Search

We report a rapid hyperspectral fluorescence lifetime imaging (FLIM) instrument that exploits high-speed FLIM technology in a line-scanning microscope. We demonstrate the acquisition of whole-field optically sectioned hyperspectral fluorescence lifetime image stacks (with 32 spectral bins) in less than 40 s and illustrate its application to unstained biological tissue. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

Funding for this project was provided by the ERC grant StG242991. GNRs are emerging as a new class of probes for theradiagnostic applications thanks to their unique optical properties. However, the achievement of proper nanoconstructs requires the synthesis of highly pure GNRs with well-defined aspect ratio (AR), in addition to extensive surface chemistry modification to provide them with active targeting and, possibly, multifunctionality. In this work, we refined the method of the seed mediated growth and developed a robust procedure for the fabrication of GNRs with specific AR. We also revealed and characterized unexplored aging phenomena that follow the synthesis and consistently alter GNRs' final AR. Such advances appreciably improved the feasibility of GNRs fabrication and offered useful insights on the growth mechanism. We next produced fluorescent, biocompatible, aptamer-conjugated GNRs by performing ligand exchange followed by bioconjugation to anti-cancer oligonucleotide AS1411. In vitro studies showed that our nanoconstructs selectively target cancer cells while showing negligible cytotoxicity. As a result, our aptamer-conjugated GNRs constitute ideal cancer-selective multifunctional probes and promising candidates as photothermal therapy agents. Publisher PDF

Intraoperative monitoring of tissue oxygen saturation (StO2 ) has potentially important applications in procedures such as organ transplantation or colorectal surgery, where successful reperfusion affects the viability and integrity of repaired tissues. In this paper a liquid crystal tuneable filter-based multispectral imaging (MSI) laparoscope is described. Motion-induced image misalignments are reduced, using feature-based registration, before regression of the tissue reflectance spectra to calculate relative quantities of oxy- and deoxyhaemoglobin. The laparoscope was validated in vivo, during porcine abdominal surgery, by making parallel MSI and blood gas measurements of the small bowel vasculature. Ischaemic conditions were induced by local occlusion of the mesenteric arcade and monitored using the system. The MSI laparoscope was capable of measuring StO2 over a wide range (30-100%) with a temporal error of ± 7.5%. The imager showed sensitivity to spatial changes in StO2 during dynamic local occlusions, as well as tracking the recovery of tissues post-occlusion.

In clinical examinations, the tissue surface topology is an important feature for detecting the tissue pathology and implementing augmented reality. We have previously presented a miniaturised structured light (SL) system for recovery of tissue surface shape in minimally invasive surgery (MIS), based on a flexible multispectral structured illumination probe (1.9 mm diameter) (Clancy et al. in Biomed Opt Express 2(11):3119–3128, 2011. doi: 10.1364/BOE.2.003119 ). This paper reports further hardware and analytical developments to improve the light pattern decoding result and increase the reconstruction accuracy. The feasibility of using an 8-band multispectral camera with higher pattern-colour discrimination ability than normal RGB camera in this system was studied. Additionally, the “normalised cut” algorithm was investigated to improve pattern segmentation. The whole SL system was evaluated by phantom and in vivo experiments. Higher pattern identification performance than that of an RGB camera was recorded by using the multispectral camera (average precision >97 %, average sensitivity >62 %). An average of $$0.88\,\pm \,0.67\,\hbox {mm}$$ reconstruction error was achieved using the proposed pattern decoding method on a heart phantom at a working distance of approximately 10 cm. The experiment showed the superiority of the multispectral camera over the RGB camera in the spot identification step. The proposed pattern decoding algorithm underwent evaluations using different experiments, showing that it provided promising reconstruction results. The potential of using this system in MIS environments has been demonstrated.

