Significance: Photoacoustic (PA) tomography has demonstrated versatile biomedical applications. However, an array-based PA computed tomography (PACT) system is complex and expensive, whereas a single-element detector-based scanning PA system is too slow to detect some fast biological dynamics in vivo. New PA imaging methods are sought after.

Aim: To overcome these limitations, we developed photoacoustic topography through an ergodic relay (PATER), a novel high-speed imaging system with a single-element detector.

Approach: PATER images widefield PA signals encoded by the acoustic ergodic relay with a single-laser shot.

Results: We applied PATER in vivo to monitor changes in oxygen saturation in a mouse brain and also to demonstrate high-speed matching of vascular patterns for biometric authentication.

Conclusions: PATER has achieved a high-speed temporal resolution over a large field of view. Our results suggest that PATER is a promising and economical alternative to PACT for fast imaging.

The editorial introduces the Journal of Biomedical Optics Special Section on Selected Topics in Biophotonics: Fluorescence Lifetime Imaging and Optical Micromechanics.

Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed.

Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging.

Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology.

Results: Our imaging system enabled macroscopic FLIM of a 6.5  ×  6.5  mm² field of view at spatial resolutions <20  μm. A frame of 512  ×  512  pixels with a lifetime accuracy <1  ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology.

Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.

Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy.

Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications.

Approach: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed.

Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

Significance: Sentinel lymph node (SLN) biopsy is an important method for metastasis staging in, e.g., patients with malignant melanoma. Tools enabling prompt histopathological analysis are expected to facilitate diagnostics; optical technologies are explored for this purpose.

Aim: The objective of this exploratory study was to investigate the potential of adopting multiphoton laser scanning microscopy (MPM) together with fluorescence lifetime analysis (FLIM) for the examination of lymph node (LN) tissue ex vivo.

Approach: Five LN tissue samples (three metastasis positive and two negative) were acquired from a biobank comprising tissues from melanoma patients. Tissues were deparaffinized and subjected to MPM-FLIM using an experimental MPM set-up equipped with a time correlated single photon counting module enabling FLIM.

Results: The data confirm that morphological features similar to conventional histology were observed. In addition, FLIM analysis revealed elevated morphological contrast, particularly for discriminating between metastatic cells, lymphocytes, and erythrocytes.

Conclusions: Taken together, the results from this investigation show promise for adopting MPM-FLIM in the context of SLN diagnostics and encourage further translational studies on fresh tissue samples.

Significance: Research in tissue engineering and in vitro organ formation has recently intensified. To assess tissue morphology, the method of choice today is restricted primarily to histology. Thus novel tools are required to enable noninvasive, and preferably label-free, three-dimensional imaging that is more compatible with futuristic organ-on-a-chip models.

Aim: We investigate the potential for using multiphoton microscopy (MPM) as a label-free in vitro approach to monitor calcium-induced epidermal differentiation.

Approach: In vitro epidermis was cultured at the air–liquid interface in varying calcium concentrations. Morphology and tissue architecture were investigated using MPM based on visualizing cellular autofluorescence.

Results: Distinct morphologies corresponding to epidermal differentiation were observed. In addition, Ca^2  +-induced effects could be distinguished based on the architectural differences in stratification in the tissue cultures.

Conclusions: Our study shows that MPM based on cellular autofluorescence enables visualization of Ca^2  +-induced differentiation in epidermal skin models in vitro. The technique has potential to be further adapted as a noninvasive, label-free, and real-time tool to monitor tissue regeneration and organ formation in vitro.

Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing.

Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers.

Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio.

Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images.

Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.

Significance: Alzheimer’s disease (AD) is an irreversible and progressive disorder that damages brain cells and impairs the cognitive abilities of the affected. Developing a sensitive and cost-effective method to detect Alzheimer’s biomarkers appears vital in both a diagnostic and therapeutic perspective.

Aim: Our goal is to develop a sensitive and reliable tool for detection of amyloid β (1-42) peptide (Aβ42), a major AD biomarker, using fiber-enhanced Raman spectroscopy (FERS).

Approach: A hollow core photonic crystal fiber (HCPCF) was integrated with a conventional Raman spectroscopic setup to perform FERS measurements. FERS was then coupled with surface-enhanced Raman spectroscopy (SERS) to further amplify the Raman signal thanks to a combined FERS-SERS assay.

Results: A minimum 20-fold enhancement of the Raman signal of Aβ42 as compared to a conventional Raman spectroscopy scheme was observed using the HCPCF-based light delivery system. The signal was further boosted by decorating the fiber core with gold bipyramids generating an additional SERS effect, resulting in an overall 200 times amplification.

Conclusions: The results demonstrate that the use of an HCPCF-based platform can provide sharp and intense Raman signals of Aβ42, in turn paving the way toward the development of a sensitive label-free detection tool for early diagnosis of AD.

Significance: Photoacoustic-based visual servoing is a promising technique for surgical tool tip tracking and automated visualization of photoacoustic targets during interventional procedures. However, one outstanding challenge has been the reliability of obtaining segmentations using low-energy light sources that operate within existing laser safety limits.

Aim: We developed the first known graphical processing unit (GPU)-based real-time implementation of short-lag spatial coherence (SLSC) beamforming for photoacoustic imaging and applied this real-time algorithm to improve signal segmentation during photoacoustic-based visual servoing with low-energy lasers.

Approach: A 1-mm-core-diameter optical fiber was inserted into ex vivo bovine tissue. Photoacoustic-based visual servoing was implemented as the fiber was manually displaced by a translation stage, which provided ground truth measurements of the fiber displacement. GPU-SLSC results were compared with a central processing unit (CPU)-SLSC approach and an amplitude-based delay-and-sum (DAS) beamforming approach. Performance was additionally evaluated with in vivo cardiac data.

Results: The GPU-SLSC implementation achieved frame rates up to 41.2 Hz, representing a factor of 348 speedup when compared with offline CPU-SLSC. In addition, GPU-SLSC successfully recovered low-energy signals (i.e., ≤268  μJ) with mean ± standard deviation of signal-to-noise ratios of 11.2  ±  2.4 (compared with 3.5  ±  0.8 with conventional DAS beamforming). When energies were lower than the safety limit for skin (i.e., 394.6  μJ for 900-nm wavelength laser light), the median and interquartile range (IQR) of visual servoing tracking errors obtained with GPU-SLSC were 0.64 and 0.52 mm, respectively (which were lower than the median and IQR obtained with DAS by 1.39 and 8.45 mm, respectively). GPU-SLSC additionally reduced the percentage of failed segmentations when applied to in vivo cardiac data.

Conclusions: Results are promising for the use of low-energy, miniaturized lasers to perform GPU-SLSC photoacoustic-based visual servoing in the operating room with laser pulse repetition frequencies as high as 41.2 Hz.