Laser Speckle Imaging Technique Research Articles

Tools for classification of growing/non-growing bacterial colonies using laser speckle imaging

Prior research has indicated the feasibility of assessing growth—associated activity in bacterial colonies through the application of laser speckle imaging techniques. A subpixel correlation method was employed to identify variations in sequential laser speckle images, thereby facilitating the visualization of specific zones indicative of microbial growth within the colony. Such differentiation between active (growing) and inactive (non-growing) bacterial colonies holds considerable implications for medical applications, like bacterial response to certain drugs or antibiotics. The present study substantiates the capability of laser speckle imaging to categorize bacterial colonies as growing or non-growing, a parameter which nonvisible in colonies when observed under white light illumination.

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.

Use of the speckle imaging sub-pixel correlation analysis in revealing a mechanism of microbial colony growth

The microbial colony growth is driven by the activity of the cells located on the edges of the colony. However, this process is not visible unless specific staining or cross-sectioning of the colony is done. Speckle imaging technology is a non-invasive method that allows visualization of the zones of increased microbial activity within the colony. In this study, the laser speckle imaging technique was used to record the growth of the microbial colonies. This method was tested on three different microorganisms: Vibrio natriegens, Escherichia coli, and Staphylococcus aureus. The results showed that the speckle analysis system is not only able to record the growth of the microbial colony but also to visualize the microbial growth activity in different parts of the colony. The developed speckle imaging technique visualizes the zone of “the highest microbial activity” migrating from the center to the periphery of the colony. The results confirm the accuracy of the previous models of colony growth and provide algorithms for analysis of microbial activity within the colony.

Objective speckle pattern-based surface roughness measurement using matrix factorization.

A method for the measurement of profile parameters of both isotropic and anisotropic surfaces is presented using the objective laser speckle imaging technique. The surface parameters are characterized in terms of a singular value decomposition method-based metric derived from the initial key contributing singular values of the speckle pattern. A simulation study is performed with random Gaussian anisotropic surfaces generated as a function of the correlation lengths in both x and y directions. In the experimental demonstration, the proposed method is verified with metallic samples having distinct surface roughness processed through widely used machining operations viz., vertical milling, and grinding. A brief discussion about the extent to which the minimum number of singular values that are sufficient to evaluate the profile parameters in the context of experimental results is provided. The method supports the measurement of profile parameters of higher magnitude in the realm of non-contact topographic measurement techniques. The experimental results substantiate the practical applicability of the proposed method.

Echo speckle imaging of dynamic processes in soft materials.

We present a laser-speckle imaging technique, termed Echo speckle imaging (ESI), that quantifies the local dynamics in biological tissue and soft materials with a noise level around or below 10% of the measured signal without affecting the spatial resolution. We achieve this through an unconventional speckle beam illumination that creates changing, statistically independent illumination conditions and substantially increases the measurement accuracy. Control experiments for dynamically homogeneous and heterogeneous soft materials and tissue phantoms illustrate the performance of the method. We show that this approach enables us to precision-monitor purely dynamic heterogeneities in turbid soft media with a lateral resolution of 100 µm and better.

Lens-free motion analysis via neuromorphic laser speckle imaging.

Laser speckle imaging (LSI) is a powerful tool for motion analysis owing to the high sensitivity of laser speckles. Traditional LSI techniques rely on identifying changes from the sequential intensity speckle patterns, where each pixel performs synchronous measurements. However, a lot of redundant data of the static speckles without motion information in the scene will also be recorded, resulting in considerable resources consumption for data processing and storage. Moreover, the motion cues are inevitably lost during the "blind" time interval between successive frames. To tackle such challenges, we propose neuromorphic laser speckle imaging (NLSI) as an efficient alternative approach for motion analysis. Our method preserves the motion information while excluding the redundant data by exploring the use of the neuromorphic event sensor, which acquires only the relevant information of the moving parts and responds asynchronously with a much higher sampling rate. This neuromorphic data acquisition mechanism captures fast-moving objects on the order of microseconds. In the proposed NLSI method, the moving object is illuminated using a coherent light source, and the reflected high frequency laser speckle patterns are captured with a bare neuromorphic event sensor. We present the data processing strategy to analyze motion from event-based laser speckles, and the experimental results demonstrate the feasibility of our method at different motion speeds.

