We present a novel pipeline for robust optical analysis of fresh human brain tissue. We capture fluorescence signal and characterize optical properties with high sensitivity and high spectral resolution. These in turn allow for quantitative analysis of protoporphyrin IX (PpIX) accumulation in various types of tumor tissue. Our ex vivo protocol for tissue handling was designed to promote high-fidelity replication of in vivo conditions. The on-going consolidation of a fresh ex vivo quantitative dataset from a cohort of 20 patients plus a control cohort lays foundation for the development of imaging devices for intraoperative fluorescence guided resection.
Three major one layer tissue models (Modified Beer-Lambert,1 Jacques 1999,2 Pilon 20093) are compared to Monte Carlo simulated diffuse reflectance spectra and measured tissue phantom spectra with known ground truth. These ground truth values were obtained using inverse adding doubling and absorbance measurements and validated using a phantom with known ground truth (BioPixs). Finally, a two layer model (Pilon 2009) was evaluated against Monte Carlo simulations and used to analyse skin reflectance data (NIST4). These models were compared on goodness of fit and parameter extraction accuracy. It was found that the Pilon 2009 one layer model performed best against Monte Carlo simulations and phantom measurements, however the Pilon 2009 two layer model had significant regions of inaccuracy. These inaccurate regions correspond to circumstances where the epidermal layer has significant thickness and melanin content, while the dermal layer has low fraction of blood meaning that the haemoglobin impact is “masked”. The extraction of parameters from the NIST skin dataset using this model returns values that do not correspond well to literature values suggesting that many of these spectra lie within an inaccurate region or indicates oversimplification of the tissue modelling. This suggests both Pilon 2009 and Jacques 1999 are suitable for modelling tissue that can be approximated as a single, homogeneous, semi-infinite slab, however the Pilon 2009 two layer model is not yet effective when encountering empirical data.
PurposeHyperspectral imaging shows promise for surgical applications to non-invasively provide spatially resolved, spectral information. For calibration purposes, a white reference image of a highly reflective Lambertian surface should be obtained under the same imaging conditions. Standard white references are not sterilizable and so are unsuitable for surgical environments. We demonstrate the necessity for in situ white references and address this by proposing a novel, sterile, synthetic reference construction algorithm.ApproachThe use of references obtained at different distances and lighting conditions to the subject were examined. Spectral and color reconstructions were compared with standard measurements qualitatively and quantitatively, using ΔE and normalized RMSE, respectively. The algorithm forms a composite image from a video of a standard sterile ruler, whose imperfect reflectivity is compensated for. The reference is modeled as the product of independent spatial and spectral components, and a scalar factor accounting for gain, exposure, and light intensity. Evaluation of synthetic references against ideal but non-sterile references is performed using the same metrics alongside pixel-by-pixel errors. Finally, intraoperative integration is assessed though cadaveric experiments.ResultsImproper white balancing leads to increases in all quantitative and qualitative errors. Synthetic references achieve median pixel-by-pixel errors lower than 6.5% and produce similar reconstructions and errors to an ideal reference. The algorithm integrated well into surgical workflow, achieving median pixel-by-pixel errors of 4.77% while maintaining good spectral and color reconstruction.ConclusionsWe demonstrate the importance of in situ white referencing and present a novel synthetic referencing algorithm. This algorithm is suitable for surgery while maintaining the quality of classical data reconstruction.