Delivery of radiofrequency (RF) electrical energy is used during surgery to heat and seal tissue, such as vessels, allowing resection without blood loss. Recent work has suggested that this approach may be extended to allow surgical attachment of larger tissue segments for applications such as bowel anastomosis. In a large series of porcine surgical procedures bipolar RF energy was used to resect and re-seal the small bowel in vivo with a commercial tissue fusion device (Ligasure; Covidien PLC, USA). The tissue was then imaged with a multispectral imaging laparoscope to obtain a spectral datacube comprising both fused and healthy tissue. Maps of blood volume, oxygen saturation and scattering power were derived from the measured reflectance spectra using an optimised light-tissue interaction model. A 60% increase in reflectance of visible light (460-700 nm) was observed after fusion, with the tissue taking on a white appearance. Despite this the distinctive shape of the haemoglobin absorption spectrum was still noticeable in the 460-600 nm wavelength range. Scattering power increased in the fused region in comparison to normal serosa, while blood volume and oxygen saturation decreased. Observed fusion-induced changes in the reflectance spectrum are consistent with the biophysical changes induced through tissue denaturation and increased collagen cross-linking. The multispectral imager allows mapping of the spatial extent of these changes and classification of the zone of damaged tissue. Further analysis of the spectral data in parallel with histopathological examination of excised specimens will allow correlation of the optical property changes with microscopic alterations in tissue structure. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Colonoscopy is the gold standard screening test for colorectal cancer. However, it can miss up to 22% of lesions. One way to reduce this misrate is by guiding the endoscopist's attention towards suspicious areas (red-flagging). In this paper, we present the proof-of-concept of a novel endoscopic imaging device for the scanning of tubular organs such as the colon and the esophagus. The envisaged concept works as an accessory for any conventional flexible endoscope using it as a rail. It can generate a red-flagged map of the whole organ by orbiting and sliding a radial array of optical sensors around and along the endoscope. This concept paves the way for a semi-automated concurrent red-flagging technique for colonoscopy that enhances the endoscopist's situational awareness and reduces misrate.

Cancerous and precancerous growths often exhibit changes in scattering, and therefore depolarization, as well as collagen breakdown, causing changes in birefringent effects. Simple difference of linear polarization imaging is unable to represent anisotropic effects like birefringence, and Mueller polarimetry is time-consuming and difficult to implement clinically. This work presents a dual-polarization endoscope to collect co- and cross-polarized images for each of two polarization states, and further incorporates narrow band detection to increase vascular contrast, particularly vascular remodeling present in diseased tissue, and provide depth sensitivity. The endoscope was shown to be sensitive to both isotropic and anisotropic materials in phantom and in vivo experiments.

Intraoperative feedback on tissue function, such as blood volume and oxygenation would be useful to the surgeon in cases where current clinical practice relies on subjective measures, such as identification of ischaemic bowel or tissue viability during anastomosis formation. Also, tissue surface profiling may be used to detect and identify certain pathologies, as well as diagnosing aspects of tissue health such as gut motility. In this paper a dual modality laparoscopic system is presented that combines multispectral reflectance and 3D surface imaging. White light illumination from a xenon source is detected by a laparoscope-mounted fast filter wheel camera to assemble a multispectral image (MSI) cube. Surface shape is then calculated using a spectrally-encoded structured light (SL) pattern detected by the same camera and triangulated using an active stereo technique. Images of porcine small bowel were acquired during open surgery. Tissue reflectance spectra were acquired and blood volume was calculated at each spatial pixel across the bowel wall and mesentery. SL features were segmented and identified using a ‘normalised cut’ algoritm and the colour vector of each spot. Using the 3D geometry defined by the camera coordinate system the multispectral data could be overlaid onto the surface mesh. Dual MSI and SL imaging has the potential to provide augmented views to the surgeon supplying diagnostic information related to blood supply health and organ function. Future work on this system will include filter optimisation to reduce noise in tissue optical property measurement, and minimise spot identification errors in the SL pattern.

Tissue optical and mechanical properties are correlated to tissue pathologic changes. This manuscript describes a dual-mode ultrasound modulated optical imaging system capable of sensing local optical and mechanical properties in reflection geometry. The optical characterisation was achieved by the acoustic radiation force assisted ultrasound modulated optical tomography (ARF-UOT) with laser speckle contrast detection. Shear waves generated by the ARF were also tracked optically by the same system and the shear wave speed was used for the elasticity measurement. Tissue mimicking phantoms with multiple inclusions buried at 11 mm depth were experimentally scanned with the dual-mode system. The inclusions, with higher optical absorption and/or higher stiffness than background, were identified based on the dual results and their stiffnesses were quantified. The system characterises both optical and mechanical properties of the inclusions compared with the ARF-UOT or the elasticity measurement alone. Moreover, by detecting the backward scattered light in reflection detection geometry, the system is more suitable for clinical applications compared with transmission geometry.