Confocal laser speckle autocorrelation imaging of dynamic flow in microvasculature

Laser speckle imaging has been widely used for in-vivo visualization of blood perfusion in biological tissues. However, existing laser speckle imaging techniques suffer from limited quantification accuracy and spatial resolution. Here we report a novel design and implementation of a powerful laser speckle imaging platform to solve the two critical limitations. The core technique of our platform is a combination of line scan confocal microscopy with laser speckle autocorrelation imaging, which is termed Line Scan Laser Speckle Autocorrelation Imaging (LS-LSAI). The technical advantages of LS-LSAI include high spatial resolution (~4.4 μm) for visualizing and quantifying blood flow in microvessels, as well as video-rate imaging speed for tracing dynamic flow.

High-speed laser speckle imaging to unravel picoliter drop-on-demand to substrate interaction.

Understanding phenomena such as evaporation and imbibition of picoliter droplets into porous substrates is crucial in printing industry to achieve a higher printing quality and print speed. After printing, the residual pigment must remain fixed at the desired location on a substrate and be of a desired volume to yield a high resolution and vibrantly printed page that has become the expectation of modern printing technology. Current research entails not only chemical composition of the ink but also how this links to the dynamics and interactions that occur between the ink and the substrate at every stage of the printed spot formation, including evaporation, wetting, and imbibition. In this paper, we present an instrument that can print on-demand picoliter volume droplets of ink onto substrates and then immediately record on evolution of the resulting dynamics when these two materials interact. This high-speed laser speckle imaging (HS-LSI) technique has been developed to monitor nanometer displacement of the drying and imbibing ink droplet at a high frame rate, up to 20000Hz, given the short timescales of these interactions. We present the design of the instrument, discuss the related challenges and the theory underlying the LSI technique, specifically how photons non-evasively probe opaque objects in a multiple scattering regime, and show how this technique can unravel the dynamics of drying and imbibition. We will finish giving a validation on the instrument and an example of its usage.

Laser speckle imaging for early detection of microbial colony forming units.

In this study, an optical contactless laser speckle imaging technique for the early identification of bacterial colony-forming units was tested. The aim of this work is to compare the laser speckle imaging method for the early assessment of microbial activity with standard visual inspection under white light illumination. In presented research, the growth of Vibrio natriegens bacterial colonies on the solid medium was observed and analyzed. Both - visual examination under white light illumination and laser speckle correlation analysis were performed. Based on various experiments and comparisons with the theoretical Gompertz model, colony radius growth curves were obtained. It was shown that the Gompertz model can be used to describe both types of analysis. A comparison of the two methods shows that laser speckle contrast imaging, combined with signal processing, can detect colony growth earlier than standard CFU counting method under white light illumination.

Detection of carious lesion by laser speckle analysis using the first order moment

Carious lesion is one of the most prevalent diseases in humankind, affecting nearly all human beings at least once in a lifetime. Early carious lesion (ECL, also known as white spot lesion) is part of the caries process in which the enamel surface loses mineral but the subsurface layer overlying the mineral-poor region remains intact. In its early stages, the detection of the carious lesion is currently still a major challenge because it is based on visual inspection, thus with an inherent subjectivity. In this work, we present a method to detect and assess the severity of ECLs using a computer-assisted laser speckle imaging technique. Forty-five polished and cut bovine incisors samples allow a paired t-test by exposing half of the surface while protecting the other half. The samples, included in a PVC tube, were immersed in 50ml of a solution (pH 5.0) containing 0.05M acetate buffer solution and 50% saturated hydroxyapatite enamel powder at 37°C. The etching time is 24h; 48h and 72h; each group contain 15 samples. We illuminate each sample with a laser and analyse the first momentum of the laser speckle images with a computer. Our results shows that the first momentum of the LSI of the sound region statistically differs from the decay region of the samples for all groups (p<0.0001). We also found a strong correlation between the acid etch duration (severity of the decay) and the shift in LSI contrast (Pearson’s correlation coefficient ρ=0.9989, R²=0.9978). Detecting decay in its early stages is still a challenge in the clinical practice, however this work demonstrates that the analysis of the statistical features of the laser speckle image in the spatial domain allows for the detection of microstructural changes in the enamel associated with the presence of the lesion even before any intervention is required.

