Interstitial imaging with multiple diffusive reflectance spectroscopy projections for in vivo blood vessels detection during brain needle biopsy procedures.

Blood vessel injury during image-guided brain biopsy poses a risk of hemorrhage. Approaches that reduce this risk may minimize related patient morbidity. We present here an intraoperative imaging device that has the potential to detect the brain vasculature in situ. The device uses multiple diffuse reflectance spectra acquired in an outward-viewing geometry to detect intravascular hemoglobin, enabling the construction of an optical image in the vicinity of the biopsy needle revealing the proximity to blood vessels. This optical detection system seamlessly integrates into a commercial biopsy system without disrupting the neurosurgical clinical workflow. Using diffusive brain tissue phantoms, we show that this device can detect 0.5-mm diameter absorptive carbon rods up to ∼ 2 mm from the biopsy window. We also demonstrate feasibility and practicality of the technique in a clinical environment to detect brain vasculature in an in vivo model system. In situ brain vascular detection may add a layer of safety to image-guided biopsies and minimize patient morbidity.

Fluorescence hyperspectral imaging for live monitoring of multiple spheroids in microfluidic chips.

Tumor spheroids represent a realistic 3D in vitro cancer model because they provide a missing link between monolayer cell culture and live tissues. While microfluidic chips can easily form and assay thousands of spheroids simultaneously, few commercial instruments are available to analyze this massive amount of data. Available techniques to measure spheroid response to external stimuli, such as confocal imaging and flow cytometry, are either not appropriate for 3D cultures, or destructive. We designed a wide-field hyperspectral imaging system to analyze multiple spheroids trapped in a microfluidic chip in a single acquisition. The system and its fluorescence quantification algorithm were assessed using liquid phantoms mimicking spheroid optical properties. Spectral unmixing was tested on three overlapping spectral entities. Hyperspectral images of co-culture spheroids expressing two fluorophores were compared with confocal microscopy and spheroid growth was measured over time. The system can spectrally analyze multiple fluorescent markers simultaneously and allows multiple time-points assays, providing a fast and versatile solution for analyzing lab on a chip devices.

Radiometric model for coaxial single- and multimode optical emission from double-clad fiber.

Double-clad fibers (DCFs) are versatile waveguides supporting a single-mode core surrounded by a multimode inner cladding. DCFs are increasingly used for multimodal biomedical applications, such as imaging or therapy, for which the core is typically used for coherent illumination and the inner cladding, to support a concurrent modality. Proper optimization is, however, critical to ensure high optical performance and requires accurate modeling of coaxial single- and multimode output beams. In this paper, we present an approach based on geometrical optics and radiometry, which provides a simple and efficient modeling tool for designing and optimizing DCF-based systems. A radiometric definition of single- and multimode output beams in terms of irradiance and radiant intensity allows for the modeling of the energy distribution along the beams' propagation. We confirmed the validity of the model through comparison with experimental measurements and demonstrate the use of the model for optimizing a catheter for concurrent OCT and laser coagulation.

Development and characterization of a handheld hyperspectral Raman imaging probe system for molecular characterization of tissue on mesoscopic scales.

PURPOSE: Raman spectroscopy is a promising cancer detection technique for surgical guidance applications. It can provide quantitative information relating to global tissue properties associated with structural, metabolic, immunological, and genetic biochemical phenomena in terms of molecular species including amino acids, lipids, proteins, and nucleic acid (DNA). To date in vivo Raman spectroscopy systems mostly included probes and biopsy needles typically limited to single-point tissue interrogation over a scale between 100 and 500 microns. The development of wider field handheld systems could improve tumor localization for a range of open surgery applications including brain, ovarian, and skin cancers. METHODS: Here we present a novel Raman spectroscopy implementation using a coherent imaging bundle of fibers to create a probe capable of reconstructing molecular images over mesoscopic fields of view. Detection is performed using linear scanning with a rotation mirror and an imaging spectrometer. Different slits widths were tested at the entrance of the spectrometer to optimize spatial and spectral resolution while preserving sufficient signal-to-noise ratios to detect the principal Raman tissue features. The nonbiological samples, calcite and polytetrafluoroethylene (PTFE), were used to characterize the performance of the system. The new wide-field probe was tested on ex vivo samples of calf brain and swine tissue. Raman spectral content of both tissue types were validated with data from the literature and compared with data acquired with a single-point Raman spectroscopy probe. The single-point probe was used as the gold standard against which the new instrument was benchmarked as it has already been thoroughly validated for biological tissue characterization. RESULT: We have developed and characterized a practical noncontact handheld Raman imager providing tissue information at a spatial resolution of 115 microns over a field of view >14 mm2 and a spectral resolution of 6 cm-1 over the whole fingerprint region. Typical integration time to acquire an entire Raman image over swine tissue was set to approximately 100 s. Spectra acquired with both probes (single-point and wide-field) showed good agreement, with a Pearson correlation factor >0.85 over different tissue categories. Protein and lipid content of imaged tissue were manifested into the measured spectra which correlated well with previous findings in the literature. An example of quantitative molecular map is presented for swine tissue and calf brain based on the ratio of protein-to-lipid content showing clear delineations between white and gray matter as well as between adipose and muscle tissue. CONCLUSION: We presented the development of a Raman imaging probe with a field of view of a few millimeters and a spatial resolution consistent with standard surgical imaging methods using an imaging bundle. Spectra acquired with the newly developed system on swine tissue and calf brain correlated well with an establish single-point probe and observed spectral features agreed with previous finding in the literature. The imaging probe has demonstrated its ability to reconstruct molecular images of soft tissues. The approach presented here has a lot of potential for the development of surgical Raman imaging probe to guide the surgeon during cancer surgery.