We present a dual wavelength endoscope which uses Endoscopic Laser Speckle Contrast Analysis (ELASCA) with the aim to image tissue blood flow and perfusion during surgical procedures. In this study we measure speckle decorelation times, which are associated with flow, by imaging speckle patterns at a wide range of detector exposure times. In order to understand the effects of image collection efficiency and sample scattering properties, control experiments with different optical systems were performed by imaging of tissue mimicking phantoms and inferring their flow parameters.

A fluorescence lifetime imaging (FLIM) system is described that utilizes a new compact and low-cost ultrafast laser source based on a gain-switched laser diode-seeded all-solid-state Cr:LiSAF regenerative amplifier that has been designed for this application. The pulse parameters of this source (0.5 μJ, 827 nm, 100 ps, 5 kHz) are shown to be appropriate to time-domain FLIM using a gated optical intensifier and the application to functional imaging of biological tissue is demonstrated, as well as the first evaluation of organic light emitting diodes using FLIM.

Polymer light-emitting diode (LED) structures have been studied using fluorescence lifetime imaging (FLIM). FLIM allows one to image perturbations in the excited state of the luminescent polymer that lead to changes in its fluorescence lifetime. Observation of strong electric field induced luminescence lifetime quenching of a blue emitting polyfluorene type co-polymer enabled the spatial resolution of field strengths. For example, black spots (degradation induced defects in the LED) did not show any lifetime quenching upon applying an electric field in contrast to the surrounding polymer layer. Future applications of this technique in the field of thin film LEDs are suggested.

Wide-field fluorescence lifetime imaging with spectral resolution and optical sectioning has been performed to achieve five-dimensional fluorescence microscopy. Spectral filtering has been shown to have the potential to provide functional information about biological tissue by simultaneously measuring the spectral/lifetime signature of the sample. The potential to use multispectral imaging to separate cellular components spatially by their different emission wavelengths has also been demonstrated thus reducing artefacts in the calculated lifetime maps. The instrument is based on diode-pumped solid-state laser technology and an ultrafast gated optical image intensifier. Also reported is the use of a picosecond blue laser diode as the excitation source to produce a fluorescence lifetime microscope with a footprint of less than 0.25m2.

We have developed a compact system for wide-field fluorescence imaging, resolved in three spatial dimensions, lifetime and wavelength, that is based on a gated optical intensifier and an all-solid-state diode pumped Cr:LiSAF oscillator-amplifier system. Exploiting spectral separation, the system has been applied to human teeth, obtaining good lifetime contrast between enamel, dentin and caries. Exploiting spectral separation combined with depth resolution, the study of fluorescent microspheres led to an enhancement in both lifetime contrast and lateral resolution.

The polar decomposition method for Mueller matrices proposed by Lu–Chipman has been demonstrated and validated for many applications. However, in some situations, e.g., when analyzing the Mueller matrix of birefringent turbid media with Mie-sized scatterers acquired in reflection geometry, the method may suffer from limitations due to the assumptions required by this method. Here we extend the Lu–Chipman method and show that it can provide more reasonable results for these situations. The method has been validated experimentally with turbid phantoms. Thus, this Letter may prove useful in tissue polarimetry.

