Significance: Hyperspectral microscopy has been intensively explored in biomedical applications. However, due to its huge three-dimensional hyperspectral data cube, it typically suffers from slow data acquisition, mass data transmission and storage, and computationally expensive postprocessing.

Aim: To overcome the above limitations, a programmable hyperspectral microscopy technique was developed, which can perform hardware-based hyperspectral data postprocessing by the physical process of optical imaging in a snapshot.

Approach: A programmable hyperspectral microscopy system was developed to collect coded microscopic images from samples under multiplexed illumination. Principal component analysis followed by linear discriminant analysis scheme was coded into multiplexed illumination and realized by the physical process of optical imaging. The contrast enhancement was evaluated on two representative types of microscopic samples, i.e., tissue section and cell samples.

Results: Compared to the microscopic images collected under white light illumination, the contrasts of coded microscopic images were significantly improved by 41% and 59% for tissue section and cell samples, respectively.

Conclusions: The proposed method can perform hyperspectral data acquisition and postprocessing simultaneously by its physical process, while preserving the most important spectral information to maximize the difference between the target and background, thus opening a new avenue for high-contrast microscopic imaging in a snapshot.

Significance: The onset of several diseases is frequently marked with anomalous mechanical alteration of the affected tissue at the intersection of cells and their microenvironment. Therefore, mapping the micromechanical attributes of the tissues could enhance our understanding of the etiology of human disease, improve the diagnosis, and help stratify therapies that target these mechanical aberrations.

Aim: We review the tremendous opportunities offered through using optics for imaging the micromechanical properties, at length scales inaccessible to other modalities, in both basic research and clinical medicine. We specifically focus on laser speckle rheology (LSR), a technology that quantifies the mechanical properties of tissues in a rapid, noncontact manner.

Approach: In LSR, the shear viscoelastic modulus is measured from the time-variant speckle intensity fluctuations reflected off the tissue. The LSR technology is engineered and configured into several embodiments, including bench-top optical systems, endoscopes for minimally invasive procedures, portable point-of-care devices, and microscopes.

Results: These technological nuances have primed the LSR for widespread applications in diagnosis and therapeutic monitoring, as demonstrated here, in cardiovascular disease, coagulation disorders, and tumor malignancies.

Conclusion: The fast-paced technological advancements, elaborated here, position the LSR as a competent candidate for many more exciting opportunities in basic research and medicine.

Significance: Although the clinical potential for Raman spectroscopy (RS) has been anticipated for decades, it has only recently been used in neurosurgery. Still, few devices have succeeded in making their way into the operating room. With recent technological advancements, however, vibrational sensing is poised to be a revolutionary tool for neurosurgeons.

Aim: We give a summary of neurosurgical workflows and key translational milestones of RS in clinical use and provide the optics and data science background required to implement such devices.

Approach: We performed an extensive review of the literature, with a specific emphasis on research that aims to build Raman systems suited for a neurosurgical setting.

Results: The main translatable interest in Raman sensing rests in its capacity to yield label-free molecular information from tissue intraoperatively. Systems that have proven usable in the clinical setting are ergonomic, have a short integration time, and can acquire high-quality signal even in suboptimal conditions. Moreover, because of the complex microenvironment of brain tissue, data analysis is now recognized as a critical step in achieving high performance Raman-based sensing.

Conclusions: The next generation of Raman-based devices are making their way into operating rooms and their clinical translation requires close collaboration between physicians, engineers, and data scientists.

Significance: It is generally agreed that the corneal mechanical properties are strongly linked to many eye diseases and could be used to assess disease progression and response to therapies. Elastography is the most notable method of assessing corneal mechanical properties, but it generally requires some type of external excitation to induce a measurable displacement in the tissue.

Aim: We present Heartbeat Optical Coherence Elastography (Hb-OCE), a truly passive method that can measure the elasticity of the cornea based on intrinsic corneal displacements induced by the heartbeat.