In the brain tumour resection surgery, functional brain mapping (FBM) has been adapted in the contemporary neurosurgical workflow for improved surgical outcome. It allows neurosurgeons to identify and preserve brain regions with critical functions, thus to prevent post-surgical neurological deficits. However, there is no effective ways to directly visualize brain functions in real-time intraoperatively. A compact functional brain imaging device that can be more easily utilised during surgery is highly desired. We’ve been developing a multispectral imaging system (MSI) with the aim to fill this unmet clinical need. The device has been designed to detect brain functional activity by characterising light reflectance changes due to the blood oxygenation/flow fluctuation and neuronal membrane change. Co-localised MSI and fMRI measurements have been performed for detecting brain response to external electrical stimulations on preclinical animal models. Results suggested MSI could be able to identify the stimulation-evoked brain region as validated by the fMRI findings
Emerging optical imaging techniques such as hyperspectral imaging (HSI) provide a promising non-invasive solution for intraoperative tissue characterisation with the potential to provide rich tissue-differentiation information over the entire surgical field. Neuro-oncology surgery would especially benefit from detailed real-time in vivo tissue characterisation, improving the accuracy with which boundaries of safe surgical resection are delineated and thereby improving patient outcomes. Current systems are limited by challenges with processing the HSI data because of incomplete characterisation of the optical properties of tissue across the complete visible and near-infrared wavelength spectrum. In this study, we characterised the optical properties of various freshly-excised brain tumours and normal cadaveric human brain tissue using a dual-beam integrating sphere spectrophotometer and the inverse adding-doubling technique. We adapted an integrating sphere to analyse 2 mm-thick tissue samples measuring 4 – 7 mm in diameter and validated the experimental setup with a tissue-mimicking optical phantom. We investigated the different spectral signatures of freshly-excised tumour tissues including pituitary adenoma, meningioma and vestibular schwannoma and compared these to normal grey and white matter, pons, pituitary, dura and cranial nerve tissues across the wavelength range of 400 – 1800 nm. It was found that brain and tumour tissues could be differentiated by their optical properties but the freezing process did alter the tissues’ relative absorption and reduced scattering coefficients. In this work, we have demonstrated a method to characterise the optical properties of small human brain and tumour specimens that may be used as a reference dataset for developing optical imaging techniques.
Fluorescence-guided brain tumour resection, notably using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) for high-grade gliomas, has been demonstrated to provide better tissue differentiation, thereby improving patient outcomes when compared to white-light guidance. Novel fluorescence imaging devices aiming to increase detection specificity and sensitivity and targeting applications beyond high-grade gliomas are typically assessed by measurements using tissue-mimicking optical phantoms. The field currently lacks adequate phantoms with well-characterised tuneable optical properties. In this study, we developed soft tissue-mimicking fluorescence phantoms (TMFP) highly suitable for this purpose. We investigated: 1) the ability to independently tune optical and fluorescent properties; 2) the stability of the fluorescence signal over time; and 3) the potential of the proposed phantoms for imaging device validation. The TMFP is based on gel-wax which is an optically transparent mineral-oil based soft material. We embedded TiO2 as scattering material, carbon black oil-paint as background absorber, and CdTe Quantum Dots (QDs) as fluorophore because of its similar fluorescence spectrum to PpIX. Scattering and absorption properties were measured by a spectrophotometer, while the fluorescence was assessed by a wide-field fluorescence imaging system (WFFI) and a spectrometer. We demonstrated that: 1) the addition of QDs didn’t alter the phantom’s scattering which was only defined by the concentration of TiO2, whereas its absorption was defined by both QDs and colour oil paint; 2) the measured fluorescence intensity was linearlyproportional to the concentration of QDs; 3) the fluorescence intensity was stable over time (up to eight months); and 4) the fluorescence signal measured by the WFFI were strongly correlated to spectrometer measurements.
In high-grade glioma surgery, tumor resection is often guided by intraoperative fluorescence imaging. 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) provides fluorescent contrast between normal brain tissue and glioma tissue, thus achieving improved tumor delineation and prolonged patient survival compared with conventional white-light-guided resection. However, commercially available fluorescence imaging systems rely solely on visual assessment of fluorescence patterns by the surgeon, which makes the resection more subjective than necessary. We developed a wide-field spectrally resolved fluorescence imaging system utilizing a Generation II scientific CMOS camera and an improved computational model for the precise reconstruction of the PpIX concentration map. In our model, the tissue’s optical properties and illumination geometry, which distort the fluorescent emission spectra, are considered. We demonstrate that the CMOS-based system can detect low PpIX concentration at short camera exposure times, while providing high-pixel resolution wide-field images. We show that total variation regularization improves the contrast-to-noise ratio of the reconstructed quantitative concentration map by approximately twofold. Quantitative comparison between the estimated PpIX concentration and tumor histopathology was also investigated to further evaluate the system.
5-ALA-PpIX fluorescence-guided brain tumour resection can increase the accuracy at which cancerous tissue is removed and thereby improve patient outcomes, as compared with standard white light imaging. Novel optical devices that aim to increase the specificity and sensitivity of PpIX detection are typically assessed by measurements in tissue-mimicking optical phantoms of which all optical properties are defined. Current existing optical phantoms specified for PpIX lack consistency in their optical properties, and stability with respect to photobleaching, thus yielding an unstable correspondence between PpIX concentration and the fluorescence intensity.
