Advances in laser speckle imaging: From qualitative to quantitative hemodynamic assessment.

Laser speckle imaging (LSI) techniques have emerged as a promising method for visualizing functional blood vessels and tissue perfusion by analyzing the speckle patterns generated by coherent light interacting with living biological tissue. These patterns carry important biophysical tissue information including blood flow dynamics. The noninvasive, label-free, and wide-field attributes along with relatively simple instrumental schematics make it an appealing imaging modality in preclinical and clinical applications. The review outlines the fundamentals of speckle physics and the three categories of LSI techniques based on their degree of quantification: qualitative, semi-quantitative and quantitative. Qualitative LSI produces microvascular maps by capturing speckle contrast variations between blood vessels containing moving red blood cells and the surrounding static tissue. Semi-quantitative techniques provide a more accurate analysis of blood flow dynamics by accounting for the effect of static scattering on spatiotemporal parameters. Quantitative LSI such as optical speckle image velocimetry provides quantitative flow velocity measurements, which is inspired by the particle image velocimetry in fluid mechanics. Additionally, discussions regarding the prospects of future innovations in LSI techniques for optimizing the vascular flow quantification with associated clinical outlook are presented.

Photobiomodulation Therapy Restores Olfactory Function Impaired by Photothrombosis in Mouse Olfactory Bulb.

An ischemic stroke typically accompanies numerous disorders ranging from somatosensory dysfunction to cognitive impairments, inflicting patients with various neurologic symptoms. Among pathologic outcomes, post-stroke olfactory dysfunctions are frequently observed. Despite the well-known prevalence, therapy options for such compromised olfaction are limited, likely due to the complexity of olfactory bulb architecture, which encompasses both the peripheral and central nervous systems. As photobiomodulation (PBM) emerged for treating ischemia-associated symptoms, the effectiveness of PBM on stroke-induced impairment of olfactory function was explored. Novel mouse models with olfactory dysfunctions were prepared using photothrombosis (PT) in the olfactory bulb on day 0. The post-PT PBM was performed daily from day 2 to day 7 by irradiating the olfactory bulb via an 808 nm laser with a fluence of 40 J/cm2 (325 mW/cm2 for 2  smin per day). The buried food test (BFT) was used to score behavioral acuity in food-deprived mice to assess the olfactory function before PT, after PT, and after PBM. Histopathological examinations and cytokine assays were performed on the mouse brains harvested on day 8. The results from BFT were specific to an individual, with positive correlations between the baseline latency time measured before PT and its alteration at the ensuing stages for both the PT and PT + PBM groups. Also, the correlation analysis in both groups showed highly similar, significant positive relationships between the early and late latency time change independent of PBM, implicating a common recovery mechanism. Particularly, PBM treatment accelerated the recovery of impaired olfaction following PT by suppressing inflammatory cytokines and enhancing both glial and vascular factors (e.g., GFAP, IBA-1, and CD31). PBM therapy during the acute phase of ischemia improves the compromised olfactory function by modulating microenvironments and inflammation status of the affected tissue.

Fast volumetric imaging with line-scan confocal microscopy by electrically tunable lens at resonant frequency.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and usually suffers from a reduced acquisition rate, which is critical to properly document rapid neurophysiological dynamics. In this study, we implemented an electrically tunable lens (ETL) in the line-scan confocal microscopy (LSCM), enabling the volumetric acquisition at the rate of 20 frames per second with a maximum volume of interest of 315 × 315 × 80 µm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 µm and 90.4 ± 2.1 µm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth axial penetration by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of both cleared mouse brain ex vivo samples and in vivo brains. The current study showed a successful application of resonant ETL for constructing a high-performance 3D axially scanning LSCM (asLSCM) system. Such advances in rapid volumetric imaging would significantly enhance our understanding of various dynamic biological processes.

Detection and differentiation of semi-transparent materials simulating biological structures using optical coherence tomography: a phantom study.

Significance: Lymphatic and peripheral nervous system imaging is of prime importance for monitoring various important pathologic processes including cancer development and metastasis, and response to therapy. Aim: Optical coherence tomography (OCT) is a promising approach for this imaging task but is challenged by the near-transparent nature of these structures. Our aim is to detect and differentiate semi-transparent materials using OCT texture analysis, toward label-free neurography and lymphography. Approach: We have recently demonstrated an innovative OCT texture analysis-based approach that used speckle statistics to image lymphatics and nerves in-vivo that does not rely on negative contrast. However, these two near-transparent structures could not be easily differentiated from each other in the texture analysis parameter space. Here, we perform a rigorous follow-up study to improve upon this differentiation in controlled phantoms mimicking the optical properties of these tissues. Results: The results of the three-parameter Rayleigh distribution fit to the OCT images of six types of tissue-mimicking materials varying in transparency and biophysical properties demonstrate clear differences between them, suggesting routes for improved lymphatics-nerves differentiation. Conclusions: We demonstrate a novel OCT texture analysis-based lymphatics-nerves differentiation methodology in tissue-simulating phantoms. Future work will focus on longitudinal in-vivo lymphangiography and neurography in response to cancer therapeutics toward adaptive personalized medicine.