Approach: Hb-OCE measurements were performed in untreated and UV-A/riboflavin cross-linked porcine corneas ex vivo, and a distinct difference in strain was detected. Furthermore, a partially cross-linked cornea was also assessed, and the treated and untreated areas were similarly distinguished.

Results: Our results suggest that Hb-OCE can spatially map displacements in the cornea induced by small fluctuations in intraocular pressure, similar to what is induced by the heartbeat.

Conclusions: The described technique opens the possibility for completely passive and noncontact in vivo assessment of corneal stiffness.

Significance: Recent generation of bioengineered human skin allowed the efficient treatment of patients with severe skin defects. However, the optical and biomechanical properties of these models are not known.

Aim: Three models of bioengineered human skin based on fibrin-agarose biomaterials (acellular, dermal skin substitutes, and complete dermoepidermal skin substitutes) were generated and analyzed.

Approach: Optical and biomechanical properties of these artificial human skin substitutes were investigated using the inverse adding-doubling method and tensile tests, respectively.

Results: The analysis of the optical properties revealed that the model that most resembled the optical behavior of the native human skin in terms of absorption and scattering properties was the dermoepidermal human skin substitutes after 7 to 14 days in culture. The time-course evaluation of the biomechanical parameters showed that the dermoepidermal substitutes displayed significant higher values than acellular and dermal skin substitutes for all parameters analyzed and did not differ from the control skin for traction deformation, stress, and strain at fracture break.

Conclusions: We demonstrate the crucial role of the cells from a physical point of view, confirming that a bioengineered dermoepidermal human skin substitute based on fibrin-agarose biomaterials is able to fulfill the minimal requirements for skin transplants for future clinical use at early stages of in vitro development.

Significance: Noninvasive in vivo fast pulsatile blood flow measurement in deep tissue is important because the blood flow waveform is correlated with physiological parameters, such as blood pressure and elasticity of blood vessels. Compromised blood flow may cause diseases, such as stroke, foot ulcer, and myocardial ischemia. There is great clinical demand for a portable and cost-effective device for noninvasive pulsatile blood flow measurement.

Aim: A diffuse-optics-based method, diffuse speckle pulsatile flowmetry (DSPF), was developed for fast measurement (∼300  Hz) of deep tissue blood flow noninvasively. To validate its performance, both a phantom experiment and in vivo demonstration were conducted.

Approach: Over the past two decades, single-mode fibers have been used as detection fibers in most diffuse-optics-based deep tissue blood flow measurement modalities. We used a multimode (MM) detection fiber with a core size of 200  μm for diffused speckle pattern detection. A background intensity correction algorithm was implemented for speckle contrast calculation. The MM detection fiber helped to achieve a level of deep tissue blood flow measurement similar to that of conventional modalities, such as diffuse correlation spectroscopy and diffuse speckle contrast analysis, but it increases the measurement rate of blood flow to 300 Hz.

Results: The design and implementation of the DSPF system were introduced. The theory of the background intensity correction for the diffused speckle pattern detected by the MM fiber was explained. A flow phantom was built for validation of the performance of the DSPF system. An in vivo cuff-induced occlusion experiment was performed to demonstrate the capability of the proposed DSPF system.

Conclusions: An MM detection fiber can help to achieve fast (∼300  Hz) pulsatile blood flow measurement in the proposed DSPF method. The cost-effective device and the fiber-based flexible probe increase the usability of the DSPF system significantly.

Significance: Pulmonary vein isolation with catheter-based radiofrequency ablation (RFA) is carried out frequently to treat atrial fibrillation. However, RFA lesion creation is only guided by indirect information (e.g., temperature, impedance, and contact force), which may result in poor lesion quality (e.g., nontransmural) and can lead to reoccurrence or complications.

Aim: The feasibility of guiding intracardiac RFA with an integrated polarization-sensitive optical coherence tomography (PSOCT)-RFA catheter in the right atria (RA) of living swine is demonstrated.

