Ultrahigh resolution optical coherence tomography (UHR-OCT) for differentiating pituitary gland versus adenoma tissue has been investigated for the first time, indicating more than 80 % accuracy. For biomarker identification, OCT images of paraffine embedded tissue are correlated to histopathological slices. The identified biomarkers are verified on fresh biopsies. Additionally, an approach, based on resolution modified UHR-OCT ex vivo data, investigating optical performance parameters for the realization in an in vivo endoscope is presented and evaluated. The identified morphological features – cell groups with reticulin framework – detectable with UHR-OCT showcase a promising differentiation ability, encouraging endoscopic OCT probe development for in vivo application.
Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing. Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers. Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio. Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images. Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.
Fluorescence guided neurosurgery based on 5-aminolevulinic acid (5-ALA) has significantly increased maximal safe resections. Fluorescence lifetime imaging (FLIM) of 5-ALA could further boost this development by its increased sensitivity. However, neurosurgeons require real-time visual feedback which was so far limited in dual-tap CMOS camera based FLIM. By optimizing the number of phase frames required for reconstruction, we here demonstrate real-time 5-ALA FLIM of human high- and low-grade glioma with up to 12 Hz imaging rate over a wide field of view (11.0 x 11.0 mm). Compared to conventional fluorescence imaging, real-time FLIM offers enhanced contrast of weakly fluorescent tissue.
[This corrects the article DOI: 10.1371/journal.pone.0227312.].
Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed. Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging. Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology. Results: Our imaging system enabled macroscopic FLIM of a 6.5 × 6.5 mm2 field of view at spatial resolutions <20 μm. A frame of 512 × 512 pixels with a lifetime accuracy <1 ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology. Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.
Non-muscle-invasive bladder cancer affects millions of people worldwide, resulting in significant discomfort to the patient and potential death. Today, cystoscopy is the gold standard for bladder cancer assessment, using white light endoscopy to detect tumor suspected lesions areas, followed by resection of these areas and subsequent histopathological evaluation. Not only does the pathological examination take days, but due to the invasive nature, the perfomed biopsy can result in significant harm to the patient. Nowadays, optical modalities, such as optical coherence tomography (OCT) or Raman spectroscopy (RS), have proven to detect cancer in real-time and can provide more detailed clinical information of a lesion, e.g. its penetration depth (stage) and the differentiation of the cells (grade). In this paper, we present an ex vivo study performed with a combined piezoelectric tube-based OCT-probe and fiber optic RS-probe imaging system that allows a large field-of-view imaging of bladder biopsies, using both modalities and a co-registered visualization, detection and grading of cancerous bladder lesions. In the present study 119 examined biopsies were characterized, showing that fiber-optic based OCT provides a sensitivity of 78 % and specificity of 69 % for the detection of non-muscle-invasive bladder cancer, while RS, on the other hand, provides a sensitivity of 81 % and specificity of 61 % for the grading of low- and high-grade tissue. Moreover, the study shows that a piezoelectric tube-based OCT-probe can have a significant endurance, suitable for future long-lasting in vivo application. These results also indicate that combined OCT and RS fiber probe-based characterization offers an exciting possibility for label-free and morpho-chemical optical biopsy for bladder cancer diagnostics.
Ion channels are responsible for numerous physiological functions ranging from transport to chemical and electrical signaling. Although static ion channel structure has been studied following a structural biology approach, spatiotemporal investigation of the dynamic molecular mechanisms of operational ion channels has not been achieved experimentally. In particular, the role of water remains elusive. Here, we perform label-free spatiotemporal second harmonic (SH) imaging and capacitance measurements of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by aqueous solution on either side. We observe changes in SH intensity upon channel activation that is traced back to changes in the orientational distribution of water molecules that reorient along the field lines of transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. The time scale of these interfacial changes is influenced by the density of ion channels and is subject to a crowding mechanism. Ion transport along cell membranes is often associated with the propagation of electrical signals in neurons. As our study shows that this process is taking place over seconds, a more complex mechanism is likely responsible for the propagation of neuronal electrical signals than just the millisecond movement of ions.
Frequency doubling of an infrared laser radiation in non-linear optical crystals is a widely used technique to obtain light in the visible range. The second harmonic generation process is influenced by several well-known parameters. In this article we study the effect of group delay dispersion on the second harmonic generation process for femtosecond pulses. We show, both through simulation and experiments, that for certain parameters even a small amount of chirp can have a detrimental effect on the conversion efficiency as well as the second harmonic beam quality. We also check the effect of higher order dispersion. By properly accounting for those effects the crystal length and focusing conditions can be optimized to reach high conversion efficiency, while maintaining low sensitivity to chirp variations and good beam quality.
