Interplay of temperature,thermal‐stresses and strains in laser‐assisted modification of collagenous tissues: Speckle‐contrast and OCT‐based studies

Moderate heating of collagenous tissues such as cartilage and cornea by infrared laser irradiation can produce biologically nondestructive structural rearrangements and relaxation of internal stresses resulting in the tissue reshaping. The reshaping results and eventual changes in optical and biological properties of the tissue strongly depend on the laser‐irradiation regime. Here, a speckle‐contrast technique based on monochromatic illumination of the tissue in combination with strain mapping by means of optical coherence elastography (OCE) is applied to reveal the interplay between the temperature and thermal stress fields producing tissue modifications. The speckle‐based technique ensured en face visualization of cross correlation and contrast of speckle images, with evolving proportions between contributions of temperature increase and thermal‐stresses determined by temperature gradients. The speckle‐technique findings are corroborated by quantitative OCE‐based depth‐resolved imaging of irradiation‐induced strain‐evolution. The revealed relationships can be used for real‐time control of the reshaping procedures (e.g., for laser shaping of cartilaginous implants in otolaryngology and maxillofacial surgery) and optimization of the laser‐irradiation regimes to ensure the desired reshaping using lower and biologically safer temperatures. The figure of waterfall OCE‐image demonstrates how the strain‐rate maximum arising in the heating‐beam center gradually splits and drifts towards the zones of maximal thermal stresses located at the temperature‐profile slopes.     

Optical coherence tomography to detect acute esophageal radiation‐induced damage in mice: A validation study

Radiation therapy for patients with non‐small‐cell lung cancer is hampered by acute radiation‐induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 μm), is able to visualize and monitor acute radiation‐induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard. Irradiated mice receive a single dose of 40 Gy at proximal and distal spots of the esophagus of 10.0 mm in diameter. We scan mice using OCT at two, three, and seven days post‐irradiation. In OCT analysis, we define ARIED as a presence of distorted esophageal layering, change in backscattering signal properties, or change in the esophageal wall thickness. The average esophageal wall thickness is 0.53 mm larger on OCT when ARIED is present based on histopathology. The overall sensitivity and specificity of OCT to detect ARIED compared to histopathology are 94% and 47%, respectively. However, the overall sensitivity of OCT to assess ARIED is 100% seven days post‐irradiation. We validate the capability of OCT to detect ARIED induced by high doses in mice. Nevertheless, clinical studies are required to assess the potential role of OCT to visualize ARIED in humans.      

Bowman's topography for improved detection of early ectasia

The aim of this study was to evaluate whether OCT topography of the Bowman's layer and artificial intelligence (AI) can result in better diagnosis of forme fruste (FFKC) and clinical keratoconus (KC). Normal (n = 221), FFKC (n = 72) and KC (n = 116) corneas were included. Some of the FFKC and KC patients had the fellow eye (VAE‐NT) with normal topography (n = 30). The Scheimpflug and OCT scans of the cornea were analyzed. The curvature and surface aberrations (ray tracing) of the anterior corneal surface [air‐epithelium (A‐E) interface in OCT] and epithelium‐Bowman's layer (E‐B) interface (in OCT only) were calculated. Four random forest models were constructed: (1) Scheimpflug only; (2) OCT A‐E only; (3) OCT E‐B only; (4) OCT A‐E and E‐B combined. For normal eyes, both Scheimpflug and OCT (A‐E and E‐B combined) performed equally in identifying these eyes (P = .23). However, OCT A‐E and E‐B showed that most VAE‐NT eyes were topographically similar to normal eyes and did not warrant a separate classification based on topography alone. For identifying FFKC eyes, OCT A‐E and E‐B combined performed significantly better than Scheimpflug (P = .006). For KC eyes, both Scheimpflug and OCT performed equally (P = 1.0). Thus, OCT Topography of Bowman's layer significantly improved the detection of FFKC eyes.     