In this study, we developed a set of aqueous-based phantoms with different compositions, using deionised water or PBS buffer as background medium, intralipid as scattering material, bovine haemoglobin as background absorber, and either PpIX dissolved in DMSO or a novel nanoparticle with similar absorption and emission spectrum to PpIX as the fluorophore. We investigated the phantom stability in terms of aggregation and photobleaching by comparing with different background medium and fluorophores, respectively. We characterised the fluorescence intensity of the fluorescent nanoparticle in different concentration of intralipid and haemoglobin and its time-dependent stability, as compared to the PpIX-induced fluorescence. We corroborated that the background medium was essential to prepare a stable aqueous phantom. The novel fluorescent nanoparticle used as surrogate fluorophore of PpIX presented an improved temporal stability and a reliable correspondence between concentration and emission intensity. We proposed an optimised phantom composition and recipe to produce reliable and repeatable phantom for validation of imaging device.
In glioma resection surgery, the detection of tumour is often guided by using intraoperative fluorescence imaging notably with 5-ALA-PpIX, providing fluorescent contrast between normal brain tissue and the gliomas tissue to achieve improved tumour delineation and prolonged patient survival compared with the conventional white-light guided resection. However, the commercially available fluorescence imaging system relies on surgeon’s eyes to visualise and distinguish the fluorescence signals, which unfortunately makes the resection subjective. In this study, we developed a novel multi-scale spectrally-resolved fluorescence imaging system and a computational model for quantification of PpIX concentration. The system consisted of a wide-field spectrally-resolved quantitative imaging device and a fluorescence endomicroscopic imaging system enabling optical biopsy. Ex vivo animal tissue experiments as well as human tumour sample studies demonstrated that the system was capable of specifically detecting the PpIX fluorescent signal and estimate the true concentration of PpIX in brain specimen.
Neural microelectrodes are well established tools for delivering therapeutic electrical pulses, and recording neural electrophysiological signals. However, long term implanted neural probes often become functionally impaired by tissue encapsulation. At present, analyzing this immune reaction is only feasible with post-mortem histology; currently no means for specific in vivo monitoring exist and most applicable imaging modalities provide no sufficient resolution for a cellular measurement in deep brain regions. Optical coherence tomography (OCT) is a well developed imaging modality, providing cellular resolution and up to 1.2 mm imaging depth in brain tissue. Further more, a fiber based spectral domain OCT was shown to be capable of minimally invasive brain intervention. In the present study, we propose to use a fiber based spectral domain OCT to monitor the the progression of the tissue's immune response and scar encapsulation of microprobes in a rat animal model. We developed an integrated OCT fiber catheter consisting of an implantable ferrule based fiber cannula and a fiber patch cable. The fiber cannula was 18.5 mm long, including a 10.5 mm ceramic ferrule and a 8.0 mm long, 125 μm single mode fiber. A mating sleeve was used to fix and connect the fiber cannula to the OCT fiber cable. Light attenuation between the OCT fiber cable and the fiber cannula through the mating sleeve was measured and minimized. The fiber cannula was implanted in rat brain together with a microelectrode in sight used as a foreign body to induce the brain tissue immune reaction. Preliminary data showed a significant enhancement of the OCT backscattering signal during the brain tissue scarring process, while the OCT signal of the flexible microelectrode was getting weaker consequentially.
A well established navigation method is one of the key conditions for successful brain surgery: It should be accurate,
safe and online operable. Recent research shows that Optical Coherence Tomography is a potential solution for this
application by providing a high resolution and small probe dimension. In this study a fiber Spectral-Domain OCT system
with a super luminescent diode with the center wavelength of 840 nm providing 13.6 μm axial resolution was used. A
single mode fiber (Ø 125 μm) was employed as the detecting probe. The information acquired by OCT was reconstructed
into grayscale images by vertically aligning several A-scans from the same trajectory with different depth, i.e. forward
scanning. For scans of typical white matter, the images showed a higher reflection of light intensity with lower
penetration depth as well as a steeper attenuation rate compared to the scans typical for grey matter. Since the axial
resolution of this OCT system is very high, some microstructures lying on the striatum, hippocampus and thalamic
nucleus were visible in these images. The research explored the potential of OCT to be integrated into a stereotactic
surgical robot as a multi-modal navigation method.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.