Approach: In total, 12 sparse lesions were created in the RA of three living swine using an integrated PSOCT-RFA catheter with standard ablation protocol. PSOCT images were displayed in real time to guide catheter-tissue apposition. After experiments, post-processed PSOCT images were analyzed to assess lesion quality and were compared with triphenyltetrazolium chloride (TTC) lesion quality analysis.

Results: Five successful lesions identified with PSOCT images were all confirmed by TTC analysis. In two ablations, PSOCT imaging detected gas bubble formation, indicating overtreatment. Unsuccessful lesions observed with PSOCT imaging were confirmed by TTC analysis.

Conclusions: The results demonstrate that the PSOCT-RFA catheter provides real-time feedback to guide catheter-tissue apposition, monitor lesion quality, and possibly help avoid complications due to overtreatment, which may enable more effective and safer RFA treatment.

Significance: Collagen is a basic component of many tissues such as tendons, muscles, and skin, and its imaging helps diagnose and monitor treatments in a variety of fields, including orthopedics. However, due to the overlapping peaks of the absorption spectrum with water in the short-wave infrared region (SWIR), it is difficult to select an optimal wavelength and obtain the photoacoustic (PA) image for collagen-based tissues. Therefore, an additional approach to selecting the proper wavelength is needed.

Aim: The aim of this study is to derive an effective PA absorption spectrum of collagen to select the optimal wavelength for high-sensitive PA imaging (PAI).

Approach: We measure the absorption spectrum by acquiring the PA signal from various collagen-based samples. To derive an effective PA absorption spectrum in the SWIR band, the following two parameters should be considered: (1) the laser excitation for generating the PA signal and (2) the absorption spectrum for water in the SWIR band. This molecular intrinsic property suggests the optimal wavelength for high-sensitive PAI of collagen-based samples.

Results: PA absorption spectral peaks of collagen were found at wavelengths of 1200, 1550, and 1700 nm. Thereby, the PA signal increased by up to five times compared with the wavelength commonly used in collagen PAI. We applied a pulsed fiber laser with a center wavelength of 1560 nm, and the three-dimensional PA image of a collagen patch was obtained.

Conclusions: The effective PA absorption spectrum contributes to the improvement of the PA image sensitivity by presenting the optimal wavelength of the target samples.

Significance: Expanded use of fluorescence-guided surgery with devices approved for use with indocyanine green (ICG) has led to a range of commercial systems available. There is a compelling need to be able to independently characterize system performance and allow for cross-system comparisons.

Aim: The goal of this work is to expand on previous proposed fluorescence imaging standard designs to develop a long-term stable phantom that spectrally matches ICG characteristics and utilizes 3D printing technology for incorporating tissue-equivalent materials.

Approach: A batch of test targets was created to assess ICG concentration sensitivity in the 0.3- to 1000-nM range, tissue-equivalent depth sensitivity down to 6 mm, and spatial resolution with a USAF test chart. Comparisons were completed with a range of systems that have significantly different imaging capabilities and applications, including the Li-Cor^® Odyssey, Li-Cor^® Pearl, PerkinElmer^® Solaris, and Stryker^® Spy Elite.

Results: Imaging of the ICG-matching phantoms with all four commercially available systems showed the ability to benchmark system performance and allow for cross-system comparisons. The fluorescence tests were able to assess differences in the detectable concentrations of ICG with sensitivity differences >10× for preclinical and clinical systems. Furthermore, the tests successfully assessed system differences in the depth-signal decay rate, as well as resolution performance and image artifacts. The manufacturing variations, photostability, and mechanical design of the tests showed promise in providing long-term stable standards for fluorescence imaging.

Conclusions: The presented ICG-matching phantom provides a major step toward standardizing performance characterization and cross-system comparisons for devices approved for use with ICG. The developed hybrid manufacturing platform can incorporate long-term stable fluorescing agents with 3D printed tissue-equivalent material. Further, long-term testing of the phantom and refinements to the manufacturing process are necessary for future implementation as a widely adopted fluorescence imaging standard.

Significance: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor.

