Thanks to reviewers who served JBO in 2020.

Significance: Surgical microscopes provide adjustable magnification, bright illumination, and clear visualization of the surgical field and have been increasingly used in operating rooms. State-of-the-art surgical microscopes are integrated with various imaging modalities, such as optical coherence tomography (OCT), fluorescence imaging, and augmented reality (AR) for image-guided surgery.

Aim: This comprehensive review is based on the literature of over 500 papers that cover the technology development and applications of surgical microscopy over the past century. The aim of this review is threefold: (i) providing a comprehensive technical overview of surgical microscopes, (ii) providing critical references for microscope selection and system development, and (iii) providing an overview of various medical applications.

Approach: More than 500 references were collected and reviewed. A timeline of important milestones during the evolution of surgical microscope is provided in this study. An in-depth technical overview of the optical system, mechanical system, illumination, visualization, and integration with advanced imaging modalities is provided. Various medical applications of surgical microscopes in neurosurgery and spine surgery, ophthalmic surgery, ear-nose-throat (ENT) surgery, endodontics, and plastic and reconstructive surgery are described.

Results: Surgical microscopy has been significantly advanced in the technical aspects of high-end optics, bright and shadow-free illumination, stable and flexible mechanical design, and versatile visualization. New imaging modalities, such as hyperspectral imaging, OCT, fluorescence imaging, photoacoustic microscopy, and laser speckle contrast imaging, are being integrated with surgical microscopes. Advanced visualization and AR are being added to surgical microscopes as new features that are changing clinical practices in the operating room.

Conclusions: The combination of new imaging technologies and surgical microscopy will enable surgeons to perform challenging procedures and improve surgical outcomes. With advanced visualization and improved ergonomics, the surgical microscope has become a powerful tool in neurosurgery, spinal, ENT, ophthalmic, plastic and reconstructive surgeries.

Significance: Deep-tissue penetration by x-rays to induce optical responses of specific molecular reporters is a new way to sense and image features of tissue function in vivo. Advances in this field are emerging, as biocompatible probes are invented along with innovations in how to optimally utilize x-ray sources.

Aim: A comprehensive review is provided of the many tools and techniques developed for x-ray-induced optical molecular sensing, covering topics ranging from foundations of x-ray fluorescence imaging and x-ray tomography to the adaptation of these methods for sensing and imaging in vivo.

Approach: The ways in which x-rays can interact with molecules and lead to their optical luminescence are reviewed, including temporal methods based on gated acquisition and multipoint scanning for improved lateral or axial resolution.

Results: While some known probes can generate light upon x-ray scintillation, there has been an emergent recognition that excitation of molecular probes by x-ray-induced Cherenkov light is also possible. Emission of Cherenkov radiation requires a threshold energy of x-rays in the high kV or MV range, but has the advantage of being able to excite a broad range of optical molecular probes. In comparison, most scintillating agents are more readily activated by lower keV x-ray energies but are composed of crystalline inorganic constituents, although some organic biocompatible agents have been designed as well. Methods to create high-resolution structured x-ray-optical images are now available, based upon unique scanning approaches and/or a priori knowledge of the scanned x-ray beam geometry. Further improvements in spatial resolution can be achieved by careful system design and algorithm optimization. Current applications of these hybrid x-ray-optical approaches include imaging of tissue oxygenation and pH as well as of certain fluorescent proteins.

Conclusions: Discovery of x-ray-excited reporters combined with optimized x-ray scan sequences can improve imaging resolution and sensitivity.

Significance: There is an essential need to develop wearable multimodality technologies that can continuously measure both blood flow and oxygenation in deep tissues to investigate and manage various vascular/cellular diseases.

Aim: To develop a wearable dual-wavelength diffuse speckle contrast flow oximetry (DSCFO) for simultaneous measurements of blood flow and oxygenation variations in deep tissues.

Approach: A wearable fiber-free DSCFO probe was fabricated using 3D printing to confine two small near-infrared laser diodes and a tiny CMOS camera in positions for DSCFO measurements. The spatial diffuse speckle contrast and light intensity measurements at the two different wavelengths enable quantification of tissue blood flow and oxygenation, respectively. The DSCFO was first calibrated using tissue phantoms and then tested in adult forearms during artery cuff occlusion.

Results: Phantom tests determined the largest effective source–detector distance (15 mm) and optimal camera exposure time (10 ms) and verified the accuracy of DSCFO in measuring absorption coefficient variations. The DSCFO detected substantial changes in forearm blood flow and oxygenation resulting from the artery occlusion, which meet physiological expectations and are consistent with previous study results.

Conclusions: The wearable DSCFO may be used for continuous and simultaneous monitoring of blood flow and oxygenation variations in freely behaving subjects.

Significance: Oxygenation is one of the skin tissue physiological properties to follow for patient care management. Furthermore, long-term monitoring of such parameters is needed at the patient bed as well as outside the hospital. Diffuse reflectance spectroscopy has been widely used for this purpose.

Aim: The aim of the study is to propose a low-cost system for the long-term measurement of skin physiological parameters in contact.