International audience; Intra-operative imaging techniques for obtaining the shape and morphology of soft-tissue surfaces in vivo are a key enabling technology for advanced surgical systems. Different optical techniques for 3-D surface reconstruction in laparoscopy have been proposed, however, so far no quantitative and comparative validation has been performed. Furthermore, robustness of the methods to clinically important factors like smoke or bleeding has not yet been assessed. To address these issues, we have formed a joint international initiative with the aim of validating different state-of-the-art passive and active reconstruction methods in a comparative manner. In this comprehensive in vitro study, we investigated reconstruction accuracy using different organs with various shape and texture and also tested reconstruction robustness with respect to a number of factors like the pose of the endoscope as well as the amount of blood or smoke present in the scene. The study suggests complementary advantages of the different techniques with respect to accuracy, robustness, point density, hardware complexity and computation time. While reconstruction accuracy under ideal conditions was generally high, robustness is a remaining issue to be addressed. Future work should include sensor fusion and in vivo validation studies in a specific clinical context. To trigger further research in surface reconstruction, stereoscopic data of the study will be made publically available at www.open-CAS.com upon publication of the paper.

Shear wave propagation provides rich information for material mechanical characterization, including elasticity and viscosity. This Letter reports tracking of shear wave propagation in turbid media by laser-speckle-contrast analysis. The theory is described, and a Monte Carlo simulation of light shear wave interaction was developed. Simulation and experiments on tissue-mimicking phantoms agree well and show tracking of shear wave at the phantom surface and at depth as well as multiple shear waves interacting within the object. The relationship between speckle contrast value and shear wave amplitude is also investigated.

Uterine transplantation surgery has been proposed as a treatment for permanent absolute uterine factor infertility (AUFI) in the case of loss of the uterus. Due to the complexity of the vasculature correct reanastomosis of the blood supply during transplantation surgery is a crucial step to ensure reperfusion and viability of the organ. While techniques such as fluorescent dye imaging have been proposed to visualise perfusion there is no gold standard for intraoperative visualisation of tissue oxygenation. In this paper results from a liquid crystal tuneable filter (LCTF)-based multispectral imaging (MSI) laparoscope are described. The system was used to monitor uterine oxygen saturation (SaO2) before and after transplantation. Results from surgeries on two animal models (rabbits and sheep) are presented. A feature-based registration algorithm was used to correct for misalignment induced by breathing or peristalsis in the tissues of interest prior to analysis. An absorption spectrum was calculated at each spatial pixel location using reflectance data from a reference standard, and the relative contributions from oxy- and deoxyhaemoglobin were calculated using a least squares regression algorithm with non-negativity constraints. Results acquired during animal surgeries show that cornual oxygenation changes are consistent with those observed in point measurements taken using a pulse oximeter, showing reduced SaO2 following reanastomosis. Values obtained using the MSI laparoscope were lower than those taken with the pulse oximeter, which may be due to the latter’s use of the pulsatile arterial blood signal. Future work incorporating immunological test results will help to correlate SaO2 levels with surgical outcomes.

Oxygen supply is an important predictor of the integrity and viability of luminal structures after surgery. Multispectral imaging is used to study haemodynamics during small bowel anastomosis and arterio-venous fistula formation surgery.

I will review our development and application of fluorescence lifetime imaging implemented in microscopy, tomography and endoscopy to provide molecular readouts across the scales from super-resolved microscopy through imaging of disease models to clinical applications.

Mueller polarimetric imaging is a promising technique which is able to provide additional polarisation contrast for surgical imaging. In this work, improved 3t3 and 4t4 Mueller matrix endoscopic systems were constructed and tested with tissue samples.

Endoscopic procedures provide important information about the internal patient anatomy but are currently restricted to a 2D texture analysis of the visible organ surfaces. Spectral imaging has high potential in generating valuable complementary information about the molecular tissue composition but suffers from long image acquisition times. As the technique requires an aligned stack of images, its benefit in endoscopic procedures is still very limited due to continuous motion of the camera and the tissue. In this paper, we present an information theory based approach to band selection for endoscopic spectral imaging. In contrast to previous approaches, our concept does not require labelled training data or an elaborate light-tissue interaction model. According to a validation study using phantom data as well as in vivo spectral data obtained from five surgeries in a porcine model, only few bands selected by our method are sufficient to reconstruct the tissue composition with a similar accuracy as obtainable with the full spectrum.