Aim: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining.

Approach: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy.

Results: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues.

Conclusions: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.

Significance: Nanosecond-pulsed laser has proven to be used to obtain the velocity of blood using the speckle contrast method. Without the scanning time, it has potential for achieving fast two-dimensional blood flow images in a photoacoustic imaging system with the same pulsed laser.

Aim: Our study aimed to evaluate the qualities of regional cerebral blood flow (rCBF) obtained in a laser speckle contrast imaging (LSCI) system using continuous wave (cw) and nanosecond pulse laser sources.

Approach: First, a LSCI system consisting of a cw laser with a wavelength of 632.8 nm and a cw laser/nanosecond pulse laser with a wavelength of 532 nm was developed. This system was used to obtain rCBF images of mouse in vivo with two different laser sources.

Results: Continuous wave lasers (532 and 632.8 nm) show different imaging characteristics for rCBF imaging. The rCBF images obtained using 532-nm nanosecond pulse laser showed higher resolution than those using 532-nm cw laser. There was no significant difference in the results using nanosecond pulse laser among various pulse widths or repetition rates.

Conclusions: It is proved that a nanosecond pulse laser could be used for LSCI.

Significance: It is commonly assumed that using the objective lens to create a tightly focused light spot for illumination provides a twofold resolution improvement over the Rayleigh resolution limit and that resolution improvement is independent of object properties. Nevertheless, such an assumption has not been carefully examined. We examine this assumption by analyzing the performance of two super-resolution methods, known as image scanning microscopy (ISM) and illumination-enhanced sparsity (IES).

Aim: We aim to identify the fundamental differences between the two methods, and to provide examples that help researchers determine which method to utilize for different imaging conditions.

Approach: We input the same image datasets into the two methods and analyze their restorations. In numerical simulations, we design objects of distinct brightness and sparsity levels for imaging. We use biological imaging experiments to verify the simulation results.

Results: The resolution of IES often exceeds twice the Rayleigh resolution limit when imaging sparse objects. A decrease in object sparsity negatively affects the resolution improvement in both methods.

Conclusions: The IES method is superior for imaging sparse objects with its main features being bright and small against a dark, large background. For objects that are largely bright with small dark features, the ISM method is favorable.

Significance: Biological tissues are typically characterized by high anisotropic scattering and may also exhibit linear form birefringence. Both scattering and birefringence bias the phase shift between transverse electric field components of polarized light. These phase alterations are associated with particular structural malformations in the tissue. In fact, the majority of polarization-based techniques are unable to distinguish the nature of the phase shift induced by birefringence or scattering of light.

Aim: We explore the distinct contributions of scattering and birefringence in the phase retardation of circularly polarized light propagated in turbid tissue-like scattering medium.

Approach: The circularly polarized light in frame of Stokes polarimetry approach is used for the screening of biotissue phantoms and chicken skin samples. The change of optical properties in chicken skin is accomplished by optical clearing, which reduces scattering, and mechanical stretch, which induces birefringence. The change of optical properties of skin tissue is confirmed by spectrophotometric measurements and second-harmonic generation imaging.

Results: The contributions of scattering and birefringence in the phase retardation of circularly polarized light propagated in biological tissues are distinguished by the locus of the Stokes vector mapped on the Poincaré sphere. The phase retardation of circularly polarized light due to scattering alterations is assessed. The value of birefringence in chicken skin is estimated as 0.3  ×  10^− 3, which agrees with alternative studies. The change of birefringence of skin tissue due to mechanical stretch in the order of 10^− 6 is detected.

Conclusions: While the polarimetric parameters on their own do not allow distinguishing the contributions of scattering and birefringence, the resultant Stokes vector trajectory on the Poincaré sphere reveals the role of scattering and birefringence in the total phase retardation. The described approach, applied independently or in combination with Mueller polarimetry, can be beneficial for the advanced characterization of various types of malformations within biological tissues.

The erratum concerns amendment of Section 2.5 of the published article.