Hybrid method combining orthogonal projection Fourier transform profilometry and laser speckle imaging for 3D visualization of flow profile

This paper reports a straightforward technique for three-dimensional (3D) visualization of a flow profile by a hybrid algorithm combining Fourier transform orthogonal fringe projection and laser speckle imaging techniques. The use of orthogonal projection aims to suppress the zero order allowing surface reconstruction with high spatial resolution and accuracy while analyzing the intensity fluctuations of diffuse backscattered laser light providing 2D flow information. Once both are achieved, 3D flow visualization can be displayed. The method is experimentally validated first with a plastic tube filled with scattering liquid (milk) running at various controlled flow rates and then with the tube embedded under scattering layers (chicken breast) of varying thickness. The system includes a single, common camera, a commercial projector (profilometry channel), a laser light source (flow channel), and a computer station. In addition, orthogonal projection processing was combined with Hilbert transform, increasing the visualization and resolution of the measured flow profile.

Reply: Intraoperative Tissue Perfusion Measurement by Laser Speckle Imaging: A Potential Aid for Reducing Postoperative Complications in Free Flap Breast Reconstruction

Reply: Intraoperative Tissue Perfusion Measurement by Laser Speckle Imaging: A Potential Aid for Reducing Postoperative Complications in Free Flap Breast Reconstruction

Establishment of transparent cranial window after cerebral subarachnoid hemorrhage and studying of cerebral cortical blood flow in rats

Objective Establishment of a transparent cranial window model of subarachnoid hemorrhage in rats for 7 days of continuous monitoring of cortical blood flow after subarachnoid hemorrhage Methods Thirty SD male rats were randomly divided into normal control group (n=10), saline group (n=10)and blood injection group (n=10). The transcranial subarachnoid hemorrhage transparent cranial window observation model was established by injecting autologous non-anticoagulation tail artery blood 0.3 ml twice in the occipital large pool and using a transparent flexible material made of silicon-based polydimethylsiloxane. Seven-day dynamic changes in cerebral cortical blood flow within the cranial window were monitored by laser speckle imaging. Results The imaging of 27 rats were clear; the blood flow velocity of the cortex decreased after SAH, which was statistically different from the control group and the saline group (P<0.01), and the blood flow on the 3rd, 5th, and 7th day after SAH. The percentage speeds were (83.20±4.16)%, (64.00±3.05)%, (75.70±3.79)%, and the 5 d was statistically different from the 3rd and 7 d (P<0.01); cortical blood perfusion after SAH The amount decreased, which was statistically different from the control group and the saline group (P<0.05). The percentage blood perfusion of the cortex on the 3rd, 5th, and 7th after SAH was (75.40±4.18)%, (31.80±5.34)%, (68.90±6.31)%, the 5th was statistically different from the 3rd and 7 d (P<0.01). Conclusion Through the use of a new flexible transparent material to establish a subarachnoid hemorrhage transparent cranial window imaging model, combined with laser speckle imaging technology can clearly detect the decline of cortical blood flow velocity and cortical blood perfusion in rats after subarachnoid hemorrhage 5 d is the peak of decline, and recovery begins on the 7th day, providing a new method for studying the pathophysiological changes in the acute and subacute phases after subarachnoid hemorrhage. Key words: Subarachnoid hemorrhage; Transparent cranial window imaging; Cerebral cortex blood flow; Laser speckle imaging technique; Rat

A Robust Method for Adjustment of Laser Speckle Contrast Imaging during Transcranial Mouse Brain Visualization