One of the main challenges for computer-assisted surgery (CAS) is to determine the intra-opera- tive morphology and motion of soft-tissues. This information is prerequisite to the registration of multi-modal patient-specific data for enhancing the surgeon’s navigation capabilites by observ- ing beyond exposed tissue surfaces and for providing intelligent control of robotic-assisted in- struments. In minimally invasive surgery (MIS), optical techniques are an increasingly attractive approach for in vivo 3D reconstruction of the soft-tissue surface geometry. This paper reviews the state-of-the-art methods for optical intra-operative 3D reconstruction in laparoscopic surgery and discusses the technical challenges and future perspectives towards clinical translation. With the recent paradigm shift of surgical practice towards MIS and new developments in 3D opti- cal imaging, this is a timely discussion about technologies that could facilitate complex CAS procedures in dynamic and deformable anatomical regions.

Mueller matrix polarimetric imaging has shown potential in tissue diagnosis but is challenging to implement endoscopically. In this work, a narrow band 3 × 3 Mueller matrix polarimetric endoscope was designed by rotating the endoscope to generate 0°, 45° and 90° linearly polarized illumination and positioning a rotating filter wheel in front of the camera containing three polarisers to permit polarization state analysis for backscattered light. The system was validated with a rotating linear polarizer and a diffuse reflection target. Initial measurements of 3 × 3 Mueller matrices on a rat are demonstrated, followed by matrix decomposition into the depolarization and retardance matrices for further analysis. Our work shows the feasibility of implementing polarimetric imaging in a rigid endoscope conveniently and economically in order to reveal diagnostic information.

Heat-induced tissue fusion via radio-frequency (RF) energy has gained wide acceptance clinically and here we present the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study provides direct insights into tissue constituent and structural changes on the molecular level, exposing spectroscopic evidence for the loss of distinct collagen fibre rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. These findings open the door for more advanced optical feedback-control methods and characterization during heat-induced tissue fusion, which will lead to new clinical applications of this promising technology.

Laser speckle contrast analysis (LASCA) is limited to being a qualitative method for the measurement of blood flow and tissue perfusion as it is sensitive to the measurement configuration. The signal intensity is one of the parameters that can affect the contrast values due to the quantization of the signals by the camera and analog-to-digital converter (ADC). In this paper we deduce the theoretical relationship between signal intensity and contrast values based on the probability density function (PDF) of the speckle pattern and simplify it to a rational function. A simple method to correct this contrast error is suggested. The experimental results demonstrate that this relationship can effectively compensate the bias in contrast values induced by the quantized signal intensity and correct for bias induced by signal intensity variations across the field of view.

Spectral filtration systems based around digital micromirror devices (DMD) have made possible the creation of illumination light with custom spectral profiles for microscopy and endoscopy. However, due to the out-of-plane deviation caused by the DMD these systems have previously required complex optics, and in addition the diffraction effects of the DMD have not been well understood. We constructed a system to simplify the optics and present a full two-dimensional analysis of the diffraction patterns from the DMD together with an analysis of and method to correct the spectral nonlinearity of the system. A comparison between the experimental and theoretical results is provided and full spectral coverage between 430 and 720 nm was demonstrated with the output coupled into a liquid light guide for easy coupling into surgical devices such as laparoscopes.

Sequential multispectral imaging is an acquisition technique that involves collecting images of a target at different wavelengths, to compile a spectrum for each pixel. In surgical applications it suffers from low illumination levels and motion artefacts. A three-channel rigid endoscope system has been developed that allows simultaneous recording of stereoscopic and multispectral images. Salient features on the tissue surface may be tracked during the acquisition in the stereo cameras and, using multiple camera triangulation techniques, this information used to align the multispectral images automatically even though the tissue or camera is moving. This paper describes a detailed validation of the set-up in a controlled experiment before presenting the first in vivo use of the device in a porcine minimally invasive surgical procedure. Multispectral images of the large bowel were acquired and used to extract the relative concentration of haemoglobin in the tissue despite motion due to breathing during the acquisition. Using the stereoscopic information it was also possible to overlay the multispectral information on the reconstructed 3D surface. This experiment demonstrates the ability of this system for measuring blood perfusion changes in the tissue during surgery and its potential use as a platform for other sequential imaging modalities.

