Guest editors introduce the special section of Journal of Biomedical Optics Volume 25, Issue 3, entitled “Biomedical Imaging and Sensing II,” a collection of papers related to the topics of the conference “Biomedical Imaging and Sensing Conference 2019” (BISC’19), which was held in April 2019, in Yokohama, Japan.

Our work details the development and characterization of a portable luminescence imaging device for detecting inflammatory responses and infection in skin wounds. The device includes a CCD camera and close-up lens integrated into a customizable 3D printed imaging chamber to create a portable light-tight imager for luminescence imaging. The chamber has an adjustable light portal that permits ample ambient light for white light imaging. This imager was used to quantify in real time the extent of two-dimensional reactive oxygen species (ROS) activity distribution using a porcine wound infection model. The imager was used to successfully visualize ROS-associated luminescent activities in vitro and in vivo. Using a pig full-thickness cutaneous wound model, we further demonstrate that this portable imager can detect the change of ROS activities and their relationship with vasculature in the wound environment. Finally, by analyzing ROS intensity and distribution, an imaging method was developed to distinguish infected from uninfected wounds. We discovered a distinct ROS pattern between bacteria-infected and control wounds corresponding to the microvasculature. The results presented demonstrate that this portable luminescence imager is capable of imaging ROS activities in cutaneous wounds in a large animal model, indicating suitability for future clinical applications.

We present coregistered images of tissue vasculature that allow a direct comparison between the performance of narrow-band imaging (NBI) and optical coherence tomography angiography (OCTA). Images were generated with a bimodal endomicroscope having a size of 15  ×  2.4  ×  3.3  mm³   (  l  ,  w  ,  h  )   that combines two imaging channels. The white light imaging channel was used to perform NBI, the current gold standard for endoscopic visualization of vessels. The second channel allowed the simultaneous acquisition of optical coherence tomography (OCT) and OCTA images, enabling a three-dimensional (3-D) visualization of morphological as well as functional tissue information. In order to obtain 3-D OCT images scanning of the light-transmitting fiber was implemented by a small piezoelectric tube. A field of view of ∼1.1  mm was achieved for both modalities. Under the assumption that OCTA can address current limitations of NBI, their performance was studied and compared during in vivo experiments. The preliminary results show the potential of OCT regarding an improved visualization and localization of vessel beds, which can be beneficial for diagnosis of pathological conditions.

We propose a nonscanning three-dimensional (3-D) fluorescence imaging technique using the transport of intensity equation (TIE) and free-space Fresnel propagation. In this imaging technique, a phase distribution corresponding to defocused fluorescence images with a point-light-source-like shape is retrieved by a TIE-based phase retrieval algorithm. From the obtained phase distribution, and its corresponding amplitude distribution, of the defocused fluorescence image, various images at different distances can be reconstructed at the desired plane after Fresnel propagation of the complex wave function. Through the proposed imaging approach, the 3-D fluorescence imaging can be performed in multiple planes. The fluorescence intensity images are captured with the help of an electrically tunable lens; hence, the imaging technique is free from motion artifacts. We present experimental results corresponding to microbeads and a biological sample to demonstrate the proposed 3-D fluorescence imaging technique.

A fiber-based endoscopic imaging system combining narrowband red-green-blue (RGB) reflectance with optical coherence tomography (OCT) and autofluorescence imaging (AFI) has been developed. The system uses a submillimeter diameter rotary-pullback double-clad fiber imaging catheter for sample illumination and detection. The imaging capabilities of each modality are presented and demonstrated with images of a multicolored card, fingerprints, and tongue mucosa. Broadband imaging, which was done to compare with narrowband sources, revealed better contrast but worse color consistency compared with narrowband RGB reflectance. The measured resolution of the endoscopic system is 25  μm in both the rotary direction and the pullback direction. OCT can be performed simultaneously with either narrowband RGB reflectance imaging or AFI.

We introduced and validated a method to miniaturize graded-index (GRIN) fiber-based optical coherence tomography (OCT) probes down to 70  μm in diameter. The probes consist in an assembly of single-mode (SM), coreless (CL), and graded-index (GRIN) fibers. We opted for a probe design enabling controlled size reduction by hydrogen fluoride etching. The fabrication approach prevents nonuniform etching for both the GRIN and SM fiber components, while it requires no probe polishing postetching. We found that the miniaturized probes present insignificant loss of sensitivity (∼1  dB) compared to their thicker (125  μm) counterparts. We also showed that their focusing capabilities remain tunable and highly predictable. The fabrication process is simple and can be carried out by using inexpensive telecom equipment. Both the fabrication process and the developed probes can benefit the prototyping of minimally invasive endoscopic tools.