Laser speckle imaging (LSI) is a well-known and useful approach for the non-invasive visualization of flows and microcirculation localized in turbid scattering media, including biological tissues (such as brain vasculature, skin capillaries etc.). Despite an extensive use of LSI for brain imaging, the LSI technique has several critical limitations. One of them is associated with inability to resolve a functionality of vessels. This limitation also leads to the systematic error in the quantitative interpretation of values of speckle contrast obtained for different vessel types, such as sagittal sinus, arteries, and veins. Here, utilizing a combined use of LSI and fluorescent intravital microscopy (FIM), we present a simple and robust method to overcome the limitations mentioned above for the LSI approach. The proposed technique provides more relevant, abundant, and valuable information regarding perfusion rate ration between different types of vessels that makes this method highly useful for in vivo brain surgical operations.

Evaluation of Formalin Fixation for Tissue Biopsies Using Shear Wave Laser Speckle Imaging System.

Chemical fixation is the slowest and often the most uncontrolled step in the multi-step process of preparing tissue for histopathology. In order to reduce the time from taking a core needle biopsy to making a diagnosis, a new approach is proposed that optically monitors the common formalin fixation process. A low-cost and highly-sensitive laser speckle imaging technique is developed to measure shear wave velocity in a biospecimen as small as 0.5 mm in thickness submerged in millifluidic channels. Shear wave velocity, which is the indicator of tissue mechanical property and induced by piezoelectric-actuation, was monitored using gelatin phantom and chicken breast during fixation, as well as post-fixed liver and colon tissues from human. Fixation levels in terms of shear wave velocity increased by approximately 271.0% and 130.8% in gelatin phantom and chicken breast, respectively, before reaching the plateaus at 10.91 m/s and 7.88 m/s. Within these small specimens, the plateaus levels and times varied with location of measurement, and between gelatin and chicken breast. This optical-based approach demonstrates the feasibility of fine-tuning preanalytical variables, such as fixation time, for a rapid and accurate histopathological evaluation; provides a quality metric during the tissue preparation protocol performed in most pathology labs; and introduces the millifluidic chamber that can be engineered to be a future disposable device that automates biopsy processing and imaging.

Assessment of microscopic repair dynamics in self-healing polymer by modeling laser speckle images

Self-healing in polymers plays an important role, since such polymers can spontaneously repair any damage introduced into them, and this in turn can enhance the durability and reliability of functional materials. Employment of the laser speckle imaging (LSI) technique can picture the microscopic repair dynamics of the healing process in real time. To assess the repair dynamics during the healing process, one needs to acquire a large amount of LSI data, and because of this the visual inspection of healing dynamics using such a data set can be a lengthy and tedious task. In order to support LSI processing, we demonstrate the application of a view-based temporal template method, the motion history image (MHI), to easily study the microscopic mechanisms of the self-healing process of polymers.The micromechanical dynamics of the healing process of a cut in both self-healing polymers as well as a non-healing version of a polymer was assessed using the MHI algorithm. The results of MHI are obtained much more easily than those of a visual inspection of all the LSI frames acquired during the healing process of the polymer.The MHI algorithm produced a visual modeling of the LSI images recorded during the healing process. The MHI algorithm could become an alternative tool to generate online results on the healing dynamics in polymers and other functional materials.

Laser speckle contrast imaging of cerebral autoregulation in rats at a macro- and microcirculation level

Using the method of laser speckle imaging for the simultaneous study of macro- and microcirculation in cerebral vessels of healthy rats, we show that the mechanisms underlying cerebral autoregulation depend on the initial condition of the organism and the sex of individual animals. The pharmacological dose-dependent stimulation of the peripheral arterial pressure increase is not accompanied by the cerebral circulation responses of analogous intensity, but manifests itself as ‘compensating’ reactions, namely, the redistribution of the blood flow at the level of macro- (in females) and microcirculation (in females and males). The obtained results extend our understanding of the capabilities of laser speckle imaging technique in neurophysiological studies of reserve abilities of cerebral circulation autoregulation under the conditions of hypertensive status formation.