Approach: We have developed a low-cost, wearable, CMOS-based device. We propose an original method for processing diffuse reflectance data to calculate the tissue oxygen saturation (StO₂).

Results: We tested the device for the assessment of tissue oxygenation during a first-in-human clinical trial that took place at the Grenoble University Hospital France.

Conclusions: The results of this clinical trial show a good accordance between our sensor and commercial devices used a reference.

Significance: Colorectal cancer incidence has decreased largely due to detection and removal of polyps. Computer-aided diagnosis development may improve on polyp detection and discrimination.

Aim: To advance detection and discrimination using currently available commercial colonoscopy systems, we developed a deep neural network (DNN) separating the color channels from images acquired under narrow-band imaging (NBI) and white-light endoscopy (WLE).

Approach: Images of normal colon mucosa and polyps from colonoscopies were studied. Each color image was extracted based on the color channel: red/green/blue. A multilayer DNN was trained using one-channel, two-channel, and full-color images. The trained DNN was then tested for performance in detection of polyps.

Results: The DNN performed better using full-colored NBI over WLE images in the detection of polyps. Furthermore, the DNN performed better using the two-channel red + green images when compared to full-color WLE images.

Conclusions: The separation of color channels from full-color NBI and WLE images taken from commercially available colonoscopes may improve the ability of the DNN to detect and discriminate polyps. Further studies are needed to better determine the color channels and combination of channels to include and exclude in DNN development for clinical use.

Significance: Adaptive optics optical coherence tomography (AO-OCT) technology enables non-invasive, high-resolution three-dimensional (3D) imaging of the retina and promises earlier detection of ocular disease. However, AO-OCT data are corrupted by eye-movement artifacts that must be removed in post-processing, a process rendered time-consuming by the immense quantity of data.

Aim: To efficiently remove eye-movement artifacts at the level of individual A-lines, including those present in any individual reference volume.

Approach: We developed a registration method that cascades (1) a 3D B-scan registration algorithm with (2) a global A-line registration algorithm for correcting torsional eye movements and image scaling and generating global motion-free coordinates. The first algorithm corrects 3D translational eye movements to a single reference volume, accelerated using parallel computing. The second algorithm combines outputs of multiple runs of the first algorithm using different reference volumes followed by an affine transformation, permitting registration of all images to a global coordinate system at the level of individual A-lines.

Results: The 3D B-scan algorithm estimates and corrects 3D translational motions with high registration accuracy and robustness, even for volumes containing microsaccades. Averaging registered volumes improves our image quality metrics up to 22 dB. Implementation in CUDA™ on a graphics processing unit registers a 512  ×  512  ×  512 volume in only 10.6 s, 150 times faster than MATLAB™ on a central processing unit. The global A-line algorithm minimizes image distortion, improves regularity of the cone photoreceptor mosaic, and supports enhanced visualization of low-contrast retinal cellular features. Averaging registered volumes improves our image quality up to 9.4 dB. It also permits extending the imaging field of view (∼2.1  ×  ) and depth of focus (∼5.6  ×  ) beyond what is attainable with single-reference registration.

Conclusions: We can efficiently correct eye motion in all 3D at the level of individual A-lines using a global coordinate system.

Significance: The study has confirmed the feasibility of using ultraviolet (UV) excitation to visualize and quantify desmoplasia in fresh tumor tissue of pancreatic adenocarcinoma (PDAC) in an orthotopic xenograft mouse model, which provides a useful imaging platform to evaluate acute therapeutic responses.

Aim: Stromal network of collagen prominent in PDAC tumors is examined by imaging fresh tissue samples stained with histological dyes. Fluorescence signals are color-transferred to mimic Masson’s trichrome staining.

Approach: Murine tumor samples were stained with Hoechst, eosin, and rhodamine B and excited at 275-nm. Fluorescence signals in the visible spectrum were captured by a CMOS color camera with high contrast and resolution at whole-tumor slice field of view.

Results: Fluorescence imaging using UV excitation is capable of visualizing collagen deposition in PDAC tumors. Both fluorescence and histology data showed collagen content of up to 30%. The collagen modulation effect due to photodynamic priming treatment was observed showing 13% of collagen reduction. Necrosis area is visible and perfusion imaging using Texas Red dextran is feasible.

Conclusions: The study demonstrates collagen visualization in fresh PDAC tumor samples using UV excitation. This imaging platform also provides quantitative stromal information from fiber analysis and visibility of necrosis and perfusion, suitable for therapeutic response assessment of photodynamic therapy.

Significance: Assessment of disease using optical coherence tomography is an actively investigated problem, owing to many unresolved challenges in early disease detection, diagnosis, and treatment response monitoring. The early manifestation of disease or precancer is typically associated with subtle alterations in the tissue dielectric and ultrastructural morphology. In addition, biological tissue is known to have ultrastructural multifractality.

Aim: Detection and characterization of nanosensitive structural morphology and multifractality in the tissue submicron structure. Quantification of nanosensitive multifractality and its alteration in progression of tumor.

Approach: We have developed a label free nanosensitive multifractal detrended fluctuation analysis(nsMFDFA) technique in combination with multifractal analysis and nanosensitive optical coherence tomography (nsOCT). The proposed method deployed for extraction and quantification of nanosensitive multifractal parameters in mammary fat pad (MFP).