Pituitary adenomas are neoplasia of the anterior pituitary gland and can be subdivided into hormone-producing tumors (lactotroph, corticotroph, gonadotroph, somatotroph, thyreotroph or plurihormonal) and hormone-inactive tumors (silent or null cell adenomas) based on their hormonal status. We therefore developed a line scan Raman microspectroscopy (LSRM) system to detect, discriminate and hyperspectrally visualize pituitary gland from pituitary adenomas based on molecular differences. By applying principal component analysis followed by a k-nearest neighbor algorithm, specific hormone states were identified and a clear discrimination between pituitary gland and various adenoma subtypes was achieved. The classifier yielded an accuracy of 95% for gland tissue and 84–99% for adenoma subtypes. With an overall accuracy of 92%, our LSRM system has proven its potential to differentiate pituitary gland from pituitary adenomas. LSRM images based on the presence of specific Raman bands were created, and such images provided additional insight into the spatial distribution of particular molecular compounds. Pathological states could be molecularly differentiated and characterized with texture analysis evaluating Grey Level Cooccurrence Matrices for each LSRM image, as well as correlation coefficients between LSRM images.
We present coregistered images of tissue vasculature that allow a direct comparison between the performance of narrow-band imaging (NBI) and optical coherence tomography angiography (OCTA). Images were generated with a bimodal endomicroscope having a size of 15 × 2.4 × 3.3 mm<sup>3</sup> ( l , w , h ) that combines two imaging channels. The white light imaging channel was used to perform NBI, the current gold standard for endoscopic visualization of vessels. The second channel allowed the simultaneous acquisition of optical coherence tomography (OCT) and OCTA images, enabling a three-dimensional (3-D) visualization of morphological as well as functional tissue information. In order to obtain 3-D OCT images scanning of the light-transmitting fiber was implemented by a small piezoelectric tube. A field of view of ∼1.1 mm was achieved for both modalities. Under the assumption that OCTA can address current limitations of NBI, their performance was studied and compared during in vivo experiments. The preliminary results show the potential of OCT regarding an improved visualization and localization of vessel beds, which can be beneficial for diagnosis of pathological conditions. .
Multimodal imaging platforms offer a vast array of tissue information in a single image acquisition by combining complementary imaging techniques. By merging different systems, better tissue characterization can be achieved than is possible by the constituent imaging modalities alone. The combination of optical coherence tomography (OCT) with non-linear optical imaging (NLOI) techniques such as two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) provides access to detailed information of tissue structure and molecular composition in a fast, label-free and non-invasive manner. We introduce a multimodal label-free approach for morpho-molecular imaging and spectroscopy and validate the system in mouse skin demonstrating the potential of the system for colocalized acquisition of OCT and NLOI signals.
We present a combination of optical coherence tomography (OCT) and Raman spectroscopy (RS) for improved diagnosis and discrimination of different stages and grades of bladder cancer ex vivo by linking the complementary information provided by these two techniques. Bladder samples were obtained from biopsies dissected via transurethral resection of the bladder tumor (TURBT). As OCT provides structural information rapidly, it was used as a red-flag technology to scan the bladder wall for suspicious lesions with the ability to discriminate malignant tissue from healthy urothelium. Upon identification of degenerated tissue via OCT, RS was implemented to determine the molecular characteristics via point measurements at suspicious sites. Combining the complementary information of both modalities allows not only for staging, but also for differentiation of low-grade and high-grade cancer based on a multivariate statistical analysis. OCT was able to clearly differentiate between healthy and malignant tissue by tomogram inspection and achieved an accuracy of 71% in the staging of the tumor, from pTa to pT2, through texture analysis followed by k-nearest neighbor classification. RS yielded an accuracy of 93% in discriminating low-grade from high-grade lesions via principal component analysis followed by k-nearest neighbor classification. In this study, we show the potential of a multi-modal approach with OCT for fast pre-screening and staging of cancerous lesions followed by RS for enhanced discrimination of low-grade and high-grade bladder cancer in a non-destructive, label-free and non-invasive way.
An endoscopic optical coherence tomography (OCT) system with a wide field-of-view of 8 mm is presented, which combines the image capability of endoscopic imaging at the middle ear with the advantages of functional OCT imaging, allowing a morphological and functional assessment of the human tympanic membrane. The endoscopic tube has a diameter of 3.5 mm and contains gradient-index optics for simultaneous forward-viewing OCT and video endoscopy. The endoscope allows the three-dimensional visualization of nearly the entire tympanic membrane. In addition, the oscillation of the tympanic membrane is measured spatially resolved and in the frequency range between 500 Hz and 5 kHz with 125 Hz resolution, which is realized by phase-resolved Doppler OCT imaging during acoustical excitation with chirp signals. The applicability of the OCT system is demonstrated in vivo. Due to the fast image acquisition, structural and functional measurements are only slightly affected by motion artifacts.
