) imaging and sheds light on the complex relationship between RBC aggregation and oxygen delivery.

Aim: To conduct in vivo assessments of the sO₂ in the radial artery of healthy volunteers and simultaneously probe the relation between the sO₂ and hemodynamic behavior such as red blood cell (RBC) aggregation.

Approach: The effects of PA-based measurements of blood hemodynamics were studied as a function of the subjects’ age (20s, 30s, and 40s). The pulsatile blood flow in the human radial artery of 12 healthy subjects was imaged in the 700 to 900 nm optical wavelength range using a linear array-based PA system.

Results: The PA power when blood velocity is minimum (Pa_max) was larger than the one attained at maximum blood velocity (Pa_min), consistent with predictions based on the cyclical variation of RBC aggregation during pulsatile flow. The difference between Pa_min and Pa_max at 800 nm (ΔPa₈₀₀) increased with age (1.7, 2.2, and 2.6 dB for age group of 20s, 30s, and 40s, respectively). The sO₂ computed from Pa_max was larger than the one from Pa_min.

Conclusions: The ΔPa₈₀₀ increased with participant age. The ΔPa₈₀₀ metric could be a surrogate of noninvasively monitoring the age-induced changes in RBC aggregation. The sO₂ change during a cycle of pulsatile blood flow also increased with age, demonstrating that RBC aggregation can affect the sO₂ change.

Significance: Frequency domain diffuse optical spectroscopy (FD-DOS) uses intensity modulated light to measure the absorption and reduced scattering coefficients of turbid media such as biological tissue. Some FD-DOS instruments utilize a single modulation frequency, whereas others use hundreds of frequencies. The effect of modulation frequency choice and measurement bandwidth on optical property (OP) extraction accuracy has not yet been fully characterized.

Aim: We aim to assess the effect of modulation frequency selection on OP extraction error and develop a high-speed look-up table (LUT) approach for OP estimation.

Approach: We first used noise-free simulations of light transport in homogeneous media to determine optimized iterative inversion model parameters and developed a new multi-frequency LUT method to increase the speed of inversion. We then used experimentally derived noise models for two FD-DOS instruments to generate realistic simulated data for a broad range of OPs and modulation frequencies to test OP extraction accuracy.

Results: We found that repeated measurements at a single low-frequency (110 MHz) yielded essentially identical OP errors as a broadband frequency sweep (35 evenly spaced frequencies between 50 and 253 MHz) for these noise models. The inclusion of modulation frequencies >300  MHz diminished overall performance for one of the instruments. Additionally, we developed a LUT inversion algorithm capable of increasing inversion speeds by up to 6  ×  , with 1000  inversions  /  s and ∼1  %   error when a single modulation frequency was used.

Conclusion: These results suggest that simpler single-frequency systems are likely sufficient for many applications and pave the way for a new generation of simpler digital FD-DOS systems capable of rapid, large-volume measurements with real-time feedback.

Significance: Intracranial pressure (ICP), variability in perfusion, and resulting ischemia are leading causes of secondary brain injury in patients treated in the neurointensive care unit. Continuous, accurate monitoring of cerebral blood flow (CBF) and ICP guide intervention and ultimately reduce morbidity and mortality. Currently, only invasive tools are used to monitor patients at high risk for intracranial hypertension.

Aim: Diffuse correlation spectroscopy (DCS), a noninvasive near-infrared optical technique, is emerging as a possible method for continuous monitoring of CBF and critical closing pressure (CrCP or zero-flow pressure), a parameter directly related to ICP.

Approach: We optimized DCS hardware and algorithms for the quantification of CrCP. Toward its clinical translation, we validated the DCS estimates of cerebral blood flow index (CBF_i) and CrCP in ischemic stroke patients with respect to simultaneously acquired transcranial Doppler ultrasound (TCD) cerebral blood flow velocity (CBFV) and CrCP.

Results: We found CrCP derived from DCS and TCD were highly linearly correlated (ipsilateral R²  =  0.77, p  =  9  ×  ^{10  −  7}; contralateral R²  =  0.83, p  =  7  ×  ^{10  −  8}). We found weaker correlations between CBF_i and CBFV (ipsilateral R²  =  0.25, p  =  0.03; contralateral R²  =  0.48, p  =  1  ×  10^−  3) probably due to the different vasculature measured.

Conclusion: Our results suggest DCS is a valid alternative to TCD for continuous monitoring of CrCP.