Plagiarism is a form of scientific misconduct defined as authoring ideas or words produced by someone else or from one’s own previous publications and attempting to publish such work without properly citing the original author and publication. Plagiarism is easy for writers to avoid by simply not copying any printed sources and by writing original text in one’s own words, and, if paraphrasing, citing the source. Unfortunately, these simple rules seem to be either forgotten or ignored by many authors, as instances of plagiarism have become all too common throughout the scientific world. 1 A charitable view is that authors who are guilty of plagiarism are ignorant of what constitutes plagiarism. Science adheres to certain standards of investigation that are invariant with respect to geography, social convention or personal opinion. Because publishing and disseminating ideas are central to the scientific endeavor and the rules governing writing and publishing data are sacrosanct, it is crucial that we all agree and adhere to common standards regarding plagiarism. As editors and publishers of the Annals of Biomedical Engineering we take seriously our role as a primary ‘‘gatekeeper’’ of scientific integrity. We do not tolerate plagiarism or any kind of associated misconduct, and urge our reviewers and readers to bring to our attention ethical questions or simply queries of what constitutes plagiarism in our manuscripts and published articles. We also expect our authors to exercise stringent care to avoid plagiarism. 1. Don’t copy. Mimicking verbatim words from

Background. Minimally invasive surgical techniques such as single access and natural orifice translumenal endoscopic surgery (NOTES) aim to reduce the number of external scars on the patient but impose restrictions on the space available for the light source within the endoscope and, therefore, the size of the field of view that can be sufficiently illuminated. Materials and Methods. This article presents and compares a number of illumination methods (xenon, light-emitting diodes, laser/phosphor, supercontinuum laser) that could be applied in single-access, robotic, and NOTES procedures. The luminance, spectral content, and intensity profile of each source was measured. Standardized images of each illuminating an abdominal simulator were assessed by a group of surgeons to provide an initial clinical impression. Results. The xenon source was found to have the highest luminance when used with a standard laparoscopic light cable, but this was significantly reduced when used with a small cable suitable for single-access applications. The supercontinuum laser–based light source had brightness comparable to the xenon, which was supported by the surgical test group observations. Conclusions. The supercontinuum fiber probe is a potential alternative to xenon light sources for use in single-access surgery with its comparable luminance, small diameter, flexibility, and even illumination. An initial in vivo test is described, providing a guide for future development.

Organ transplant success is dependent on blood supply health. A multispectral imaging laparoscope has been used to monitor tissue oxygenation changes during a rabbit uterine transplant. A feature tracking algorithm was used to compensate for movement. © OSA 2012.

Light-emitting diode (LED) based endoscopic illumination devices have been shown to have several benefits over arclamp systems. LEDs are energy-efficient, small, durable, and inexpensive, however their use in endoscopy has been limited by the difficulty in efficiently coupling enough light into the endoscopic light cable. We have demonstrated a highly homogenised lightpipe LED light source that combines the light from four Luminus LEDs emitting in the red, green, blue and violet using innovative dichroics that maximise light throughput. The light source spectrally combines light from highly divergent incoherent sources that have a Lambertian intensity profile to provide illumination matched to the acceptance numerical aperture of a liquid light guide or fibre bundle. The LED light source was coupled to a standard laparoscope and performance parameters (power, luminance, colour temperature) compared to a xenon lamp. Although the total illuminance from the endoscope was lower, adjustment of the LEDs' relative intensities enabled contrast enhancement in biological tissue imaging. The LED light engine was also evaluated in a minimally invasive surgery (MIS) box trainer and in vivo during a porcine MIS procedure where it was used to generate 'narrowband' images. Future work using the violet LED could enable photodynamic diagnosis of bladder cancer.

In this paper we present a method to visualize the pressure field of an ultrasound beam in a single shot of the CCD and to image the shear wave propagation based on acousto-optic laser speckle contrast analysis. The contrast images show features in the near field, far field and central region of the ultrasound beam and the pressure profile fits with that measured with a hydrophone. The shear wave propagation was acquired by changing the imaging delay time after the ultrasound burst. This method can be used to study the shear wave properties of common tissue phantoms to guide experiments on tissue.

