KEYWORDS: Linear filtering, Signal to noise ratio, Signal detection, Electronic filtering, Amplifiers, Filtering (signal processing), Digital signal processing, Convolution, Data acquisition, Visualization
Lock-in amplification is a technique used in many applications such as non-linear optics, spectroscopy and biomedical imaging. It is used to recover weak signals from noisy environments using a known carrier wave. Historically, this has been achieved using dedicated instruments, which suffer from slow connections to computer for further analysis. Here we present a technique that uses a PCIe waveform digitizer to stream data to a graphics processing unit, where digital lock-in amplification occurs. Briefly, the input signal is multiplied by an in-phase and quadrature reference before undergoing low-pass filtering with a sinc filter. The DC components of the in-phase and quadrature signals are then used to give the magnitude and phase of the signal. The bottleneck in this digital implementation is low-pass filtering due to the long filter length required to efficiently remove higher frequencies. To achieve the highest data throughput, convolution of the input signal with the low-pass filter was implemented in both time domain and frequency domain. Furthermore, decimation of the filtered data can further reduce processing time. Preliminary results show that the implemented digital lock-in amplification can sustain a continuous sampling rate of 3 billion samples per second. Using a 12 bit resolution analog to digital converter with an 800 mV input range, signals under 3 μV are reliably detectable. This GPU-based lock-in amplification implementation provides an ideal and low-cost solution for continuous and gapless signals acquisition.
Aging is accompanied by complex structural changes in the heart. To explore this remodeling, we used a serial optical coherence tomography scanner to image the entire heart at a microscopic resolution. The imaging platform combines optical coherence microscopy to vibratome sectioning to automatically image every subsection of the sample. Post-processing algorithms were then used to stitch back together the sample in a large 3D dataset. We imaged the heart of 7 young (4 months) and 5 old (24 months) wildtype mice (C57B16) with the imaging platform. Optical coherence tomography of the myocardium reveals myofiber orientation that changes linearly from the endocardium to the epicardium. This change in orientation also varies with the distance from the apex of the heart : close to the apex, the change in myofiber orientation with respect to wall depth is larger. In old mice, this change was lower when compared to young mice due to remodelling. As reported in other works, the average volume of old mice hearts (97 ± 3 mm3) was significantly larger (p<0.05) when compared to young hearts (87 ± 3 mm3). Myocardial wall thickening was accompanied by a reduction of light attenuation in the endocardium. Attenuation coefficient in old mice endocardium was measured at 15.4 ± 0.4 cm-1, compared to 18.6 ± 0.5 cm-1 in young mice, which was significantly lower (p<0.05). The use of a serial optical coherence tomography allows new insight into fine changes of the whole heart.
In this study, an automated serial two-photon microscope was used to image a fluorescent gelatin filled rodent’s brain in 3D. A method to compute vascular density using automatic segmentation was combined with coregistration techniques to build group-level vasculature metrics. By studying the medial prefrontal cortex and the hippocampal formation of 3 age groups (2, 4.5 and 8 months old), we compared vascular density for both WT and an Alzheimer model transgenic brain (APP/PS1). We observe a loss of vascular density caused by the ageing process and we propose further analysis to confirm our results.
High resolution imaging of whole rodent brains using serial OCT scanners is a promising method to investigate microstructural changes in tissue related to the evolution of neuropathologies. Although micron to sub-micron sampling resolution can be obtained by using high numerical aperture objectives and dynamic focusing, such an imaging system is not adapted to whole brain imaging. This is due to the large amount of data it generates and the significant computational resources required for reconstructing such volumes. To address this limitation, a dual resolution serial OCT scanner was developed. The optical setup consists in a swept-source OCT made of two sample and reference arms, each arm being coupled with different microscope objectives (3X / 40X). Motorized flip mirrors were used to switch between each OCT arm, thus allowing low and high resolution acquisitions within the same sample. The low resolution OCT volumes acquired with the 3X arm were stitched together, providing a 3D map of the whole mouse brain. This brain can be registered to an OCT brain template to enable neurological structures localization. The high resolution volumes acquired with the 40X arm were also stitched together to create local high resolution 3D maps of the tissue microstructure. The 40X data can be acquired at any arbitrary location in the sample, thus limiting storage-heavy high resolution data to application restricted to specific regions of interest. By providing dual-resolution OCT data, this setup can be used to validate diffusion MRI with tissue microstructure derived metrics measured at any location in ex vivo brains.
An automated serial histology setup combining optical coherence tomography (OCT) imaging with vibratome sectioning was used to image eight wild type mouse brains. The datasets resulted in thousands of volumetric tiles resolved at a voxel size of (4.9×4.9×6.5) μm3 stitched back together to give a three-dimensional map of the brain from which a template OCT brain was obtained. To assess deformation caused by tissue sectioning, reconstruction algorithms, and fixation, OCT datasets were compared to both in vivo and ex vivo magnetic resonance imaging (MRI) imaging. The OCT brain template yielded a highly detailed map of the brain structure, with a high contrast in white matter fiber bundles and was highly resemblant to the in vivo MRI template. Brain labeling using the Allen brain framework showed little variation in regional brain volume among imaging modalities with no statistical differences. The high correspondence between the OCT template brain and its in vivo counterpart demonstrates the potential of whole brain histology to validate in vivo imaging.