Results: Initially, the nsOCT approach is numerically validated on synthetic submicron axial structures. The nsOCT technique was applied to pathologically characterized MFP of murine breast tissue to extract depth-resolved nanosensitive submicron structures. Subsequently, two-dimensional MFDFA were deployed on submicron structural en face images to extract nanosensitive tissue multifractality. We found that nanosensitive multifractality increases in transition from healthy to tumor.

Conclusions: This method for extraction of nanosensitive tissue multifractality promises to provide a noninvasive diagnostic tool for early disease detection and monitoring treatment response. The novel ability to delineate the dominant submicron scale nanosensitive multifractal properties may also prove useful for characterizing a wide variety of complex scattering media of non-biological origin.

Significance: Line scanning-based temporal focusing multiphoton microscopy (TFMPM) has superior axial excitation confinement (AEC) compared to conventional widefield TFMPM, but the frame rate is limited due to the limitation of the single line-to-line scanning mechanism. The development of the multiline scanning-based TFMPM requires only eight multiline patterns for full-field uniform multiphoton excitation and it still maintains superior AEC.

Aim: The optimized parallel multiline scanning TFMPM is developed, and the performance is verified with theoretical simulation. The system provides a sharp AEC equivalent to the line scanning-based TFMPM, but fewer scans are required.

Approach: A digital micromirror device is integrated in the TFMPM system and generates the multiline pattern for excitation. Based on the result of single-line pattern with sharp AEC, we can further model the multiline pattern to find the best structure that has the highest duty cycle together with the best AEC performance.

Results: The AEC is experimentally improved to 1.7  μm from the 3.5  μm of conventional TFMPM. The adopted multiline pattern is akin to a pulse-width-modulation pattern with a spatial period of four times the diffraction-limited line width. In other words, ideally only four π  /  2 spatial phase-shift scans are required to form a full two-dimensional image with superior AEC instead of image-size-dependent line-to-line scanning.

Conclusions: We have demonstrated the developed parallel multiline scanning-based TFMPM has the multiline pattern for sharp AEC and the least scans required for full-field uniform excitation. In the experimental results, the temporal focusing-based multiphoton images of disordered biotissue of mouse skin with improved axial resolution due to the near-theoretical limit AEC are shown to clearly reduce background scattering.

Significance: Particle field holography is a versatile technique to determine the size and distribution of moving or stationary particles in air or in a liquid without significant disturbance of the sample volume. Although this technique is applied in biological sample analysis, it is limited to small sample volumes, thus increasing the number of measurements per sample. In this work, we characterize the maximum achievable volume limit based on the specification of a given sensor to realize the development of a potentially low-cost, single-shot, large-volume holographic microscope.

Aim: We present mathematical formulas that will aid in the design and development and improve the focusing speed for the numerical reconstruction of registered holograms in particle field holographic microscopes. Our proposed methodology has potential application in the detection of Schistosoma haematobium eggs in human urine samples.

Approach: Using the Fraunhofer holography theory for opaque objects, we derived an exact formula for the maximum diffraction-limited volume for an in-line holographic setup. The proof-of-concept device built based on the derived formulas was experimentally validated with urine spiked with cultured Schistosoma haematobium eggs.

Results: Results obtained show that for urine spiked with Schistosoma haematobium eggs, the volume thickness is limited to several millimeters due to scattering properties of the sample. The distances of the target particles could be estimated directly from the hologram fringes.

Conclusion: The methodology proposed will aid in the development of large-volume holographic microscopes.

Significance: Water is a primary absorber of infrared (IR) laser energy, and urinary stones are immersed in fluid in the urinary tract and irrigated with saline during IR laser lithotripsy. Laser-induced vapor bubbles, formed during lithotripsy, contribute to the stone ablation mechanism and stone retropulsion effects.

Aim: Introduction of a surfactant may enable manipulation of vapor bubble dimensions and duration, potentially for more efficient laser lithotripsy.

Approach: A surfactant with concentrations of 0%, 5%, and 10% was tested. A single pulse from a thulium fiber laser with wavelength of 1940 nm was delivered to the surfactant through a 200-μm-core optical fiber, using a wide range of laser parameters, including energies of 0.05 to 0.5 J and pulse durations of 250 to 2500  μs.

Results: Bubble length, width, and duration with surfactant increased on average by 29%, 17%, and 120%, compared with water only.

Conclusions: Our study demonstrated successful manipulation of laser-induced vapor bubble dimensions and duration using a biocompatible and commercially available surfactant. With further study, use of a surfactant may potentially improve the “popcorn” technique of laser lithotripsy within the confined space of the kidney, enable non-contact laser lithotripsy at longer working distances, and provide more efficient laser lithotripsy.

The erratum corrects a grant number listed in Acknowledgments section of the original article.

The errata correct errors that appeared in Table 2 of the published article.

The note corrects an error that appeared in Equation 14 of the originally published article.

The erratum corrects an error in the originally provided equation for ΔT.

An error in the 2nd author’s name is corrected.