Optical coherence tomography (OCT) to image active and inactive retinoblastomas as well as retinomas.

PURPOSE: To illustrate Optical Coherence Tomography (OCT) images of active and inactive retinoblastoma (Rb) tumours. METHODS: Current observational study included patients diagnosed with retinoblastoma and retinoma who were presented at Amsterdam UMC and Jules-Gonin Eye Hospital, between November 2010 and October 2017. Patients aged between 0 and 4 years were imaged under general anaesthesia with handheld OCT in supine position. Patients older than 4 years were imaged with the conventional OCT (Heidelberg Engineering, Heidelberg Spectralis, Germany). All patients included were divided into two groups: active and inactive tumours (retinoma and regression patterns). Patients' medical records and OCT images were analysed during meetings via discussions by ophthalmologists and physicists. RESULTS: Twelve Dutch and 8 Swiss patients were divided into two groups: 2 patients with active tumour versus 18 patients with inactive tumour. Subsequently, inactive group could be divided in two groups, which consisted of 10 patients with retinoma and 8 patients with different regression pattern types. Of all included patients, 15 were male (75%). Median age at diagnosis was 18.0 months (range 0.19-715.2 months). A total of 12 retinoblastoma (active and inactive) and 8 retinoma foci were investigated by OCT. No distinction could be made between active and inactive tumours using only OCT. CONCLUSION: Optical coherence tomography alone cannot distinguish between active and inactive Rbs. However, handheld OCT adds useful information to the established imaging techniques in the monitoring and follow-up of retinoblastoma patients. With this study, we provide an overview of OCT images of active and inactive Rbs.

Optical coherence tomography velocimetry based on decorrelation estimation of phasor pair ratios (DEPPAIR).

Quantitative velocity estimations in optical coherence tomography requires the estimation of the axial and lateral flow components. Optical coherence tomography measures the depth resolved complex field reflected from a sample. While the axial velocity component can be determined from the Doppler shift or phase shift between a pair of consecutive measurements at the same location, the estimation of the lateral component for in vivo applications is still challenging. One approach to determine lateral velocity is multiple simultaneous measurements at different angles. In another approach the lateral component can be retrieved through repeated measurements at (nearly) the same location by an analysis of the decorrelation over time. In this paper we follow the latter approach. We describe a model for the complex field changes between consecutive measurements and use it to predict the uncertainties for amplitude-based, phase-based and complex algorithms. The uncertainty of the flow estimations follows from a statistical analysis and is determined by the number of available measurements and the applied analysis method. The model is verified in phantom measurements and the dynamic range of velocity estimations is investigated. We demonstrate that phase-based and complex (phasor) based lateral flow estimation methods are superior to amplitude-based algorithms.

Phase-based OCT angiography in diagnostic imaging of pediatric retinoblastoma patients: abnormal blood vessels in post-treatment regression patterns.

Phase-based OCT angiography of retinoblastoma regression patterns with a novel handheld 1050 nm clinical imaging system is demonstrated for the first time in children between 0 and 4 years old under general anesthesia. Angiography is mapped at OCT resolution by flow detection at every pixel with en-face projection from the volume between nerve fiber layer and retinal pigment epithelium. We show a striking difference between blood vasculature of healthy retina, and retinoblastoma regression patterns after chemotherapy, as well as varying complexity of abnormal vasculature in regression patterns types 2, 3, and 4. We demonstrate abnormal, tortuous and prominent vasculature in type 3 regression patterns having the highest risk of tumor recurrences and a lower probability to reduction into flat scars. The ability to visualize 3-D angiography might offer new insights in understanding of retinoblastoma development and its response to therapy.

3D texture analysis for classification of second harmonic generation images of human ovarian cancer.

Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ovarian cancer. To quantify these alterations we implemented a form of 3D texture analysis to delineate the fibrillar morphology observed in 3D Second Harmonic Generation (SHG) microscopy image data of normal (1) and high risk (2) ovarian stroma, benign ovarian tumors (3), low grade (4) and high grade (5) serous tumors, and endometrioid tumors (6). We developed a tailored set of 3D filters which extract textural features in the 3D image sets to build (or learn) statistical models of each tissue class. By applying k-nearest neighbor classification using these learned models, we achieved 83-91% accuracies for the six classes. The 3D method outperformed the analogous 2D classification on the same tissues, where we suggest this is due the increased information content. This classification based on ECM structural changes will complement conventional classification based on genetic profiles and can serve as an additional biomarker. Moreover, the texture analysis algorithm is quite general, as it does not rely on single morphological metrics such as fiber alignment, length, and width but their combined convolution with a customizable basis set.

Texture analysis applied to second harmonic generation image data for ovarian cancer classification.

Remodeling of the extracellular matrix has been implicated in ovarian cancer. To quantitate the remodeling, we implement a form of texture analysis to delineate the collagen fibrillar morphology observed in second harmonic generation microscopy images of human normal and high grade malignant ovarian tissues. In the learning stage, a dictionary of "textons"­frequently occurring texture features that are identified by measuring the image response to a filter bank of various shapes, sizes, and orientations­is created. By calculating a representative model based on the texton distribution for each tissue type using a training set of respective second harmonic generation images, we then perform classification between images of normal and high grade malignant ovarian tissues. By optimizing the number of textons and nearest neighbors, we achieved classification accuracy up to 97% based on the area under receiver operating characteristic curves (true positives versus false positives). The local analysis algorithm is a more general method to probe rapidly changing fibrillar morphologies than global analyses such as FFT. It is also more versatile than other texture approaches as the filter bank can be highly tailored to specific applications (e.g., different disease states) by creating customized libraries based on common image features.

Estimating the risk of squamous cell cancer induction in skin following nonlinear optical imaging.

High power femto-second (fs) laser pulses used for in-vivo nonlinear optical (NLO) imaging can form cyclobutane pyrimidine dimers (CPD) in DNA, which may lead to carcinogenesis via subsequent mutations. Since UV radiation from routine sun exposure is the primary source of CPD lesions, we evaluated the risk of CPD-related squamous cell carcinoma (SCC) in human skin due to NLO imaging relative to that from sun exposure. We developed a unique cancer risk model expanding previously published estimation of risk from exposure to continuous wave (CW) laser. This new model showed that the increase in CPD-related SCC in skin from NLO imaging is negligible above that due to regular sun exposure.

Carcinogenic damage to deoxyribonucleic acid is induced by near-infrared laser pulses in multiphoton microscopy via combination of two- and three-photon absorption.

Nonlinear optical imaging modalities (multiphoton excited fluorescence, second and third harmonic generation) applied in vivo are increasingly promising for clinical diagnostics and the monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited resolution at near-infrared (IR) wavelengths. However, high peak intensity of femtosecond laser pulses required for two-photon processes causes formation of cyclobutane-pyrimidine-dimers (CPDs) in cellular deoxyribonucleic acid (DNA) similar to damage from exposure to solar ultraviolet (UV) light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis. In this study, we investigate CPD damage that results in Chinese hamster ovary cells in vitro from imaging them with two-photon excited autofluorescence. The CPD levels are quantified by immunofluorescent staining. We further evaluate the extent of CPD damage with respect to varied wavelength, pulse width at focal plane, and pixel dwell time as compared with more pronounced damage from UV sources. While CPD damage has been expected to result from three-photon absorption, our results reveal that CPDs are induced by competing twoand three-photon absorption processes, where the former accesses UVA absorption band. This finding is independently confirmed by nonlinear dependencies of damage on laser power, wavelength, and pulse width.

Structural changes in mixed Col I/Col V collagen gels probed by SHG microscopy: implications for probing stromal alterations in human breast cancer.