Achieving a maximal safe extent of resection during brain tumor surgery is the goal for improved patient prognosis. Fluorescence‐guided neurosurgery using 5‐aminolevulinic acid (5‐ALA) induced Protoporphyrin IX has thereby become a valuable tool enabling a high frequency of complete resections and a prolonged progression‐free survival in glioblastoma patients. We present a widefield fluorescence lifetime imaging device with 250 mm working distance working under similar conditions like surgical microscopes based on a time‐of‐flight based dual tap CMOS camera. In contrast to intensity‐based fluorescence imaging our method is invariant to light scattering and absorption while being sensitive to the molecular composition of the tissue. We evaluate the feasibility of lifetime imaging of Protoporphyrin IX using our system to analyze brain tumor phantoms and fresh 5‐ALA labeled human tissue samples. The results demonstrate the potential of our lifetime sensing device to go beyond the limitation of current intensity‐based fluorescence‐guided neurosurgery. This article is protected by copyright. All rights reserved.
The interaction of oils and lipids is relevant for membrane biochemistry since the cell uses bilayer membranes, lipid droplets and oily substances in its metabolic cycle. In addition, a variety of model lipid membrane systems, such as freestanding horizontal membranes and droplet interface bilayers are made using oil to facilitate membrane monolayer apposition. We characterize the behavior of excess oil inside horizontal freestanding lipid bilayers using different oils, focusing on hexadecane and squalene. Using a combination of second harmonic (SH) and white-light imaging, we measure how oil redistributes within the membrane bilayer after formation. SH imaging shows that squalene forms a wider annulus compared to hexadecane suggesting that there is a higher quantity of squalene remaining in the bilayer compared to hexadecane. Excess oil droplets that appear right after membrane formation are tracked with white-light microscopy. Hexadecane droplets move directionally to the edge of the membrane with diffusion constants similar to single lipids, while squalene oil droplets move randomly with lower diffusion speeds similar to lipid condensed domains and remain trapped in the center of the bilayer for ~1-3 hours. We discuss the observed differences in terms of different coupling mechanism between the oil and lipid molecules induced by the different chemical structures of the oils.
The interfacial structure and dynamics of water in a microscopically confined geometry is imaged in three dimensions and on millisecond time scales. We developed a 3D wide-field second harmonic microscope that employs structured illumination. We image pH induced chemical changes on the curved and confined inner and outer surfaces of a cylindrical glass micro-capillary immersed in aqueous solution. The image contrast reports on the orientational order of interfacial water, induced by charge-dipole interactions between water molecules and surface charges. The images constitute surface potential maps. Spatially resolved surface pKa,s values are determined for the silica deprotonation reaction. Values range from 2.3<pKa,s<10.7, highlighting the importance of surface heterogeneities. Water molecules that rotate along an oscillating external electric field are also imaged. With this approach, real time movies of surface processes that involve flow, heterogeneities and potentials can be made, which will further developments in electrochemistry, geology, catalysis, biology, and microtechnology.
This paper presents a method for hand-eye camera calibration via an optical tracking system (OTS) faciltating robotic applications. The camera pose cannot be directly tracked via the OTS. Because of this, a transformation matrix between a marker-plate pose, tracked via the OTS, and the camera pose needs to be estimated. To this end, we evaluate two different approaches for hand-eye calibration. In the first approach, the camera is in a fixed position and a 2D calibration plate is displaced. In the second approach, the camera is also fixed, but now a 3D calibration object is moved. The first step of our method consists of collecting N views of the marker-plate pose and the calibration plates, acquired via OTS. This is achieved by keeping the camera fixed and moving the calibration plate, while taking a picture of the calibration plate using the camera. A dataset is constructed that contains marker-plate poses and the relative camera poses. Afterwards, the transformation matrix is then computed, following a least-squares minimization. Accuracy in hand-eye calibration is computed in terms of re-projection error, calculated based on camera homography transformations. For both approaches, we measure the changes in accuracy as a function of the number of poses used for each calibration, while we define the minimum number of poses required to obtain a good camera calibration. Results of the experiments show similar performances for the two evaluated methods, achieving a median value of the re-projection error at N = 25 poses of 0.76 mm for the 2D calibration plate and 0.70 mm for the 3D calibration object. Also, we have found that minimally 15 poses are required to achieve a good camera calibration.
A bone healing assessment is crucial for the successful treatment of fractures, particularly in terms of the timing of support devices. However, in clinical practice, this assessment is only made qualitatively through bone manipulation and X-rays, and hence cannot be repeated as often as might be required. The present study reconsiders the quantitative method of frequency response analysis for healing assessments, and specifically for fractures treated with an external fixator. The novelty consists in the fact that bone excitation and response are achieved through fixator pins, thus overcoming the problem of transmission through soft-tissues and their damping effect. The main objective was to develop and validate a test procedure in order to characterize the treated bone. More than 80 tests were performed on a tibia phantom alone, a phantom with pins, and a phantom with a complete fixator. Different excitation techniques and input–output combinations were compared. The results demonstrated the effectiveness of a procedure based on impact tests using a micro-hammer. Pins and fixator were demonstrated to influence the frequency response of the phantom by increasing the number of resonant frequencies. This procedure will be applied in future studies to monitor healing both in in vitro and in vivo conditions.