基于OCT技术的鼠皮肤激光热损伤修复过程的监测

采用Er:YAG激光(波长为2 940 nm,能量密度为:2.5 J/cm2单光斑,扫描次数为4)照射活体小白鼠皮肤,利用光学相干层析成像(optical coherence tomography,OCT)技术在活体小鼠上观察其皮肤组织在激光作用之前及作用之后光热损伤修复的整个过程,得到了激光光热作用下引起损伤的皮肤组织在此过程中皮肤光学特性参数的变化情况,发现皮肤修复过程中光学参数有显著差异,并分析了这些差异引起的原因,以揭示激光美容中并发症主要因素。     

Biocompatibility evaluation of bioprinted decellularized collagen sheet implanted in vivo cornea using swept‐source optical coherence tomography

Corneal transplantation by full‐thickness penetrating keratoplasty with human donor tissue is a widely accepted treatment for damaged or diseased corneas. Although corneal transplantation has a high success rate, a shortage of high‐quality donor tissue is a considerable limitation. Therefore, bioengineered corneas could be an effective solution for this limitation, and a decellularized extracellular matrix comprises a promising scaffold for their fabrication. In this study, three‐dimensional bioprinted decellularized collagen sheets were implanted into the stromal layer of the cornea of five rabbits. We performed in vivo noninvasive monitoring of the rabbit corneas using swept‐source optical coherence tomography (OCT) after implanting the collagen sheets. Anterior segment OCT images and averaged amplitude‐scans were acquired biweekly to monitor corneal thickness after implantation for 1 month. The averaged cornea thickness in the control images was 430.3 ± 5.9 μm, while the averaged thickness after corneal implantation was 598.5 ± 11.8 μm and 564.5 ± 12.5 μm at 2 and 4 weeks, respectively. The corneal thickness reduction of 34 μm confirmed the biocompatibility through the image analysis of the depth‐intensity profile base. Moreover, hematoxylin and eosin staining supported the biocompatibility evaluation of the bioprinted decellularized collagen sheet implantation. Hence, the developed bioprinted decellularized collagen sheets could become an alternative solution to human corneal donor tissue, and the proposed image analysis procedure could be beneficial to confirm the success of the surgery.      

Multi‐modal optical imaging characterization of atherosclerotic plaques

We combined cross‐polarization optical coherence tomography (CP OCT) and non‐linear microscopy based on second harmonic generation (SHG) and two‐photon‐excited fluorescence (2PEF) to assess collagen and elastin fibers and other vascular structures in the development of atherosclerosis, including identification of vulnerable plaques, which remains an important clinical problem and imaging application. CP OCT's ability to visualize tissue birefringence and cross‐scattering adds new information about the microstructure and composition of the plaque. However its interpretation can be ambiguous, because backscattering contrast may have a similar appearance to the birefringence related fringes. Our results represent a step towards minimally invasive characterization and monitoring of different stages of atherosclerosis, including vulnerable plaques. CP OCT image of intimal thickening in the human coronary artery. The dark stripe in the cross‐polarization channel (arrow) is a polarization fringe related to the phase retardation between two eigen polarization states. It is histologically located in the area of the lipid pool, however this stripe is a polarization artifact, rather than direct visualization of the lipid pool.

     

Corneal tomographic features of postrefractive surgery ectasia

The purpose of this study was to evaluate the tomographic features of postrefractive surgery eyes. This was a retrospective evaluation of clinical data. Three patients with post‐LASIK (laser‐assisted in situ keratomileusis) and two patients with post‐SMILE (small incision lenticule extraction) ectasia were imaged with Scheimpflug imaging (SI, Pentacam) and optical coherence tomography (OCT, RTVue). Curvature and wavefront aberrations of the air‐epithelium interface (A‐E) and epithelium‐Bowman's layer interface (E‐B) were derived. OCT of normal and keratoconic eyes from an earlier study were compared with the data of the ectasia eyes. Curvature and aberrometry of the A‐E interfaces were statistically similar between SI and OCT. However, OCT revealed a steeper and more aberrated E‐B interface than A‐E though correlation between them was inferior to the correlation for keratoconic eyes. Furthermore, the magnitude of differences between the A‐E and E‐B interfaces was greater in the ectasia eyes than the keratoconic eyes. OCT could possibly assist better in selecting appropriate treatment plan for postrefractive surgery ectasia eyes than conventional tomographers.      

Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air‐puff optical coherence tomography and corneal hysteresis

Application of the air‐puff swept source optical coherence tomography (SS‐OCT) instrument to determine the influence of viscoelasticity on the relation between overall the air‐puff force and corneal apex displacement of porcine corneas ex vivo is demonstrated. Simultaneous recording of time‐evolution of the tissue displacement and air pulse stimulus allows obtaining valuable information related in part to the mechanical properties of the cornea. A novel approach based on quantitative analysis of the corneal hysteresis of OCT data is presented. The corneal response to the air pulse is assessed for different well‐controlled intraocular pressure (IOP) levels and for the progression of cross‐linking‐induced stiffness of the cornea. Micrometer resolution, fast acquisition and noncontact character of the air‐puff SS‐OCT measurements have potential to improve the in vivo assessment of mechanical properties of the human corneas.      