Second Harmonic Generation (SHG) microscopy has been previously used to describe the morphology of collagen in the extracellular matrix (ECM) in different stages of invasion in breast cancer. Here this concept is extended by using SHG to provide quantitative discrimination of self-assembled collagen gels, consisting of mixtures of type I (Col I) and type V (Col V) isoforms which serve as models of changes in the ECM during invasion in vivo. To investigate if SHG is sensitive to changes due to Col V incorporation into Col I fibrils, gels were prepared with 0-20% Col V with the balance consisting of Col I. Using the metrics of SHG intensity, fiber length, emission directionality, and depth-dependent intensities, we found similar responses for gels comprised of 100% Col I, and 95% Col I/5% Col V, where these metrics were all significantly different from those of the 80% Col I/20% Col V gels. Specifically, the gels of lower Col V content produce brighter SHG, are characterized by longer fibers, and have a higher forward/backward emission ratio. These attributes are all consistent with more highly organized collagen fibrils/fibers and are in agreement with previous TEM characterization as well as predictions based on phase matching considerations. These results suggest that SHG can be developed to discriminate Col I/Col V composition in tissues to characterize and follow breast cancer invasion.

Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy.

BACKGROUND: Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion. METHODS: For this investigation we use Second Harmonic Generation (SHG) imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties. RESULTS: We find the SHG emission attributes (directionality and relative intensity) and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen. CONCLUSIONS: Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.

Retention of polarization signatures in SHG microscopy of scattering tissues through optical clearing.

Polarization responses in Second Harmonic Generation (SHG) imaging microscopy are a valuable method to quantify aspects of tissue structure, and may be a means to differentiate normal and diseased tissues. Due to multiple scattering, the polarization data is lost in turbid tissues. Here we investigate if this information can be retained through the use of optical clearing which greatly reduces the scattering coefficient and increases the corresponding mean free path. To this end, we have measured the SHG intensity as a function of laser polarization and the SHG signal anisotropy in murine tendon and striated muscle over a depth range of 200 microns. We find that the laser polarization is highly randomized in the uncleared tissues at depths corresponding to only 2-3 scattering collisions (50- 10 microns). This depolarization of the laser is also reflected in the randomized anisotropy of the SHG signal as it is created over a range of polarization states. In strong contrast, both polarization signatures are significantly retained through ~200 microns of tissue thickness following treatment with 50% glycerol. Moreover, the measured polarization responses for both tendon and striated muscle are consistent with the extent of reduction of the respective scattering coefficients upon clearing. We suggest the method will be applicable to SHG imaging of connective disorders as well as cancer through several hundred microns of extracellular matrix.

Quantitative second harmonic generation imaging and modeling of the optical clearing mechanism in striated muscle and tendon.

We have investigated the mechanisms and capabilities of optical clearing in conjunction with second harmonic generation (SHG) imaging in tendon and striated muscle. Our approach combines three-dimensional (3-D) SHG imaging of the axial attenuation and directional response with Monte Carlo simulation (based on measured bulk optical properties) of the creation intensity and propagation through the tissues. Through these experiments and simulations, we show that reduction of the primary filter following glycerol treatment dominates the axial attenuation response in both muscle and tendon. However, these disparate tissue types are shown to clear through different mechanisms of the glycerol-tissue interaction. In the acellular tendon, glycerol application reduces scattering by both index matching as well as increasing the interfibril separation. This results in an overall enhancement of the 3-D SHG intensity, where good agreement is found between experiment and simulation. Through analysis of the axial response as a function of glycerol concentration in striated muscle, we conclude that the mechanism in this tissue arises from matching of the refractive index of the cytoplasm of the muscle cells with that of the surrounding higher-index collagenous perimysium. We further show that the proportional decrease in the scattering coefficient mu(s) with increasing glycerol fraction can be well-approximated by Mie theory.

Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation.

We report the integrated use of 3D second harmonic generation (SHG) imaging microscopy and Monte Carlo simulation as a combined metric to quantifiably differentiate normal and diseased tissues based on the physical properties of the respective extracellular matrix. To achieve this, we have identified a set of parameters comprised of the SHG creation attributes and the bulk optical parameters, which are used collectively via comparative analysis. Monte Carlo simulations of the SHG axial directional and attenuation responses allow their decomposition into the underlying factors that are not readily obtainable through experimental techniques. Specifically, this approach allows for estimation of the SHG creation attributes (directionality and relative conversion efficiency) and separation of primary and secondary filter effects, collectively that form the observed SHG contrast. The quantitative metric is shown for the connective tissue disorder Osteogenesis Imperfecta (characterized by abnormal assembly of type I collagen) using a murine model that expresses the disease in the dermis layer of skin. Structural dissimilarities between the osteogenesis imperfecta mouse and wild-type tissues lead to significant differences in the SHG depth-dependent directionality and signal attenuation. The Monte Carlo simulations of these responses using measured bulk optical parameters reproduce the experimental data trends, and the extracted emission directionality and conversion efficiencies are consistent with independent determinations. The simulations also illustrate the dominance of primary filter affects on overall SHG generation and attenuation. Thus, the combined method of 3D SHG imaging and modeling forms an essential foundation for parametric description of the matrix properties that are not distinguishable by sole consideration of either bulk optical parameters or SHG alone. Moreover, due to the quasi-coherence of the SHG process in tissues, we submit that this approach contains unique information not possible by purely scattering based methods and that these methods will be applicable in the general case where the complex fibrillar structure is difficult to fully quantify via morphological analysis.

Second harmonic generation imaging microscopy studies of osteogenesis imperfecta.

We have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization in the oim mouse model for human osteogenesis imperfecta (OI). OI is a heritable disease in which the type I collagen fibrils are either abnormally organized or small, resulting in a clinical presentation of recurrent bone fractures and other pathologies related to collagen-comprised tissues. Exploiting the exquisite sensitivity of SHG to supramolecular assembly, we investigated whether this approach can be utilized to differentiate normal and oim tissues. By comparing SHG intensity, fibrillar morphology, polarization anisotropy, and signal directionality, we show that statistically different results are obtained for the wild type (WT) and disease states in bone, tendon, and skin. All these optical signatures are consistent with the collagen matrix in the oim tissues being more disordered, and these results are further consistent with the known weaker mechanical properties of the oim mouse. While the current work shows the ability of SHG to differentiate normal and diseased states in a mouse model, we suggest that our results provide a framework for using SHG as a clinical diagnostic tool for human OI. We further suggest that the SHG metrics described could be applied to other connective tissue disorders that are characterized by abnormal collagen assembly.

Coherent and incoherent SHG in fibrillar cellulose matrices.

Second Harmonic Generation (SHG) microscopy probes the organization of tissue or material structure through morphological and polarization analyses. In terms of diagnostic or analytical potential, it is important to understand the coherent and incoherent aspects of the emission in highly scattering environments. It is also of fundamental importance whether the SHG polarization signatures are retained in such turbid media. We examine these issues for purified cellulose specimens, which, in analogy to structural proteins, comprise highly birefringent and chiral fibrillar structures. In these matrices we observe predominantly coherent forward directed emission as well as backwards contrast consisting of direct, coherent emission and an incoherent component arising from multiply scattered forward directed SHG. These processes display a pronounced depth dependence evidenced by changes in morphology as well in the measured forward-backwards ratio (F/B). Specifically, from regions near the surface the backwards channel displays small fibrils not present in the forward emission. In addition, at depths beyond one mean free path, the fibril morphologies become highly similar, suggesting the observed backwards contrast is also comprised of a component that arises from multiple scattering of the initially forward directed wave. The depth dependence of the forward to backward ratio is consistent with Monte Carlo simulations of photon diffusion based on the measured scattering coefficient mus of 75 cm-1 and anisotropy factor, g=0.94 at the SHG wavelength. Consistent with the experimental observations, these simulations indicate that the backwards channel becomes increasingly incoherent with increasing depth into the specimen. We also demonstrate that the polarization dependence of the SHG can be measured through 500 microm of thickness. Similarly, the SHG signal anisotropy is largely preserved through this depth with only a slight depolarization being observed.