An automated massive histology setup combined with an optical coherence tomography (OCT) microscope was used to image a total of n=5 whole mouse brains. Each acquisition generated a dataset of thousands of OCT volumetric tiles at a sampling resolution of 4.9×4.9×6.5 μm. This paper describes techniques for reconstruction and segmentation of the sliced brains. In addition to the measured OCT optical reflectivity, a single scattering photon model was used to compute the attenuation coefficients within each tissue slice. Average mouse brain templates were generated for both the OCT reflectivity and attenuation contrasts and were used with an n-tissue segmentation algorithm. To better understand the brain tissue OCT contrast origin, one of the mouse brains was acquired using dMRI and coregistered to its corresponding assembled brain. Our results indicate that the optical reflectivity in a fiber bundle varies with its orientation, its fiber density, and the number of fiber orientations it contains. The OCT mouse brain template generation and coregistration to dMRI data demonstrate the potential of this massive histology technique to pursue cross-sectional, multimodal, and multisubject investigations of small animal brains.
A whole rodent brain was imaged using an automated massive histology setup and an Optical Coherence Tomography (OCT) microscope. Thousands of OCT volumetric tiles were acquired, each covering a size of about 2.5x2.5x0.8 mm3 with a sampling resolution of 4.9x4.9x6.5 microns. This paper shows the techniques for reconstruction, attenuation compensation and segmentation of the sliced brains. The tile positions within the mosaic were evaluated using a displacement model of the motorized stage and pairwise coregistration. Volume blending was then performed by solving the 3D Laplace equation, and consecutive slices were assembled using the cross-correlation of their 2D image gradient. This reconstruction algorithm resulted in a 3D map of optical reflectivity for the whole brain at micrometric resolution. OCT tissue slices were then used to estimate the local attenuation coefficient based on a single scattering photon model. The attenuation map obtained exhibits a high contrast for all white matter fibres, regardless of their orientation. The tissue optical attenuation from the intrinsic OCT reflectivity contributes to better white matter tissue segmentation. The combined 3D maps of reflectivity and attenuation is a step toward the study of white matter at a microscopic scale for the whole brain in small animals.
A combined serial OCT/confocal scanner was designed to image large sections of biological tissues at microscopic resolution. Serial imaging of organs embedded in agarose blocks is performed by cutting through tissue using a vibratome which sequentially cuts slices in order to reveal new tissue to image, overcoming limited light penetration encountered in microscopy. Two linear stages allow moving the tissue with respect to the microscope objective, acquiring a 2D grid of volumes (1x1x0.3 mm) with OCT and a 2D grid of images (1x1mm) with the confocal arm. This process is repeated automatically, until the entire sample is imaged. Raw data is then post-processed to re-stitch each individual acquisition and obtain a reconstructed volume of the imaged tissue. This design is being used to investigate correlations between white matter and microvasculature changes with aging and with increase in pulse pressure following transaortic constriction in mice. The dual imaging capability of the system allowed to reveal different contrast information: OCT imaging reveals changes in refractive indices giving contrast between white and grey matter in the mouse brain, while transcardial perfusion of FITC or pre-sacrifice injection of Evans Blue shows microsvasculature properties in the brain with confocal imaging.
Despite recent findings on the possible role of age-related cerebral microvasculature changes in cognition decline, previous studies of capillary blood flow in aging (using animal models) are scarce and limited to anesthetized conditions. Since anesthesia can have different effects in young and old animals, it may introduce a confounding effect in aging studies. The present study aimed to eliminate the potential confound introduced by anesthesia by measuring capillary blood flow parameters in both awake conditions and under isoflurane anesthesia. We used 2-photon laser scanning fluorescence microscopy to measure capillary diameter, red blood cell velocity and flux, hematocrit and capillary volumetric flow in individual capillaries in the barrel cortex of 6- and 24-month old C57Bl/6 mice. It was observed that microvascular properties are significantly affected by anesthesia leading to different trends in capillary blood flow parameters with aging when measured under awake or anesthetized conditions. The findings in this study suggest taking extra care in interpreting aging studies from anesthetized animals.
Here, we present a serial OCT/confocal scanner for histological study of the mouse brain. Three axis linear stages combined with a sectioning vibratome allows to cut thru the entire biological tissue and to image every section at a microscopic resolution. After acquisition, each OCT volume and confocal image is re-stitched with adjacent acquisitions to obtain a reconstructed, digital volume of the imaged tissue. This imaging platform was used to investigate correlations between white matter and microvasculature changes in aging mice. Three age groups were used in this study (4, 12, 24 months). At sacrifice, mice were transcardially perfused with a FITC containing gel. The dual imaging capability of the system allowed to reveal different contrast information: OCT imaging reveals changes in refractive indices giving contrast between white and grey matter in the mouse brain, while transcardial perfusion of a FITC shows microsvasculature in the brain with confocal imaging.
The thorough characterization of skin samples is a critical step in investigating dermatological diseases. The combination of depth-sensitive anatomical imaging with molecular imaging has the potential to provide vast information about the skin. In this proof-of-concept work we present high-resolution mosaic images of skin biopsies using Optical Coherence Tomography (OCT) manually co-registered with standard microscopy, bi-dimensional Raman spectral mapping and fluorescence imaging. A human breast skin sample, embedded in paraffin, was imaged with a swept-source OCT system at 1310 nm. Individual OCT volumes were acquired in fully automated fashion in order to obtain a large field-of-view at high resolution (~10μm). Based on anatomical features, the other three modalities were manually co-registered to the projected OCT volume, using an affine transformation. A drawback is the manual co-registration, which may limit the utility of this method. However, the results indicate that multiple imaging modalities provide complementary information about the sample. This pilot study suggests that multi-modal microscopy may be a valuable tool in the characterization of skin biopsies.
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.