Transformation of the dermal collagen framework under laser heating

The aim of this study was to compare between the changes undergone by the dermal collagen framework when heated by IR laser radiation and by traditional means and to reveal the specific features of the dermal matrix modification under moderate IR laser irradiation. Rabbit skin specimens were heated to 50°C, 55°C, 60°C and 65°C in a calorimeter furnace and with a 1.68‐μm fiber Raman laser. The proportion of the degraded collagen macromolecules was determined by differential scanning calorimetry. Changes in the architectonics of the collagen framework were revealed by using standard, phase‐contrast, polarization optical and scanning electron microscopy techniques. The collagen denaturation and dermal matrix amorphization temperature in the case of laser heating proved to be lower by 10°C than that for heating in the calorimeter furnace. The IR laser treatment of the skin was found to cause a specific low‐temperature (45°C‐50°C) transformation of its collagen framework, with some collagen macromolecules remaining intact. The transformation reduces to the splitting of collagen bundles and distortion of the course of collagen fibers. The denaturation of collagen macromolecules in the case of traditional heating takes its course in a threshold manner, so that their pre‐denaturation morphological changes are insignificant.     

Clinical assessment of non carious cervical lesion using swept‐source optical coherence tomography

Non‐carious cervical lesions (NCCLs) involve various forms of tooth loss with different etiologies. This study aimed to utilize swept‐source optical coherence tomography (SS‐OCT) at 1300 nm wavelength range in vitro and in vivo to evaluate and clarify the mechanism of NCCLs. In the in vitro phase, a dentin attenuation coefficient (μ_t) derived from the SS‐OCT signal at NCCL was compared with mineral loss obtained from transverse microradiography (TMR) to determine a μ_t threshold to discriminate demineralization of cervical dentin in vivo. In the clinical study, 242 buccal surfaces were investigated in 35 subjects. Presence and dimensions of NCCLs, cervical cracking and the degree of demineralization at the exposed cervical dentin were determined using SS‐OCT. Dentin demineralization was observed in 69% of NCCLs. SS‐OCT results confirm that dentin mineral loss and occlusal attrition were associated with larger NCCLs, and can be considered as an etiological factor in formation and progress of these lesions.

( A ) We determined the attenuation coeffcient (μ_t) threshold of SS‐OCT signal for the detection of demineralization (1.21) from in vitro study. DEM: demineralized dentin, sound: sound dentin. ( B ) Using the μ_t threshold, we observed NCCLs in vivo to detect the demineralization in cervical dentin. SS‐OCT scanning was performed along the red line. ( C ) SS‐OCT image obtained along the red line in B. In SS‐OCT, brightness of dentin beneath the NCCL was increased (arrow) compared with intact zone. The cervical dentin was slightly demineralized (μ_t: 1.25). e: enamel, d: dentin, g: gingiva.     

Imaging directed photothermolysis through two‐photon absorption demonstrated on mouse skin – a potential novel tool for highly targeted skin treatment

One‐photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un‐intended tissue damage. Our objective is to test whether two‐photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second‐harmonic‐generation (SHG) and two‐photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non‐invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA‐based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two‐photon absorption photothermolysis method could potentially be applied in clinical dermatology. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

One‐to‐one registration of en‐face optical coherence tomography attenuation coefficients with histology of a prostatectomy specimen

Optical coherence tomography (OCT), enables high‐resolution 3D imaging of the morphology of light scattering tissues. From the OCT signal, parameters can be extracted and related to tissue structures. One of the quantitative parameters is the attenuation coefficient; the rate at which the intensity of detected light decays in depth. To couple the quantitative parameters with the histology one‐to‐one registration is needed. The primary aim of this study is to validate a registration method of quantitative OCT parameters to histological tissue outcome through one‐to‐one registration of OCT with histology. We matched OCT images of unstained fixated prostate tissue slices with corresponding histology slides, wherein different histologic types were demarcated. Attenuation coefficients were determined by a supervised automated exponential fit (corrected for point spread function and sensitivity roll‐off related signal losses) over a depth of 0.32 mm starting from 0.10 mm below the automatically detected tissue edge. Finally, the attenuation coefficients corresponding to the different tissue types of the prostate were compared. From the attenuation coefficients, we produced the squared relative residue and goodness‐of‐fit metric R². This article explains the method to perform supervised automated quantitative analysis of OCT data, and the one‐to‐one registration of OCT extracted quantitative data with histopathological outcomes.      