Significance: Large space-bandwidth product is highly desirable in many biomedical imaging. Fourier ptychographic microscopy (FPM) is a computational imaging technique that can significantly increase the space-bandwidth product of a standard microscope. The illuminator of a Fourier ptychographic microscope is not flexible at present, and it is inconvenient to meet different imaging needs.

Aim: An illuminator based on a two-axis motorized rotation stage was presented to provide a more flexible illuminating way with the goal of meeting different imaging needs.

Approach: The illuminator adopts a concentric illuminating method to provide coherent illumination in any direction on the sample plane. The sampling pattern can be freely designed and changed according to the parameters of the imaging system. A dither removing algorithm was proposed to remove the potential dither influence introduced in the image acquisition process.

Results: The illuminator could be conveniently integrated into different imaging systems. The feasibility and flexibility were demonstrated by applying it to imaging systems with numerical aperture of 0.045 and 0.01. The resolution gain is about 4- and 13-fold, respectively. The effectiveness of the dither removing algorithm was validated in both simulation and experiment.

Conclusions: A more flexible illuminator for FPM was presented to meet different imaging needs. A dither removing algorithm was proposed to remove dither influence.

Significance: Fourier ptychography (FP) is a computational imaging approach that achieves high-resolution reconstruction. Inspired by neural networks, many deep-learning-based methods are proposed to solve FP problems. However, the performance of FP still suffers from optical aberration, which needs to be considered.

Aim: We present a neural network model for FP reconstructions that can make proper estimation toward aberration and achieve artifact-free reconstruction.

Approach: Inspired by the iterative reconstruction of FP, we design a neural network model that mimics the forward imaging process of FP via TensorFlow. The sample and aberration are considered as learnable weights and optimized through back-propagation. Especially, we employ the Zernike terms instead of aberration to decrease the optimization freedom of pupil recovery and perform a high-accuracy estimation. Owing to the auto-differentiation capabilities of the neural network, we additionally utilize total variation regularization to improve the visual quality.

Results: We validate the performance of the reported method via both simulation and experiment. Our method exhibits higher robustness against sophisticated optical aberrations and achieves better image quality by reducing artifacts.

Conclusions: The forward neural network model can jointly recover the high-resolution sample and optical aberration in iterative FP reconstruction. We hope our method that can provide a neural-network perspective to solve iterative-based coherent or incoherent imaging problems.

Significance: Optoacoustic-induced vibrations of the hearing organ can potentially be used for a hearing device. To increase the efficiency of such a hearing device, the conversion of the light energy into vibration energy within each type of irradiated tissue has to be optimized.

Aim: To analyze the wavelength-dependency of optoacoustic-induced vibrations within the tympanic membrane (TM), and to define the most efficient and best-suited optical stimulation parameters for a novel auditory prosthesis.

Approach: Single nanosecond laser pulses, continuously tunable in a range of visible to near-infrared, were used to excite the guinea pig TM. The induced vibrations of the hearing organ were recorded at the malleus using a laser Doppler vibrometer.

Results: Our results indicate a strong wavelength-dependency of the vibration’s amplitude correlating with the superposition of the absorption spectra of the different specific tissue components.

Conclusions: We investigated the spectrum of the vibrations of the hearing organ that were induced optoacoustically within a biological membrane embedded in air, in an animal model. First applications for these results can be envisioned for the optical stimulation of the peripheral hearing organ as well as for research purposes.

Significance: Active implants require batteries as power supply. Their lifetime is limited and may require a second surgical intervention for replacement. Intracorporal energy harvesting techniques generate power within the body and supply the implant. Solar cells below the skin can be used to harvest energy from light.

Aim: To investigate the potential of subdermal solar energy harvesting.

Approach: We evaluated global radiation data for defined time slots and calculated the output power of a subdermal solar module based on skin and solar cell characteristics. We assumed solar exposure profiles based on daily habits for an implanted solar cell. The output power was calculated for skin types VI and I/II.

Results: We show that the yearly mean power in most locations on Earth is sufficient to power modern cardiac pacemakers if 10 min midday solar irradiation is assumed. All skin types are suitable for solar harvesting. Moreover, we provide a software tool to predict patient-specific output power.

Conclusions: Subdermal solar energy harvesting is a viable alternative to primary batteries. The comparison to a human case study showed a good agreement of the results. The developed code is available open source to enable researchers to investigate further applications of subdermal solar harvesting.