Combined optical coherence tomography and hyperspectral imaging using a double-clad fiber coupler.

This work demonstrates the combination of optical coherence tomography (OCT) and hyperspectral imaging (HSI) using a double-clad optical fiber coupler. The single-mode core of the fiber is used for OCT imaging, while the inner cladding of the double-clad fiber provides an efficient way to capture the reflectance spectrum of the sample. The combination of both methods enables three-dimensional acquisition of the sample morphology with OCT, enhanced with complementary molecular information contained in the hyperspectral image. The HSI data can be used to highlight the presence of specific molecules with characteristic absorption peaks or to produce true color images overlaid on the OCT volume for improved tissue identification by the clinician. Such a system could be implemented in a number of clinical endoscopic applications and could improve the current practice in tissue characterization, diagnosis, and surgical guidance.

Tri-modal microscope for head and neck tissue identification.

A novel tri-modal microscope combining optical coherence tomography (OCT), spectrally encoded confocal microscopy (SECM) and fluorescence imaging is presented. This system aims at providing a tool for rapid identification of head and neck tissues during thyroid surgery. The development of a dual-wavelength polygon-based swept laser allows for synchronized, co-registered and simultaneous imaging with all three modalities. Further ameliorations towards miniaturization include a custom lens for optimal compromise between orthogonal imaging geometries as well as a double-clad fiber coupler for increased throughput. Image quality and co-registration is demonstrated on freshly excised swine head and neck tissue samples to illustrate the complementarity of the techniques for identifying signature cellular and structural features.

Double-clad fiber coupler for partially coherent detection.

Double-clad fibers (DCF) have many advantages in fibered confocal microscopes as they allow for coherent illumination through their core and partially coherent detection through their inner cladding. We report a double-clad fiber coupler (DCFC) made from small inner cladding DCF that preserves optical sectioning in confocal microscopy while increasing collection efficiency and reducing coherent effects. Due to the small inner cladding, previously demonstrated fabrication methods could not be translated to this coupler's fabrication. To make such a coupler possible, we introduce in this article three new design concepts. The resulting DCFC fabricated using two custom fibers and a modified fusion-tapering technique achieves high multimodal extraction (≥70 %) and high single mode transmission (≥80 %). Its application to reflectance confocal microscopy showed a 30-fold increase in detected signal intensity, a 4-fold speckle contrast reduction with a penalty in axial resolution of a factor 2. This coupler paves the way towards more efficient confocal microscopes for clinical applications.

Toward an automated method for optical coherence tomography characterization.

With the increasing use of optical coherence tomography (OCT) in biomedical applications, robust yet simple methods for calibrating and benchmarking a system are needed. We present here a procedure based on a calibration object complemented with an algorithm that analyzes three-dimensional OCT datasets to retrieve key characteristics of an OCT system. The calibration object combines state-of-the-art tissue phantom material with a diamond-turned aluminum multisegment mirror. This method is capable of determining rapidly volumetric field-of-view, axial resolution, and image curvature. Moreover, as the phantom material mimics biological tissue, the system's signal and noise levels can be evaluated in conditions close to biological experiments. We believe this method could improve OCT quantitative data analysis and help OCT data comparison for longitudinal or multicenter studies.

Optical coherence tomography for the identification of musculoskeletal structures of the spine: a pilot study.

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine requiring in severe cases invasive surgery. Here, we explore the potential of optical coherence tomography (OCT) as a guiding tool for novel fusionless minimally invasive spinal surgeries on an ex vivo porcine model. We show that OCT, despite its limited penetration depth, may be used to precisely locate structures such as growth plate, bone and intervertebral disk using relative attenuation coefficients. We further demonstrate a segmentation algorithm that locates growth plates automatically on en-face OCT reconstructions.

Double-clad fiber with a tapered end for confocal endomicroscopy.

We present a double-clad fiber coupler (DCFC) for use in confocal endomicroscopy to reduce speckle contrast, increase signal collection while preserving optical sectioning. The DCFC is made by incorporating a double-clad tapered fiber (DCTF) to a fused-tapered DCFC for achromatic transmission (from 1265 nm to 1325 nm) of > 95% illumination light trough the single mode (SM) core and collection of > 40% diffuse light through inner cladding modes. Its potential for confocal endomicroscopy is demonstrated in a spectrally-encoded imaging setup which shows a 3 times reduction in speckle contrast as well as 5.5 × increase in signal collection compared to imaging with a SM fiber.