We report a small exploratory study of a methodology for real-time imaging of chemical and physical changes in spinal cords in the immediate aftermath of a localized contusive injury. One hundred separate experiments involving scanning NIR images, one-dimensional, two-dimensional (2-D), and point measurements, obtained in vivo, within a 3  ×  7  mm field, on spinal cords surgically exposed between T9 and T10 revealed differences between injured and healthy cords. The collected raw data, i.e., elastic and inelastic emission from the laser probed tissues, combined via the PV[O]H algorithm, allow construction of five images over the first 5 h post injury. Within the larger study, a total of 13 rats were studied using 2-D images, i.e., injured and control. A single 830-nm laser (100-μm diameter round spot) was spatially line-scanned across the cord to reveal photobleaching effects and surface profiles possibly locating a near surface longitudinal artery/vein. In separate experiments, the laser was scanned in two dimensions across the exposed cord surface relative to the injury in a specific pattern to avoid uneven photobleaching of the imaged tissue. The 2-D scanning produced elastic and inelastic emission that allowed construction of PV[O]H images that had good fidelity with the visually observed surfaces and separate line scans and suggested differences between the volume fractions of fluid and turbidity of injured and healthy cord tissue.

We present a system combining optical coherence tomography (OCT) and multispectral imaging (MSI) for coregistered structural imaging and surface color imaging. We first describe and numerically validate an optimization model to guide the selection of the MSI wavelengths and their relative intensities. We then demonstrate the integration of this model into an all-fiber bench-top system. We implement frequency-domain multiplexing for the MSI to enable concurrent acquisition of both OCT and MSI at OCT acquisition rates. Such a system could be implemented in endoscopic practices to provide multimodal, high-resolution imaging of deep organ structures that are currently inaccessible to standard video endoscopes.

Blood flow is a parameter used to diagnose vascular diseases based on flow speed, blood pressure, and vessel size. Different techniques have been developed to estimate the relative blood flow speed and to improve the visualization of deep blood vessels; one such technique is laser speckle contrast imaging (LSCI). LSCI images contain a high level of noise mainly when deep blood vessels are imaged. To improve their visualization, several approaches for contrast computation have been developed. However, there is a compromise between noise attenuation and temporal resolution. On the one hand, spatial approaches have low spatial resolution, high temporal resolution, and significant noise attenuation, while temporal approaches have the opposite. A recent approach combines a temporal base with a directional process that allows improving the visualization of blood vessels. Nevertheless, it still contains a high level of noise and requires a high number of raw frames for its base. We propose, a space-directional approach focused on improving noise attenuation and temporal resolution for contrast computation. The results of reference approaches and the proposed one are compared quantitatively. Moreover, it is shown that the visualization of blood vessels in LSCI images can be improved by a general morphological process when the noise level is reduced.

A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach–Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss Physcomitrella patens. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.

As solid-state laser technology continues to mature, high-energy lasers operating in the near-infrared (NIR) band have seen increased utilization in manufacturing, medical, and military applications. Formulations of maximum permissible exposure limits establish guidelines for the safe use of these systems for a given set of laser parameters, based on past experimental and analytical studies of exposure thresholds causing injury to the skin and eyes. The purpose of our study is to characterize the skin response to multiple-pulsed laser exposures at the NIR wavelength of 1070 nm, at a constant beam diameter of 1 cm, using anesthetized Yucatan mini-pig subjects. Our study explores three constant total laser-on times of 0.01, 0.1, and 10 s as single- and multiple-pulse sequences. Exposures consisting of 10, 30, and 100 pulses have identical individual pulse durations but different duty cycles in order to include variable degrees of thermal additivity. A plurality of three observers quantifies skin damage with the minimally visible lesion metric, judged at the 1- and 24-h intervals postexposure. Calculation of the median effective dose (ED₅₀) provides injury thresholds for all exposure conditions, based on varying laser power across subjects. The results of this study will provide a quantitative basis for the incorporation of multiple-pulsed laser exposure into standards and augment data contained in the existing ED₅₀ database.

Significance: Many studies in colorectal cancer (CRC) use murine ectopic tumor models to determine response to treatment. However, these models do not replicate the tumor microenvironment of CRC. Physiological information of treatment response derived via diffuse reflectance spectroscopy (DRS) from murine primary CRC tumors provide a better understanding for the development of new drugs and dosing strategies in CRC.