Laser speckle decorrelation time-based platelet function testing in microfluidic system.

Platelet aggregation and adhesion are critically involved in both normal hemostasis and thrombosis during vascular injury. Before any surgery, it is important to identify the number of platelets and their functionality to reduce the risk of bleeding; therefore, platelet function testing is a requirement. We introduce a novel evaluation method of assessing platelet function with laser speckle contrast imaging. The speckle decorrelation time (SDT) of the blood flowing through a microfluidic channel chip provides a quantitative measure of platelet aggregation. We compared SDTs of whole blood and platelet-poor blood, i.e., whole blood stripped of its buffy coat region, and found a marked reduction in decorrelation time for platelet-poor blood. The measured SDT of platelet-poor blood was 1.04 ± 0.21 ms, while that of whole blood was 2.64 ± 0.83 ms. To further characterize the sensitivity of our speckle decorrelation time-based platelet function testing (SDT-PFT), we added various agonists involved in platelet aggregation, including adenosine diphosphate (ADP), epinephrine (EPI), and arachidonic acid (AA). In this study, the results show that whole blood with ADP resulted in the largest SDT, followed by whole blood with AA, whole blood with EPI, whole blood without agonist, and platelet-poor blood with or without agonist. These findings show that SDT-PFT has the potential for rapid screening of bleeding disorders and monitoring of anti-platelet therapies with only a small volume of blood.

Optofluidic laser speckle image decorrelation analysis for the assessment of red blood cell storage.

Red blood cells (RBCs) undergo irreversible biochemical and morphological changes during storage, contributing to the hemorheological changes of stored RBCs, which causes deterioration of microvascular perfusion in vivo. In this study, a home-built optofluidic system for laser speckle imaging of flowing stored RBCs through a transparent microfluidic channel was employed. The speckle decorrelation time (SDT) provides a quantitative measure of RBC changes, including aggregation in the microchannel. The SDT and relative light transmission intensity of the stored RBCs were monitored for 42 days. In addition, correlations between the decorrelation time, RBC flow speed through the channel, and relative light transmission intensity were obtained. The SDT of stored RBCs increased as the storage duration increased. The SDTs of the RBCs stored for 21 days did not significantly change. However, for the RBCs stored for over 35 days, the SDT increased significantly from 1.26 ± 0.27 ms to 6.12 ± 1.98 ms. In addition, we measured the relative light transmission intensity and RBC flow speed. As the RBC storage time increased, the relative light transmission intensity increased, whereas the RBC flow speed decreased in the microchannel. The optofluidic laser speckle image decorrelation time provides a quantitative measure of assessing the RBC condition during storage. Laser speckle image decorrelation analysis may serve as a convenient assay to monitor the property changes of stored RBCs.

Deep brain optical coherence tomography angiography in mice: in vivo, noninvasive imaging of hippocampal formation.

The hippocampus is associated with memory and navigation, and the rodent hippocampus provides a useful model system for studying neurophysiology such as neural plasticity. Vascular changes at this site are closely related to brain diseases, such as Alzheimer's disease, dementia, and epilepsy. Vascular imaging around the hippocampus in mice may help to further elucidate the mechanisms underlying these diseases. Optical coherence tomography angiography (OCTA) is an emerging technology that can provide label-free blood flow information. As the hippocampus is a deep structure in the mouse brain, direct in vivo visualisation of the vascular network using OCTA and other microscopic imaging modalities has been challenging. Imaging of blood vessels in the hippocampus has been performed using multiphoton microscopy; however, labelling with fluorescence probes is necessary when using this technique. Here, we report the use of label-free and noninvasive microvascular imaging in the hippocampal formation of mice using a 1.7-µm swept-source OCT system. The imaging results demonstrate that the proposed system can visualise blood flow at different locations of the hippocampus corresponding with deep brain areas.

In vivo study of optical speckle decorrelation time across depths in the mouse brain.

The strong optical scattering of biological tissue confounds our ability to focus light deeply into the brain beyond depths of a few hundred microns. This challenge can be potentially overcome by exploiting wavefront shaping techniques which allow light to be focused through or inside scattering media. However, these techniques require the scattering medium to be static, as changes in the arrangement of the scatterers between the wavefront recording and playback steps reduce the fidelity of the focus that is formed. Furthermore, as the thickness of the scattering medium increases, the influence of the dynamic nature becomes more severe due to the growing number of scattering events experienced by each photon. In this paper, by examining the scattering dynamics in the mouse brain in vivo via multispeckle diffusing wave spectroscopy (MSDWS) using a custom fiber probe that simulates a point-like source within the brain, we investigate the relationship between this decorrelation time and the depth of the point-like light source inside the living mouse brain at depths up to 3.2 mm.

Erratum: In vivo study of optical speckle decorrelation time across depths in the mouse brain: erratum.

[This corrects the article on p. 4855 in vol. 8.].