Computer-aided diagnosis of ophthalmic diseases using optical coherence tomography (OCT) relies on the extraction of thickness and size measures from the OCT images, but such defined layers are usually not observed in emerging OCT applications aimed at "optical biopsy" such as pulmonology or gastroenterology. Mathematical methods such as Principal Component Analysis (PCA) or textural analyses including both spatial textural analysis derived from the two-dimensional discrete Fourier transform (DFT) and statistical texture analysis obtained independently from center-symmetric auto-correlation (CSAC) and spatial grey-level dependency matrices (SGLDM), as well as, quantitative measurements of the attenuation coefficient have been previously proposed to overcome this problem. We recently proposed an alternative approach consisting of a region segmentation according to the intensity variation along the vertical axis and a pure statistical technology for feature quantification. OCT images were first segmented in the axial direction in an automated manner according to intensity. Afterwards, a morphological analysis of the segmented OCT images was employed for quantifying the features that served for tissue classification. In this study, a PCA processing of the extracted features is accomplished to combine their discriminative power in a lower number of dimensions. Ready discrimination of gastrointestinal surgical specimens is attained demonstrating that the approach further surpasses the algorithms previously reported and is feasible for tissue classification in the clinical setting.

Biophotonics encompasses the fundamental interactions of light and biological materials. The field has produced optical imaging, diagnostic and therapeutic devices that have impacted modern medicine. It has provided major advances in biomedical research through development of technologies for high resolution microscopy, and contributed to the progress in molecular labelling through the development of fluorescent proteins, and medically relevant contrast and therapeutic agents. Many of these interdisciplinary activities have involved bioengineers and have grown out of bioengineering departments. This special issue of ABME encompasses research papers at the interface of biophotonics and bioengineering which we have solicited from internationally renowned researchers. This includes a total of 16 review articles to introduce the field to a general audience of bioengineers, and the topics have been selected to cover a broad range of biophotonics research areas. The subjects of endoscopy and tissue optics are covered in reviews by Stoyanov on how computational

A gaze-contingent autofocus system using an eye-tracker and liquid lens has been constructed for use with a surgical robot, making it possible to rapidly (within tens of milliseconds) change focus using only eye-control. This paper reports the results of a user test comparing the eye-tracker to a surgical robot's in-built mechanical focusing system. In the clinical environment, this intuitive interface removes the need for an external mechanical control and improves the speed at which surgeons can make decisions, based on the visible features. Possible applications include microsurgery and gastrointestinal procedures where the object distance changes due to breathing and/or peristalsis.

Nanoparticles are viewed as a promising tool for numerous medical applications, for instance imaging and photothermal therapy (PTT) has been proposed using gold nanorods. We are developing multi-functional gold nanorods (m-GNRs) which have potential for image guided endoscopic surgery of tumour tissue with a modified laparoscope system. A new synthesis method potentially allows any useful acid functionalised molecules to be bonded at the surface. We have created fluorescent m-GNRs which can be used for therapy as they absorb light in the infrared, which may penetrate deep into the tissue and produce localised heating. We have performed a tissue based experiment to demonstrate the feasibility of fluorescence guided PTT using m- GNRs. Ex vivo tests were performed using sheep heart. This measurement, correlated with the fluorescence signal of the m-GNRs measured by the laparoscope allows the clear discrimination of the artery system containing m-GNRs. A laser diode was used to heat the m-GNRs and a thermal camera was able to record the heat distribution. These images were compared to the fluorescence images for validation.

We investigate polarized multispectral endoscopic imaging to detect the micron-sized scatterers, tested with microspheres and animal livers. It is feasible to endoscopically image scatterer size with potential for in vivo diagnosis of dysplasia.

Intraoperative 3D imaging during minimally invasive surgery (MIS) is possible using structured lighting and has applications in the quantification of tissue morphology. However, projection schemes containing various patterns and colours can be disruptive to the surgeon's field of view. In this paper, a stroboscopic system is proposed in which structured lighting and white light images are interleaved during a high-speed camera acquisition so that the patterned light is not perceived and white light can be used solely for navigation and visual assessment. A beam chopper synchronised with the camera switches rapidly between the two lighting modes while still providing video rate display. A spectrally-encoded structured lighting system is provided by an optical fibre-based probe developed in our lab and is suitable for use in endoscopic biopsy channels. In this dual acquisition mode it is possible to display an augmented view so that the centroids of the structured lighting features are visible on the white light image. Sequential acquisition of varying exposure time images with the high speed camera also allowed the generation of high dynamic range images of the wavelength-encoded structured lighting pattern. Possible applications of this work include classification of polyp morphology as an indicator of pathology.