Aim: Tumor response to chemotherapy in a primary CRC model was quantified via DRS to extract total hemoglobin content (tHb), oxygen saturation (StO₂), oxyhemoglobin, and deoxyhemoglobin in tissue.

Approach: A multimodal DRS and imaging probe (0.78 mm outside diameter) was designed and validated to acquire diffuse spectra longitudinally—via endoscopic guidance—in developing colon tumors under 5-fluoruracil (5-FU) maximum-tolerated (MTD) and metronomic regimens. A filtering algorithm was developed to compensate for positional uncertainty in DRS measurements

Results: A maximum increase in StO₂ was observed in both MTD and metronomic chemotherapy-treated murine primary CRC tumors at week 4 of neoadjuvant chemotherapy, with 21  ±  6  %   and 17  ±  6  %   fold changes, respectively. No significant changes were observed in tHb.

Conclusion: Our study demonstrates the feasibility of DRS to quantify response to treatment in primary CRC models.

Laser energy sealing systems have attracted much attention over the past decade given the general shift in surgical paradigm toward less invasive surgical approaches. Given this, it is paramount to have an objective method with which the quality of energy seals can be evaluated. Current methodologies used for this purpose can be problematic in the evaluation of small vessel seals. A methodology employing Doppler optical coherence tomography (DOCT) for the evaluation of energy seals is introduced. Avian chorioallantoic membrane vessels were subjected to thulium laser irradiation and were then scanned via OCT. Outcomes were classified based on several markers, predominantly the presence or absence of flow postirradiation. Vessel diameter and general morphology were also taken into consideration. Vessels were classified into four groups: seal (29%), rupture (30%), partial seal (19%), and unaffected (22%). All vessels were also evaluated visually by a trained neurovascular surgeon, and these visually classified outcomes were compared with DOCT evaluated outcomes. It was found that whether the vessel was considered sealed or not sealed was dependent on the evaluation method (p  =  0.01) where visual classification resulted in 18% more seals than DOCT classification. Further, the specificity of visual classification was found to be strongly dependent on the number of partial seals (p  <  0.0001). DOCT has shown to be an indispensable method for the evaluation of energy seals not only solely due to its high velocity resolution but also due to valuable microscopic morphological insight regarding the biological mechanisms responsible for energy sealing.

Significance: Shear wave optical coherence elastography is an emerging technique for characterizing tissue biomechanics that relies on the generation of elastic waves to obtain the mechanical contrast. Various techniques, such as contact, acoustic, and pneumatic methods, have been used to induce elastic waves. However, the lack of higher-frequency components within the elastic wave restricts their use in thin samples. The methods also require moving parts and/or tubing, which therefore limits the extent to which they can be miniaturized.

Aim: To overcome these limitations, we propose an all-optical approach using photothermal excitation. Depending on the absorption coefficient of the sample and the laser pulse energy, elastic waves are generated either through a thermoelastic or an ablative process. Our study aimed to experimentally determine the boundary between the thermoelastic and the ablative regimes for safe all-optical elastography applications.

Approach: Tissue-mimicking graphite-doped phantoms and chicken liver samples were used to investigate the boundary between thermoelastic and ablative regimes. A pulsed laser at 532 nm was used to induce elastic waves in the samples. Laser-induced elastic waves were detected using a line field low coherence holography instrument. The shape of the elastic wave amplitude was analyzed and used to determine the transition point between thermoelastic and ablative regimes.

Results: The transition from the thermoelastic to the ablative regime is accompanied by the nonlinear increase in surface wave amplitude as well as the transformation of the wave shape. Correlation between the absorption coefficient and the transition point energy was experimentally determined using graphite-doped phantoms and applied to biological samples ex vivo.

Conclusions: Our study described a methodology for determining the boundary region between thermoelastic and ablative regimes of elastic wave generation. These can be used for the development of a safe method for completely noncontact, all-optical microscale assessment of tissue biomechanics using laser-induced elastic waves.

We demonstrate dual modality of free-space fluorescence diffuse optical tomography (FDOT) and handheld ultrasound (US) imaging to reveal both functional and structural information in small animals. FDOT is a noninvasive method for examining the fluorophore inside an object from the light distribution of the surface. In FDOT, a 660-nm continuous wave diode laser was used as an excitation source and an electron-multiplying charge-coupled device (EMCCD) was used for fluorescence data acquisition. Both the laser and EMCCD were mounted on a 360-deg rotation gantry for the transmission optical data collection. The structural information is obtained from a 6- to 17-MHz handheld US linear transducer by single-side access and conducts in the reconstruction as soft priors. The rotation ranges from 0 deg to 360 deg; different rotation degrees, object positions, and parameters were determined for comparison. Both phantom and tissue phantom results demonstrate that fluorophore distribution can be recovered accurately and quantitatively using this imaging system. Finally, an animal study confirms that the system can extract a dual-modality image, validating its feasibility for further in vivo experiments. In all experiments, the error and standard deviation decrease as the rotation degree is increased and the error was reduced to 10% when the rotation degree was increased over 135 deg.