Endomicroscopic optical coherence tomography for cellular resolution imaging of gastrointestinal tracts

Our ability to detect neoplastic changes in gastrointestinal (GI) tracts is limited by the lack of an endomicroscopic imaging tool that provides cellular‐level structural details of GI mucosa over a large tissue area. In this article, we report a fiber‐optic‐based micro‐optical coherence tomography (μOCT) system and demonstrate its capability to acquire cellular‐level details of GI tissue through circumferential scanning. The system achieves an axial resolution of 2.48 μm in air and a transverse resolution of 4.8 μm with a depth‐of‐focus (DOF) of ~150 μm. To mitigate the issue of limited DOF, we used a rigid sheath to maintain a circular lumen and center the distal‐end optics. The sensitivity is tested to be 98.8 dB with an illumination power of 15.6 mW on the sample. With fresh swine colon tissues imaged ex vivo, detailed structures such as crypt lumens and goblet cells can be clearly resolved, demonstrating that this fiber‐optic μOCT system is capable of visualizing cellular‐level morphological features. We also demonstrate that time‐lapsed frame averaging and imaging speckle reduction are essential for clearly visualizing cellular‐level details. Further development of a clinically viable μOCT endomicroscope is likely to improve the diagnostic outcome of GI cancers.      

Non‐invasive optical method for real‐time assessment of intracorneal riboflavin concentration and efficacy of corneal cross‐linking

Keratoconus is the primary cause of corneal transplantation in young adults worldwide. Riboflavin/UV‐A corneal cross‐linking may effectively halt the progression of keratoconus if an adequate amount of riboflavin enriches the corneal stroma and is photo‐oxidated by UV‐A light for generating additional cross‐linking bonds between stromal proteins and strengthening the biomechanics of the weakened cornea. Here we reported an UV‐A theranostic prototype device for performing corneal cross‐linking with the ability to assess corneal intrastromal concentration of riboflavin and to estimate treatment efficacy in real time. Seventeen human donor corneas were treated according to the conventional riboflavin/UV‐A corneal cross‐linking protocol. Ten of these tissues were probed with atomic force microscopy in order to correlate the intrastromal riboflavin concentration recorded during treatment with the increase in elastic modulus of the anterior corneal stroma. The intrastromal riboflavin concentration and its consumption during UV‐A irradiation of the cornea were highly significantly correlated (R = 0.79; P = .03) with the treatment‐induced stromal stiffening effect. The present study showed an ophthalmic device that provided an innovative, non‐invasive, real‐time monitoring solution for estimating corneal cross‐linking treatment efficacy on a personalized basis.      

Pixel classification method in optical coherence tomography for tumor segmentation and its complementary usage with OCT microangiography

A novel machine‐learning method to distinguish between tumor and normal tissue in optical coherence tomography (OCT) has been developed. Pre‐clinical murine ear model implanted with mouse colon carcinoma CT‐26 was used. Structural‐image‐based feature sets were defined for each pixel and machine learning classifiers were trained using “ground truth” OCT images manually segmented by comparison with histology. The accuracy of the OCT tumor segmentation method was then quantified by comparing with fluorescence imaging of tumors expressing genetically encoded fluorescent protein KillerRed that clearly delineates tumor borders. Because the resultant 3D tumor/normal structural maps are inherently co‐registered with OCT derived maps of tissue microvasculature, the latter can be color coded as belonging to either tumor or normal tissue. Applications to radiomics‐based multimodal OCT analysis are envisioned.      