Imaging functional blood vessels by the laser speckle imaging (LSI) technique using Q-statistics of the generalized differences algorithm

In this work, we report about q statistics concept to improve the performance of generalized differences algorithm based on intensity histogram for imaging functional blood vessel structures in a rodent window chamber of a mice. The method uses the dynamic speckle signals obtained by transilluminating the rodent window chamber to create activity maps of vasculatures. The proposed method of generalized differences with q statistics (GDq) is very sensitive to the values of defined parameters such as: camera exposure time, the q value and the camera frame number. Appropriate choice of q values enhances the visibility (contrast) of functional blood vessels but at the same time without sacrificing the spatial resolution, which is of utmost importance for in-vivo vascular imaging.

Monitoring of cerebral blood flow during hypoxia-ischemia and resuscitation in the neonatal rat using laser speckle imaging.

Neonatal hypoxic‐ischemic encephalopathy (HIE) is associated with alterations in cerebral blood flow (CBF) as a result of perinatal asphyxia. The extent to which CBF changes contribute to injury, and whether treatments that ameliorate these changes might be neuroprotective, is still unknown. Higher throughput techniques to monitor CBF changes in rodent models of HIE can help elucidate the underlying pathophysiology. We developed a laser speckle imaging (LSI) technique to continuously monitor CBF in six postnatal‐day 10 (P10) rats simultaneously before, during, and after unilateral hypoxia‐ischemia (HI, ligation of the left carotid artery followed by hypoxia in 8% oxygen). After ligation, CBF to the ligated side fell by 30% compared to the unligated side (P < 0.0001). Hypoxia induced a bilateral 55% reduction in CBF, which was partially restored by resuscitation. Compared to resuscitation in air, resuscitation in 100% oxygen increased CBF to the ligated side by 45% (P = 0.033). Individual variability in CBF response to hypoxia between animals accounted for up to 24% of the variability in hemispheric area loss to the ligated side. In both P10 and P7 models of unilateral HI, resuscitation in 100% oxygen did not affect hemispheric area loss, or hippocampal CA1 pyramidal neuron counts, after 1‐week survival. Continuous CBF monitoring using LSI in multiple rodents simultaneously can screen potential treatment modalities that affect CBF, and provide insight into the pathophysiology of HI.

Dual-wavelength laser speckle imaging for monitoring brain metabolic and hemodynamic response to closed head traumatic brain injury in mice.

Abstract. The measurement of dynamic changes in brain hemodynamic and metabolism events following head trauma could be valuable for injury prognosis and for planning of optimal medical treatment. Specifically, variations in blood flow and oxygenation levels serve as important biomarkers of numerous pathophysiological processes. We employed the dual-wavelength laser speckle imaging (DW-LSI) technique for simultaneous monitoring of changes in brain hemodynamics and cerebral blood flow (CBF) at early stages of head trauma in a mouse model of intact head injury (n=10). For induction of head injury, we used a weight-drop device involving a metal mass (∼50 g ) striking the mouse’s head in a regulated manner from a height of ∼90 cm. In comparison to baseline measurements, noticeable dynamic variations were revealed immediately and up to 1 h postinjury, which indicate the severity of brain damage and highlight the ability of the DW-LSI arrangement to track brain pathophysiology induced by injury. To validate the monitoring of CBF by DW-LSI, measurements with laser Doppler flowmetry (LDF) were also performed (n=5), which confirmed reduction in CBF following injury. A secondary focus of the study was to investigate the effectiveness of hypertonic saline as a neuroprotective agent, inhibiting the development of complications after brain injury in a subgroup of injured mice (n=5), further demonstrating the ability of DW-LSI to monitor the effects upon brain dynamics of drug treatment. Overall, our findings further support the use of DW-LSI as a noninvasive, cost-effective tool to assess changes in hemodynamics under a variety of pathological conditions, suggesting its potential contribution to the biomedical field. To the best of our knowledge, this work is the first to make use of the DW-LSI modality in a small animal model to (1) investigate brain function during the critical first hour of closed head injury trauma, (2) correlate between injury parameters of LDF measurements, and (3) monitor brain hemodynamic and metabolic response to neuroprotective drug treatment.