Significance: Detection and characterization of circulating tumor cells (CTCs), a key determinant of metastasis, are critical for determining risk of disease progression, understanding metastatic pathways, and facilitating early clinical intervention.

Aim: We aim to demonstrate label-free imaging of suspected melanoma CTCs.

Approach: We use a linear-array-based photoacoustic tomography system (LA-PAT) to detect melanoma CTCs, quantify their contrast-to-noise ratios (CNRs), and measure their flow velocities in most of the superficial veins in humans.

Results: With LA-PAT, we successfully imaged suspected melanoma CTCs in patients in vivo, with a CNR >9. CTCs were detected in 3 of 16 patients with stage III or IV melanoma. Among the three CTC-positive patients, two had disease progression; among the 13 CTC-negative patients, 4 showed disease progression.

Conclusions: We suggest that LA-PAT can detect suspected melanoma CTCs in patients in vivo and has potential clinical applications for disease monitoring in melanoma.

Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology.

Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light.

Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature.

Results: A specific combination of wavelengths at low irradiances (250 to 400  mW  /  cm² for 1064 nm and 55 to 65  mW  /  cm² for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes.

Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.

Significance: The large background, narrow dynamic range, and detector saturation have been the common limiting factors in stimulated emission (SE)-based pump-probe microscopy, attributed to the very small signal overriding the very intense laser probe beam. To better differentiate the signal of interest from the background, lock-in detection is used to measure the fluorescence quenching, which is termed spontaneous loss (SL). The advantages are manifold. The spontaneous fluorescence signal can be well separated from both the pump and the probe beams with filters, thus eliminating the background, enlarging the dynamic range, and avoiding the saturation of the detector.

Aim: We propose and demonstrate an integrated pump-probe microscopy technique based on lock-in detection for background removal and dynamic range enhancement through SL detection.

Approach: The experimental setup is configured with a pulsed diode laser at a wavelength λ_pu  =  635  nm, acting as a pump (excitation) and a mode-locked Ti:sapphire laser at a central wavelength λ_pr  =  780  nm, serving as the probe beam (stimulation). Both pulse trains are temporally synchronized through high precision delay control by adjusting the length of the triggering cables. The pump and probe beams are alternatively modulated at different frequencies f₁ and f₂ to extract the stimulated gain (SG) and SL signal.

Results: SG signal shows saturation due to the irradiation of the intense probe beam onto the photodetector. However, the detector saturation does not occur at high probe beam power for SL detection. The fluorescence lifetime images are acquired with reduced background. The theoretical signal-to-noise ratios for SG and SL are also estimated by photon statistics.

Conclusion: We have confirmed that the detection of SL allows the elimination of the large background without photodetector saturation, which commonly exists in SG configuration. This modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

Significance: Glioblastoma multiforme (GBM) is the most frequently diagnosed adult primary brain malignancy with poor patient prognosis. GBM can recur despite aggressive treatment due to therapeutically resistant glioblastoma stem cells (GSCs) that may exhibit metabolic plasticity.

Aim: Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence can be acquired with fluorescence lifetime imaging microscopy (FLIM) to examine its bound and free metabolic states in GSC and GBM tissues.

Approach: We compared the mean NADH fluorescence lifetime in live human GSCs and normal neural stem cells and validated those results by measuring oxygen consumption rates (OCRs). We also examined the role that invasive versus less-invasive GSCs had on tumor metabolism by measuring the mean NADH lifetimes and the relative amount of the longer-lived component of NADH and correlated these results with survival in an orthotopic mouse xenograft model.

Results: Mean NADH lifetime, amount of bound NADH, and OCR were increased in GSCs. Compared with normal mouse brain, mean NADH lifetimes were longer for all GBM tissues. Invasive xenografts had higher relative amounts of the longer-lived NADH component, and this correlated with decreased survival.

Conclusions: FLIM offers cellular resolution quantification of metabolic flux in GBM phenotypes, potentially informing biomedical researchers on improved therapeutic approaches.