Inhibition of cortical neural networks using infrared laser

The aim of the present study is to optimize parameters for inhibiting neuronal activity safely and investigating thermal inhibition of rat cortex neural networks in vitro by continuous infrared (IR) laser. Rat cortex neurons were cultured on multi‐electrode arrays until neural networks were formed with spontaneous neural activity. Neurons were then irradiated to inhibit the activity of the networks using different powers of 1550 nm IR laser light. A finite element heating model, calibrated by the open glass pipette method, was used to calculate temperature increases at different laser irradiation intensities. A damage signal ratio (DSR) was evaluated to avoid excessive heating that may damage cells. The DSR predicted that cortex neurons should be safe at temperatures up to 49.6°C for 30 seconds, but experiments suggested that cortex neurons should not be exposed to temperatures over 46°C for 30 seconds. Neural response experiments showed that the inhibition of neural activity is temperature dependent. The normal neural activity could be inhibited safely with an inhibition degree up to 80% and induced epileptiform activity could be suppressed. These results show that continuous IR laser radiations provide a possible way to safely inhibit the neural network activity.      

Morphological analysis of pore size and connectivity in a thick mixed‐culture biofilm

Morphological parameters are commonly used to predict transport and metabolic kinetics in biofilms. Yet, quantification of biofilm morphology remains challenging because of imaging technology limitations and lack of robust analytical approaches. We present a novel set of imaging and image analysis techniques to estimate internal porosity, pore size distributions, and pore network connectivity to a depth of 1 mm at a resolution of 10 µm in a biofilm exhibiting both heterotrophic and nitrifying activities. Optical coherence tomography (OCT) scans revealed an extensive pore network with diameters as large as 110 µm directly connected to the biofilm surface and surrounding fluid. Thin‐section fluorescence in situ hybridization microscopy revealed that ammonia‐oxidizing bacteria (AOB) distributed through the entire thickness of the biofilm. AOB were particularly concentrated in the biofilm around internal pores. Areal porosity values estimated from OCT scans were consistently lower than those estimated from multiphoton laser scanning microscopy, though the two imaging modalities showed a statistically significant correlation (r = 0.49, p < 0.0001). Estimates of areal porosity were moderately sensitive to gray‐level threshold selection, though several automated thresholding algorithms yielded similar values to those obtained by manually thresholding performed by a panel of environmental engineering researchers (±25% relative error). These findings advance our ability to quantitatively describe the geometry of biofilm internal pore networks at length scales relevant to engineered biofilm reactors and suggest that internal pore structures provide crucial habitat for nitrifier growth.     

Assessment of the metabolism and morphology of the porcine cornea,lens and retina by 2‐photon imaging

Two‐photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2‐photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom‐built 2‐photon microscope for 5‐dimensional microscopy with a near infrared broadband sub‐15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.      

Noninvasive cross‐sectional imaging of proximal caries using swept‐source optical coherence tomography (SS‐OCT) in vivo

The aim of this study was to determine the diagnostic accuracy of swept‐source optical coherent tomography (SS‐OCT) in detecting and estimating the depth of proximal caries in posterior teeth in vivo. SS‐OCT images and bitewing radiographs were obtained from 86 proximal surfaces of 53 patients. Six examiners scored the locations according to a caries lesion depth scale (0–4) using SS‐OCT and the radiographs. The results were compared with clinical observations obtained after the treatment. SS‐OCT could detect the presence of proximal caries in tomograms that were synthesized based on the backscatter signal obtained from the proximal carious lesion through occlusal enamel. SS‐OCT showed significantly higher sensitivity and larger area under the receiver operating characteristic curve than radiographs for the detection of cavitated enamel lesions and dentin caries (Student's t ‐test, p < 0.05). SS‐OCT appears to be a more reliable and accurate method than bitewing radiographs for the detection and estimation of the depth of proximal lesions in the clinical environment. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

采用OCT系统对组织光学通透的研究

组织通透方法采用高折射率化学试剂对生物组织进行渗透,改变组织的光学均匀性,可以有效地改善光学成像的穿透深度,受到生物医学光学研究领域的重视。利用光学相干层析成像技术,测量通透过程中不同测量深度下组织的散射特征的变化。通过采用系统信号对数的梯度值近似地表征光学散射系数,研究了通透过程中组织的散射特征随渗透时间和测量深度的动态关系。实验证明了组织通透可以有效地增加光子的穿透深度,并改善成像质量。研究发现:不同测量深度处组织的散射系数及其变化幅度、变化过程和变化趋势等均存在一定的差异性,并与组织的微观结构、其通透效果,化学试剂在组织中的渗透行为等有密切关系,有助于组织通透过程的理解,并为组织通透机制提供可能的实验依据。