Double-clad fiber coupler for endoscopy.

We present a double-clad fiber coupler (DCFC) for use in endoscopy to reduce speckle contrast, increase signal collection and depth of field. The DCFC is made by fusing and tapering two all silica double-clad fiber (DCF) and allows achromatic transmission of >95% of core illumination (1265nm - 1325nm) as well as collection of >42% of inner cladding diffuse light. Its potential for endoscopy is demonstrated in a spectrally encoded imaging setup which shows speckle reduction by a factor 5, increased signal collection by a factor 9 and enhanced depth of field by 1.8 times. Separation by the DCFC of single- and multi-mode signals allows combining low-speckle reflectance images (25.5 fps) with interferometrically measured depth profiles (post-processed) for of small three-dimensional (3D) features through an all-fiber low loss instrument.

Towards a handheld probe based on optical coherence tomography for minimally invasive spine surgeries.

Minimally invasive surgical (MIS) techniques for the correction of scoliosis are under development. The installation of fusionless implants targeting the vertebral growth plate requires precise identification of spinal micro-structures. During ex vivo studies, we demonstrate that optical coherence tomography (OCT) allows visualization of spinal tissues including the growth plate, the intervertebral disc and the vertebral body. This study aims at designing a handheld probe using OCT and assessing its potential for use in MIS. An OCT handheld probe was built which satisfies criteria for resolution, penetration and field of view required for spinal MIS techniques. Ex vivo images of rat tail and porcine vertebrae enabled differentiating musculoskeletal tissues of the spine (growth plate, intervertebral disc and vertebral body). Pending in vivo studies on porcine models, we evaluated the probe on a human finger and demonstrated its ability to image human tissues at video rate (25 fps) with proper imaging depth and resolution. These preliminary results showed the potential of the OCT probe for dynamic and precise imaging of spinal tissues.

Rapid spectrally encoded fluorescence imaging using a wavelength-swept source.

We present rapid imaging of fluorescent samples using spectral encoding (SE). A near-IR wavelength-swept source in used to preserve the SE of the position, despite Stokes shifts. To validate this approach, we imaged fluorescent PbS quantum dot solutions at concentrations down to 0.5+/-0.1micromol/L. This simple configuration allowed acquisition rates of up to 9920 lines of 1024 pixels per second to create high-resolution images. This spectrally encoded setup could be easily miniaturized for endoscopy, thus combining high-resolution fluorescence with confocal reflectance imaging at unmatched acquisition speed.

[Recent approaches of quantification of interstitial fibrosis in renal transplantation]. / Apports récents des techniques de quantification de la fibrose pour l'examen anatomo-pathologique en transplantation rénale.

Chronic allograft injury can be diagnosed early at a pre-clinical stage by its histopathological changes. Interstitial fibrosis (IF), one of its main histopathological features, is currently assessed by semi-quantitative analysis according to the Banff classification. Subjective interpretation by the pathologist is the main limiting factor of such a method. Different morphometric approaches have been used to quantify IF. Point counting methods have been published but their use is very tedious. Thus, semi-automatic computerized measurements of IF in biopsy specimens immunostained for collagen III have been proposed. Most authors used Sirius Red-stained tissue examined under polarized or non-polarized light. However, morphometric methods are time-consuming. Automatic color segmentation image analysis is a new, rapid, and robust method to quantify IF on renal biopsies stained by light green trichrome. It can be recommended as being reliable and reproducible for routine use. Methodology based on second harmonic generation microscopy allows specific quantitative imaging of interstitial fibrosis with high reproducibility and may bring complementary informations in the future. The prognostic value of quantitative image analysis might be increased by techniques addressing the dynamics of fibrosis matrix generation.

Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling.

Interstitial fibrosis is a powerful pejorative predictor of progression of nephropathies in a variety of chronic renal diseases. It is characterized by the depletion of kidney cells and their replacement by extracellular matrix, in particular, type-I fibrillar collagen, a protein scarce in normal interstitium. However, assessment of fibrosis remains a challenge in research and clinical pathology. We develop a novel methodology based on second harmonic generation (SHG) microscopy, and we image collagen fibers in human and mouse unstained kidneys. We take into account the variability in renal shape, and we develop automated image processing for quantitative scoring of thick murine tissues. This approach allows quantitative 3-D imaging of interstitial fibrosis and arterial remodeling with high accuracy. Moreover, SHG microscopy helps to raise pathophysiological questions. First, imaging of a large volume within a mouse kidney shows that progression of fibrosis is a heterogeneous process throughout the different renal compartments. Second, SHG from fibrillar collagens does not overlap with the glomerular tuft, despite patent clinical and experimental glomerulosclerosis. Since glomerulosclerosis involves SHG-silent nonfibrillar collagens, our work supports pathophysiological differences between interstitial fibrosis and glomerulosclerosis, a clearly nonfibrotic process.

[Second- and third-harmonic generation microscopies for the structural imaging of intact tissues]. / Microscopies multi-harmoniques pour l'imagerie structurale de tissus intacts.

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.