We present a dual-wavelength endoscopic laser speckle contrast system including illumination with polarization maintaining fibres and imaging using a leached fibre image guide. This system has a frame rate of 10 Hz and can rapidly monitor changes in blood flow in vivo, including due to the heart beat, using the contrast values of the speckle images recorded with 1 ms exposure time. In addition the mean intensities can record the respiration period and can indicate changes in tissue oxygenation. This system was tested during an occlusion to a human finger and is being applied in endoscopy.

Ultrasound-mediated optical tomography (UOT) is a hybrid technique that is able to combine the high penetration depth and high spatial resolution of ultrasound imaging to overcome the limits imposed by optical scattering for deep tissue optical sensing and imaging. It has been proposed as a method to detect blood concentrations, oxygenation and metabolism at depth in tissue for the detection of vascularized tumours or the presence of absorbing or scattering contrast agents. In this paper, the basic principles of the method are outlined and methods for simulating the UOT signal are described. The main detection methods are then summarized with a discussion of the advantages and disadvantages of each. The recent focus on increasing the weak UOT signal through the use of the acoustic radiation force is explained, together with a summary of our results showing sensitivity to the mechanical shear stiffness and optical absorption properties of tissue-mimicking phantoms.

The increasing incidence of non-alcoholic fatty liver diseases (NAFLD) and the consequent progression to cirrhosis is expected to become a major cause of liver transplantation. This will exacerbate the organ donor shortage and mean that 'marginal' fatty liver grafts are more frequently used. Autofluorescence spectroscopy is a fast, objective, and non-destructive method to detect change in the endogenous fluorophores distribution and could prove to be a valuable tool for NAFLD diagnosis and transplant graft assessment.A system was constructed consisting of a fibre probe with two laser diodes that provided excitation light at 375 and 405 nm, and an imaging spectrograph system. This was used to distinguish fluorescence spectra acquired from the harvested livers from mice with NAFLD of differing severity (healthy, mild steatotic and steatohepatitic). The fluorescence data were entered into a sparse multiclass probabilistic algorithm for disease classification. Histopathology, thiobarbituric acid reactive substances (TBARS) and alanine transaminase (ALT) assays were conducted in addition to the fluorescence measurementsTBARS and ALT assays enabled differentiation of the steatohepatitic group from the mild steatosis and control groups (P ≤ 0.028) but failed to separate the mild steatotic group from the control group. The three groups were all clearly differentiated from each other using fluorescence spectroscopy, and classification accuracy was found to be 95%.Fluorescence spectroscopy appears to be a promising approach for the analysis of diseased liver tissue.

Ultrasound-modulated optical tomography (UOT) combines optical contrast with ultrasound spatial resolution and has great potential for soft tissue functional imaging. One current problem with this technique is the weak optical modulation signal, primarily due to strong optical scattering in diffuse media and minimal acoustically induced modulation. The acoustic radiation force (ARF) can create large particle displacements in tissue and has been shown to be able to improve optical modulation signals. However, shear wave propagation induced by the ARF can be a significant source of nonlocal optical modulation which may reduce UOT spatial resolution and contrast. In this paper, the time evolution of shear waves was examined on tissue mimicking-phantoms exposed to 5 MHz ultrasound and 532 nm optical radiation and measured with a CCD camera. It has been demonstrated that by generating an ARF with an acoustic burst and adjusting both the timing and the exposure time of the CCD measurement, optical contrast and spatial resolution can be improved by ~110% and ~40% respectively when using the ARF rather than 5 MHz ultrasound alone. Furthermore, it has been demonstrated that this technique simultaneously detects both optical and mechanical contrast in the medium and the optical and mechanical contrast can be distinguished by adjusting the CCD exposure time.

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis.