Image Acquisition Rate Research Articles

Comparison of phase-resolved Doppler optical coherence tomography and optical coherence tomography angiography for measuring retinal blood vessels size

The goal of this study was to compare two OCT-based methods for measuring retinal blood vessels size: Phase-resolved Doppler OCT (DOCT) and OCT angiography (OCTA). The study was conducted in rats (n= 6) using a SD-OCT system operating at 1060 nm with 92 kHz image acquisition rate. Arteries and veins were separated by the phase polarity. Results from this study showed that the venal diameters are significantly larger than the arterial diameters, and there is no significant difference in the vessel diameters measured by both methods.

A preliminary study of fetal intelligent navigation echocardiography in the normal fetuses during second and third trimester

Objective To investigate the value of fetal intelligent navigation echocardiography (FINE, 5D Heart) in displaying the normal fetal heart structure during 20 to 38 weeks′ gestation. Methods Three-dimensional data of fetal heart during 20 to 38 weeks′ gestation were acquired using volume transducer prospectively. The STIC volume images were post-processed with 5D heart software by three physicians with different levels of echocardiographic examination and the quality of the section images were analyzed. The intra- and interobserver repeatability was analyzed for the qualification of section images. The correlation between the quality of image and gestational age, body mass index, the placenta position, fetal initial plane position were evaluated using univariate and multivariable logistic regression analysis. Results Two hundreds and five cases of pregnant women were included, and 9 cases were excluded.Volume data acquisition was completed in 180 pregnant women, and the image acquisition rate was 91.8%. The data were assessed twice by three observers and qualified 9 section images were gotten in 156-180 cases (degree of qualification 86.7%-100%). The screening images were satisfied in 152 cases (84.4%), dissatisfied with 28 cases (15.6%). The intra-observer agreement by three observers was absolute concord in four chamber view and abdomen/stomach view; excellent in three vessel trachea view(3VT), five chamber view, left ventricular outflow tract view and superior and inferior vena cava view; good in right ventricular outflow tract view, ductal arch view and aortic arch view. The inter-observer agreement by each of three observers was absolute concord in four chamber view and abdomen/stomach view; excellent in superior and inferior vena cava view; excellent to good in five chamber view, left ventricular outflow tract view, right ventricular outflow tract view; good in 3VT, ductal arch view and aortic arch view. In a univariate analysis, the quality of the image was associated with obesity (χ2=15.338, P=0.000), gestational age (χ2=23.356, P=0.000). Multivariable analysis showed that the obesity (OR=5.587, P=0.001) and gestational age (OR=8.843, P=0.000) were factors affecting the quality of the image. The average time spent in post-processing the images for first time by three observers was (2.77±1.39)min, and there was no significant difference among observers (P>0.05). Conclusions Fetal intelligent navigation echocardiography (FINE, 5D Heart) based on STIC volumes can successfully generate 9 standard fetal echocardiography views in 86.7%-100% of fetuses in the 2nd and 3rd trimesters. This method can simplify examination of the fetal heart and reduce operator dependency. Key words: Echocardiography; Fetal heart; Spatiotemporal image correlation; Fetal intelligent navigation echocardiography(FINE)

Visualizing Micro-anatomical Structures of the Posterior Cornea with Micro-optical Coherence Tomography

Diagnosis of corneal disease and challenges in corneal transplantation require comprehensive understanding of corneal anatomy, particularly that of the posterior cornea. Micro-optical coherence tomography (µOCT) is a potentially suitable tool to meet this need, owing to its ultrahigh isotropic spatial resolution, high image acquisition rate and depth priority scanning mode. In this study, we explored the ability of µOCT to visualize micro-anatomical structures of the posterior cornea ex vivo and in vivo using small and large animals. µOCT clearly delineated cornea layers and revealed micro-anatomical structures, including not only polygonal endothelial cells, stellate keratocytes, collagen fibres and corneal nerve fibres but also new structures such as the dome-shaped basolateral side of endothelial cells and lattice structures at the interface between endothelium and Descemet’s membrane. Based on these observations, a short post-harvest longitudinal study was conducted on rat cornea to test the feasibility of using µOCT to monitor the quality of endothelial cells. This study successfully reveals a series of morphological features and pathological changes in the posterior cornea at the cellular level in situ and in real time with µOCT. These findings enrich knowledge of corneal anatomy and suggest that µOCT may be a promising imaging tool in corneal transplantation.

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-μm resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family.

Lossless Three-Dimensional Parallelization in Digitally Scanned Light-Sheet Fluorescence Microscopy

We introduce a concept that enables parallelized three-dimensional imaging throughout large volumes with isotropic 300–350 nm resolution. By staggering high aspect ratio illumination beams laterally and axially within the depth of focus of a digitally scanned light-sheet fluorescence microscope (LSFM), multiple image planes can be simultaneously imaged with minimal cross-talk and light loss. We present a first demonstration of this concept for parallelized imaging by synthesizing two light-sheets with nonlinear Bessel beams and perform volumetric imaging of fluorescent beads and invasive breast cancer cells. This work demonstrates that in principle any digitally scanned LSFM can be parallelized in a lossless manner, enabling drastically faster volumetric image acquisition rates for a given sample brightness and detector technology.

Are there intracellular Ca2+ oscillations correlated with flagellar beating in human sperm? A three vs. two-dimensional analysis

Are there intracellular Ca2+ ([Ca2+]i) oscillations correlated with flagellar beating in human sperm? The results reveal statistically significant [Ca2+]i oscillations that are correlated with the human sperm flagellar beating frequency, when measured in three-dimensions (3D). Fast [Ca2+]i oscillations that are correlated to the beating flagellar frequency of cells swimming in a restricted volume have been detected in hamster sperm. To date, such findings have not been confirmed in any other mammalian sperm species. An important question that has remained regarding these observations is whether the fast [Ca2+]i oscillations are real or might they be due to remaining defocusing effects of the Z component arising from the 3D beating of the flagella. Healthy donors whose semen samples fulfill the WHO criteria between the age of 18-28 were selected. Cells from at least six different donors were utilized for analysis. Approximately the same number of experimental and control cells were analyzed. Motile cells were obtained by the swim-up technique and were loaded with Fluo-4 (Ca2+ sensitive dye) or with Calcein (Ca2+ insensitive dye). Ni2+ was used as a non-specific plasma membrane Ca2+ channel blocker. Fluorescence data and flagella position were acquired in 3D. Each cell was recorded for up to 5.6 s within a depth of 16 microns with a high speed camera (coupled to an image intensifier) acquiring at a rate of 3000 frames per second, while an oscillating objective vibrated at 90 Hz via a piezoelectric device. From these samples, eight experimental and nine control sperm cells were analyzed in both 2D and 3D. We have implemented a new system that allows [Ca2+]i measurements of the human sperm flagellum beating in 3D. These measurements reveal statistically significant [Ca2+]i oscillations that correlate with the flagellar beating frequency. These oscillations may arise from intracellular sources and/or Ca2+ transporters, as they were insensitive to external Ni2+, a non-specific plasma membrane Ca2+ channel blocker. N/A. Analysis in 3D needs a very fast image acquisition rate to correctly sample a volume containing swimming sperm. This condition requires a very short exposure time per image making it necessary to use an image intensifier which also increases noise. The lengthy analysis time required to obtain reliable results limited the number of cells that could be analyzed. The possibility of recording flagellar [Ca2+]i oscillations described here may open a new avenue to better understand ciliary and flagellar beating that are fundamental for mucociliary clearance, oocyte transport, fertilization, cerebrospinal fluid pressure regulation and developmental left-right symmetry breaking in the embryonic node. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) (grants 253952 to G.C.; 156667 to F.M.M. and Fronteras 71 39908-Q to A.D. and Post-doctoral scholarships 366844 to P.H.-H. and 291028 to F.M.) and the Dirección General de Asuntos del Personal Académico of the Universidad Nacional Autónoma de México (DGAPA-UNAM) (grants CJIC/CTIC/4898/2016 to F.M. and IN205516 to A.D.). There are no conflicts of interest to declare.

High-speed polarization-resolved coherent Raman scattering imaging

Polarization-resolved coherent Raman scattering (polar-CRS) provides rich information on molecular orientational organization, with the strong advantages of being a label-free and chemically specific imaging method. Its implementation, however, strongly reduces the imaging acquisition rate, due to limits imposed by polarization tuning. Here we demonstrate fast-polar-CRS imaging based on combined electro-optic polarization and acousto-optic amplitude modulations, applicable to both stimulated Raman scattering and coherent anti-Stokes Raman scattering imaging. The proposed scheme adds polarization information without compromising the capacities of regular CRS intensity imaging; increases the speed of orientational imaging by two orders of magnitude as compared with previous approaches; and does not require post-processing analyses. We show that this method permits sub-second time-scale imaging of lipid order packing and local lipid membrane deformations in artificial lipid multilayers, but also in red blood cell ghosts, demonstrating its high sensitivity down to a single lipid bilayer membrane.

Microfluidic flow spectrometer

We present a microfluidic device which allows one to study the dynamics of oscillatory flows for a frequency range between 1 and 300 Hz. The fluid in the microdevice could be Newtonian, viscoelastic, or even a biofluid, since the device is made of PMMA, which makes it biocompatible and free of elastomeric elements. Coupling a piezoelectric to a micropiston allows one to impose periodic movement to the fluid, with zero mean flow and amplitudes of up to m, within the microchannels in which the dynamics is studied. The use of a fast camera coupled to a microscope allows one to study the dynamics of m tracer particles and interfaces at an image acquisition rate as fast as 5000 frames per second. The fabrication of the device is easy and cost-effective, since it is based on the use of a micromilling machine. The dynamics of a Newtonian fluid is studied as a proof of principle.

Fast compressive pulse-echo ultrasound imaging using random incident sound fields

In fast pulse-echo ultrasound imaging (UI), the image quality is traded off against the image acquisition rate by reducing the number of sequential wave emissions per image. To alleviate this tradeoff, the concept of compressed sensing (CS) was proposed by the authors in previous studies. CS regularizes the linear inverse scattering problem (ISP) associated with fast pulse-echo UI by postulating the existence of a nearly-sparse representation of the object to be imaged. This representation is obtained by a known linear transform, e.g., the Fourier or a wavelet transform. A central degree of freedom in the regularized ISP is the choice of the incident sound fields. Previous studies focused exclusively on steered plane waves. In this study, we investigate the usage of random incident sound fields to improve the relevant mathematical properties of the scattering operator governing the linear ISP. These sound fields are synthesized by a linear transducer array whose physical elements are excited applying combinations of random time delays and random apodization weights. Using simulated and experimentally obtained radio frequency signals, we demonstrate that these sound fields significantly reduce the recovery errors and improve the rate of convergence for low signal-to-noise ratios.

Multimodal breast cancer imaging using coregistered dynamic diffuse optical tomography and digital breast tomosynthesis.

Diffuse optical tomography (DOT) is emerging as a noninvasive functional imaging method for breast cancer diagnosis and neoadjuvant chemotherapy monitoring. In particular, the multimodal approach of combining DOT with x-ray digital breast tomosynthesis (DBT) is especially synergistic as DBT prior information can be used to enhance the DOT reconstruction. DOT, in turn, provides a functional information overlay onto the mammographic images, increasing sensitivity and specificity to cancer pathology. We describe a dynamic DOT apparatus designed for tight integration with commercial DBT scanners and providing a fast (up to 1 Hz) image acquisition rate to enable tracking hemodynamic changes induced by the mammographic breast compression. The system integrates 96 continuous-wave and 24 frequency-domain source locations as well as 32 continuous wave and 20 frequency-domain detection locations into low-profile plastic plates that can easily mate to the DBT compression paddle and x-ray detector cover, respectively. We demonstrate system performance using static and dynamic tissue-like phantoms as well as in vivo images acquired from the pool of patients recalled for breast biopsies at the Massachusetts General Hospital Breast Imaging Division.

Correlation of Visually Evoked Functional and Blood Flow Changes in the Rat Retina Measured With a Combined OCT+ERG System.

To correlate visually evoked functional and blood flow changes in the rat retina measured simultaneously with a combined optical coherence tomography and electroretinography system (OCT+ERG). Male Brown Norway (n = 6) rats were dark adapted and anesthetized with ketamine/xylazine. Visually evoked changes in the retinal blood flow (RBF) and functional response were measured simultaneously with an OCT+ERG system with 3-μm axial resolution in retinal tissue and 47-kHz image acquisition rate. Both single flash (10 and 200 ms) and flicker (10 Hz, 20% duty cycle, 1- and 2-second duration) stimuli were projected onto the retina with a custom visual stimulator, integrated into the OCT imaging probe. Total axial RBF was calculated from circular Doppler OCT scans by integrating over the arterial and venal flow. Temporary increase in the RBF was observed with the 10- and 200-ms continuous stimuli (∼1% and ∼4% maximum RBF change, respectively) and the 10-Hz flicker stimuli (∼8% for 1-second duration and ∼10% for 2-second duration). Doubling the flicker stimulus duration resulted in ∼25% increase in the RBF peak magnitude with no significant change in the peak latency. Single flash (200 ms) and flicker (10 Hz, 1 second) stimuli of the same illumination intensity and photon flux resulted in ∼2× larger peak RBF magnitude and ∼25% larger RBF peak latency for the flicker stimulus. Short, single flash and flicker stimuli evoked measureable RBF changes with larger RBF magnitude and peak latency observed for the flicker stimuli.

Dynamic measurements using digital image correlation

Digital image correlation (DIC), which enables non-contact measurement of displacements and strains, has seen widespread adoption within the geotechnical physical modelling community for the measurement of static displacements. Advances in high temporal resolution cameras now permit the use of DIC to calculate accelerations. However, it is currently unclear how the image acquisition rate and the choice of DIC algorithm influence the quality of this data. This paper describes the sources of error that affect the dynamic measurement accuracy. Numerical and physical experiments are used to demonstrate the relevance of (a) bias error in the sub-pixel interpolation scheme, (b) the ratio of sample rate to the frequency of the signal being monitored and (c) the signal-to-noise ratio on the accuracy and precision of DIC acceleration measurements. The results demonstrate that by using appropriate image texture, sampling frequencies and signal-to-noise ratios, measurements with an accuracy similar to accelerometers can be achieved. The displacement measurement error due to bias errors was found to be 0·0015 pixels. The error in the calculated velocity and acceleration was a function of the amplitude of displacement measurements with an optimum ratio between the sampling frequency to the signal frequency found to be between 25 and 50.

Usefulness of the echocardiographic paravertebral approach for the diagnosis of descending thoracic aortic dissection.

Transthoracic echocardiography (TTE) is not recommended as the first-line diagnostic modality for Stanford type B aortic dissection (type-B AD). The aims of this study were to evaluate the usefulness and factors influencing for the diagnosis of type-B AD using the transthoracic echocardiographic paravertebral approach (PVA). We compared the image acquisition rate of descending thoracic aorta (DTA) and the diagnostic rate of type-B AD using TTE versus PVA. Both tests were compared with type-B AD, which was diagnosed by enhanced computed tomography (CT), as the reference standard. We also analyzed the factors influencing adequate image acquisition and the diagnosis of type-B AD using the PVA. The length between the dorsal thoracic surface and the DTA (TDAL) and thickness of lung on the TDAL line (LTh) were measured on the CT images. No significant difference was found between the image acquisition rate of the DTA between the PVA and the TTE (70.1 vs. 64.2%, p=0.56), while the diagnostic rate of type-B AD using the PVA was significantly greater than when using the TTE (56.7 vs. 26.9%, p<0.001). Furthermore, when adequate images of DTA were obtained using the PVA, 80.9% of the patients were diagnosed with type-B AD. A multivariate analysis demonstrated that a lower LTh (p=0.001) and the existence of a pleural effusion (p=0.03) significantly influenced the diagnosis of type-B AD using the PVA. The PVA might be a useful method for diagnosis of type-B AD, when adequate images of the DTA are obtained.

Assessment of Electromagnetic Stirrer Agitated Liquid Metal Flows by Dynamic Neutron Radiography

This paper presents qualitative and quantitative characterization of two-phase liquid metal flows agitated by the stirrer on rotating permanent magnets. The stirrer was designed to fulfill various eddy flows, which may have different rates of solid particle entrapment from the liquid surface and their homogenization. The flow was characterized by visualization of the tailored tracer particles by means of dynamic neutron radiography, an experimental method well suited for liquid metal flows due to low opacity of some metals for neutrons. The rather high temporal resolution of the image acquisition (32 Hz image acquisition rate) allows for the quantitative investigation of the flows up to 30 cm/s using neutron particle image velocimetry. In situ visualization of the two-phase liquid metal flow is also demonstrated.

Use of an Image Acquisition Stabilizer Improves Sidestream Dark Field Imaging of the Serosa during Open Gastrointestinal Surgery

Aim: To investigate whether an image acquisition stabilizer (IAS) mounted on the sidestream dark field camera (SDF) during gastrointestinal surgery improves image stability and acquisition. Methods: Serosal SDF imaging was compared with SDF imaging combined with an IAS (SDF + IAS) during gastrointestinal surgery. Stability was assessed as the image drift in pixels and the time to obtain stable images. The success rate was determined as the percentage of analyzable images after recording. The effect of negative pressure from the IAS was determined during single-spot measurements and by comparing microvascular parameters between groups. Data are presented as mean ± SD. Results: Sixty serosal measurements were performed per group; 87% were successful in the SDF group and 100% in the SDF + IAS group (p = 0.003). Image drift in the SDF group was 148 ± 36 versus 55 ± 15 pixels in the SDF + IAS group; p < 0.001. Time to stable image was 96 ± 60 s in the SDF group versus 57 ± 31 s in the SDF + IAS group; p = 0.03. No effect of negative pressure was seen. Conclusion: The use of an IAS mounted on an SDF camera during serosal microvascular assessment improves the success rate of image acquisition and stability and reduces the time to stable image with no effect on the microcirculation.

Stereoscopic augmented reality for single camera endoscopy: a virtual study

Endoscopic surgery causes less tissue injury compared to open surgical techniques, thus promoting more rapid recuperation and reduced post-operative pain. Endoscopy, however, allows the surgeon to visualise only the anatomical surface of the surgical site, with a relatively narrow field of view. Moreover, the 2D video captured by the conventional endoscope does not provide depth perception of the surgical scene. In this study, these limitations have been addressed with the development of an augmented reality (AR) system with stereoscopic visualisation. A phantom and its 3D CT model were used, respectively, to form the real and virtual parts of the AR. The virtual environment camera pose was tracked using algorithms for image feature detection, feature matching and Perspective-n-Point applied on the endoscopic image and the 3D virtual model-rendered image. The endoscope video frame- and the virtual model-rendered images were superimposed to form the AR composite view. The depth buffer (z-buffer) of the rendering window was further used to make a stereo pair of the AR image. The AR system produced a stereo composite view having well-aligned real and virtual components. The RMS error of the real and virtual image contours registration was 9.6 ± 6.7 mm. Correlation coefficients between the depth map from z-buffer and a depth camera was between 0.60 and 0.96 (p < 0.05). The AR system requires further improvement to be applicable at a higher frame rate of endoscope image acquisition. It also needs to include motion and deformation models when applied to animals or patients.

4-D imaging of sub-second dynamics in pore-scale processes using real-time synchrotron X-ray tomography

Abstract. A variable volume flow cell has been integrated with state-of-the-art ultra-high-speed synchrotron X-ray tomography imaging. The combination allows the first real-time (sub-second) capture of dynamic pore (micron)-scale fluid transport processes in 4-D (3-D + time). With 3-D data volumes acquired at up to 20 Hz, we perform in situ experiments that capture high-frequency pore-scale dynamics in 5–25 mm diameter samples with voxel (3-D equivalent of a pixel) resolutions of 2.5 to 3.8 µm. The data are free from motion artefacts and can be spatially registered or collected in the same orientation, making them suitable for detailed quantitative analysis of the dynamic fluid distribution pathways and processes. The methods presented here are capable of capturing a wide range of high-frequency nonequilibrium pore-scale processes including wetting, dilution, mixing, and reaction phenomena, without sacrificing significant spatial resolution. As well as fast streaming (continuous acquisition) at 20 Hz, they also allow larger-scale and longer-term experimental runs to be sampled intermittently at lower frequency (time-lapse imaging), benefiting from fast image acquisition rates to prevent motion blur in highly dynamic systems. This marks a major technical breakthrough for quantification of high-frequency pore-scale processes: processes that are critical for developing and validating more accurate multiscale flow models through spatially and temporally heterogeneous pore networks.

NON-INVASIVE DIAGNOSTICS OF NON-TUMOR DISORDERS WITH OPTICAL COHERENCE TOMOGRAPHY

Introduction: The distinctive features of modern inflammatory diseases are their indolent course and predisposition to chronicity. Chronic inflammation is the basis for the development of serious disorders, including autoimmune and oncologic ones, and a cause of major functional abnormalities of the human body. Diagnostics of chronic inflammation is associated with a number of problems which originate from a variety of clinical phenotypes and morphological forms, on the one hand, and from the rising prevalence of their latent course, on the other. In its turn, an adequate treatment choice may be based on understanding and accurate detection of etiological and morphological types of the disease. In this connection, development of novel diagnostic techniques, such as optical coherence tomography (OCT), is of utmost importance. Aim: To demonstrate feasibility of OCT in detection of morphological abnormalities associated with chronic inflammatory disorders of various localizations. Materials and methods: OCT diagnostics was performed in addition to standard diagnostic procedures in 225 patients with chronic inflammatory disorders of various localizations, such as chronic recurrent rhinitis, chronic latent salpingitis and chronic endometritis. For OCT, an “OCT-1300U” system was used, with central wavelength of 1280 nm, spatial resolution of 15–20 μm, image acquisition rate of 8 frames per second, imaging depth of 1.4 mm and a probe diameter of 2.4 mm. The following techniques of quantitative processing were used for processing of the OCT images. Quantitative characterization of histograms and image texture was performed with further determination of values typical for various images groups corresponding to particular states of biological tissues. Neural network was also employed with histograms and 2D Fourier transforms of the images as input data. Results: OCT laparoscopy allowed to obtain three types of OCT images of fallopian tubes with features typical for healthy and inflammatory states. Presence of heterogeneity in the structure of an OCT image indicated heterogeneity in the muscle layer of the fallopian tube, which allowed for a conclusion on chronic salpingitis. With OCT hysteroscopy we demonstrated the ability of OCT to visualize pathomorphologic features of chronic endometritis. The diagnostic data obtained by OCT-rhinoscopy allowed to draw preliminary conclusions on the OCT ability to detect specific features of various types of chronic rhinitis. Numerical processing of diagnostic OCT images resulted in their higher objectivity and an improvement of diagnostic accuracy of the technique. Conclusion: OCT is able to detect morphological changes induced by a chronic inflammatory process. OCT criteria were shown to be universal for various organs. We demonstrated the possibility of non-invasive diagnostics of pathomorphologic forms of disease, showed some challenges in the development of OCT for diagnostics of non-tumor disorders and ways to overcome them are proposed, in particular, the use of image numerical processing. Further studies seem to be justified.

In Vivo Imaging and Morphometry of the Human Pre-Descemet's Layer and Endothelium With Ultrahigh-Resolution Optical Coherence Tomography.

To visualize in vivo and quantify the thickness of the posterior corneal layers: the acellular pre-Descemet's layer (PDL), Descemet's membrane (DM), and endothelium (END) in healthy subjects, using ultrahigh-resolution optical coherence tomography (UHR-OCT). A research-grade, 800-nm UHR-OCT system with 0.95-μm axial resolution in corneal tissue was used to image in vivo the posterior cornea in healthy subjects. The system offers approximately 98 dB sensitivity for 680 μW optical power incident on the cornea and 34,000 A-scans/s image acquisition rate. This study comprised 20 healthy subjects, aged 20 to 60 years. The thickness of the PDL, DM, and END layers was measured both with a custom, automatic segmentation algorithm and manually. The boundaries and structure of the posterior corneal layers were clearly visible in the UHR-OCT images. The average thickness was measured to be 6.6 ± 1.4 μm (PDL), 10.4 ± 2.9 μm (DM), and 4.8 ± 0.4 μm (END), which agrees well with published data from ex vivo studies. Both the END and DM thickness showed minor spatial variations, whereas the PDL showed up to 2× thickness change for different locations on the same cross-sectional corneal image or over the entire imaged region of the cornea. Our data indicate that all three layers of the posterior cornea can be clearly visualized in vivo and their thicknesses measured precisely with UHR-OCT. Although the PDL thickness showed large spatial variations, the thickness of the DM and END layers was consistent over the entire imaged region of the cornea.

Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis.

MALDI imaging mass spectrometry is a powerful analytical tool enabling the visualization of biomolecules in tissue. However, there are unique challenges associated with protein imaging experiments including the need for higher spatial resolution capabilities, improved image acquisition rates, and better molecular specificity. Here we demonstrate the capabilities of ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR IMS platforms as they relate to these challenges. High spatial resolution MALDI-TOF protein images of rat brain tissue and cystic fibrosis lung tissue were acquired at image acquisition rates >25 pixels/s. Structures as small as 50 μm were spatially resolved and proteins associated with host immune response were observed in cystic fibrosis lung tissue. Ultra-high speed MALDI-TOF enables unique applications including megapixel molecular imaging as demonstrated for lipid analysis of cystic fibrosis lung tissue. Additionally, imaging experiments using MALDI FTICR IMS were shown to produce data with high mass accuracy (<5 ppm) and resolving power (∼75 000 at m/z 5000) for proteins up to ∼20 kDa. Analysis of clear cell renal cell carcinoma using MALDI FTICR IMS identified specific proteins localized to healthy tissue regions, within the tumor, and also in areas of increased vascularization around the tumor.