Cellular-resolution in vivo imaging of human corneal microstructures plays an important role in the diagnosis and management of corneal disorders. It can also help evaluate the disease progression and the treatment response to different therapies. In this study, a new approach, polarization-dependent optical coherence microscope (POCM), was implemented to non-invasively image the microstructures of the human cornea in vivo. The approach leveraged the polarization propriety of light as well as the self-interference between the corneal surface and underlying layers to achieve high-contrast imaging of the human cornea. POCM achieved volumetric (500 x 500 x 2048 voxels) imaging of corneal microstructures over a field of view 0.5 x 0.5mm2 with a lateral resolution of ~2.2μm and a volume rate of 1Hz. While the system achieved a ~2.4μm axial resolution (in the cornea) with its standard reference arm, the self-interference approach enabled the highest axial resolution of 1.4μm enabled by the source and detector, making it possible to achieve high-contrast imaging of microstructures of the anterior cornea free of degradations from dispersion mismatch, eye motion, and corneal curvature.
Scientists today have powerful tools to alter a wide range of genes in animal models with ease to understand biological processes in development and disease conditions. However, there is a lack of imaging technology to assess the effects of these genetic modifications in live mammals. Without these in vivo imaging capabilities, scientists cannot fully understand how the individual organism’s response to overall process. In this work, we have developed a multi-modal optical coherence microscopy (OCM) and dual-channel fluorescence microscopy (DC-FM) to enable the evaluation of biological processes in transgenic mice models. The combined system achieved a simultaneous recording of reflectance and fluorescence signals of deep tissue layers at the speed of 250 kHz and a lateral resolution of ~ 2 μm over a field of view of 1.3 x 1.3 mm2 . OCM and DC-FM also achieved an axial resolution of 2.8 μm and 23 μm, respectively. To evaluate the performances of the system in imaging the cornea of transgenic mice, a conditional dual-reporter mouse strain, KerartTA/tet-O-Cre/RosamTmG triple transgenic mouse strain, which express membrane-bound tomato red (mT), was harnessed. Upon exposure to doxycycline (Dox) in KerartTA/tet-O-Cre/RosamTmG mice, it would then express a membrane-bound green fluorescent protein (mG) in the corneal stromal cells. While OCM evaluated the change in thickness and structures of the cornea, DC-FM provided green and red proteins expression in the cornea. This system will enable longitudinal in vivo studies on transgenic mice models to advance the understanding of developmental and disease mechanisms.
Multimodal imaging systems offer the opportunity to scrutinize different properties of biological samples. Optical coherence microscopy (OCM) is a non-invasive and high-resolution imaging technique capable of generating threedimensional images of tissue. In this work, a multimodal imaging system interleaving OCM with a dual-channel fluorescence microscopy (DC-FM) system was developed to add functional imaging capabilities to OCM. The combined system was able to simultaneously acquire both reflectance and fluorescence data from the same location of the sample at the speed of 250 kHz, and with a lateral resolution of ~ 2.1 μm. An axial resolution of 2.4 μm in sample over the imaging depth of 1 mm was achieved with OCM. The performances of the combined system were evaluated by imaging a multi-layer tape as well as a gel containing green and red fluorescent microspheres. While OCM enabled the depth localization of all fluorescent microspheres, it was not able to discriminate between green and red fluorophores, a feature that was achieved with DC-FM. Hence, the interleaved system has the potential of assessing structural as well as cellular level functional changes in biological samples. This system will be applied toward longitudinal studies in small animal models.
Bleed-through is a common problem in multi-channel fluorescence microscopy when simultaneously imaging multiple organelles tagged with different fluorophores. This is majorly due to the large emission spectra of fluorophores. The correction of bleed-through in a multi-channel fluorescence microscopy system is essential to accurately identify or track the location of multiple fluorophores simultaneously. This paper presents a method for eliminating the bleed-through in a dual-channel fluorescence microscopy system using highly sensitive photomultiplier tubes. Fluorescein and Alexa fluor 594 dyes that were diluted in phosphate buffer solution at different dilution ratios were used to establish the relationship between the intensities of both channels. The bleed-through intensity in the red channel was quantified using a nonlinear polynomial model. Bleed-through correction was performed using the derived polynomial coefficients and the detected intensity in the green channel. The approach was experimentally validated on a mixed solution of fluorescein and Alexa fluor 594 using the scanning mode dual-channel fluorescence microscopy system.
We report on a pathway for Gabor domain optical coherence microscopy (GD-OCM)-based metrology to assess the donor’s corneal endothelial layers ex vivo. Six corneas from the Lions Eye Bank at Albany and Rochester were imaged with GD-OCM. The raw 3-D images of the curved corneas were flattened using custom software to enhance the 2-D visualization of endothelial cells (ECs); then the ECs within a circle of 500-μm-diameter were analyzed using a custom corner method and a cell counting plugin in ImageJ. The EC number, EC area, endothelial cell density (ECD), and polymegethism (CV) were quantified in five different locations for each cornea. The robustness of the method (defined as the repeatability of measurement together with interoperator variability) was evaluated by independently repeating the entire ECD measurement procedure six times by three different examiners. The results from the six corneas show that the current modality reproduces the ECDs with a standard deviation of 2.3% of the mean ECD in every location, whereas the mean ECD across five locations varies by 5.1%. The resolution and imaging area provided through the use of GD-OCM may help to ultimately better assess the quality of donor corneas in transplantation.
To identify the microstructural modification of the corneal layers during the course of the disease, optical technologies have been pushing the boundary of innovation to achieve cellular resolution of deep layers of the cornea. Gabor-domain optical coherence microscopy (GD-OCM), an optical coherence tomography-based technique that can achieve an isotropic of ∼2-μm resolution over a volume of 1 mm × 1 mm × 1.2 mm, was developed to investigate the microstructural modifications of corneal layers in four common corneal diseases. Since individual layer visualization without cutting through several layers is challenging due to corneal curvature, a flattening algorithm was developed to remove the global curvature of the endothelial layer and display the full view of the endothelium and Descemet’s membrane in single en face images. As a result, GD-OCM revealed the qualitative changes in size and reflectivity of keratocytes in Fuchs endothelial corneal dystrophy (FECD), which varied by the degree of disease. More importantly, elongated shape and hyperactivation characteristics of keratocytes, associated with the early development of guttae, appeared to start in the posterior stroma very early in the disease process and move toward the anterior stroma during disease progression. This work opens a venue into the pathogenesis of FECD.
KEYWORDS: Optical coherence microscopy, Optical coherence tomography, Microelectromechanical systems, Image resolution, Scanners, 3D image processing, 3D metrology, In vivo imaging, Real time imaging, Metrology, Microscopes, Image processing, Graphics processing units
Real-time volumetric high-definition wide-field-of-view in-vivo cellular imaging requires micron-scale resolution in 3D.
Compactness of the handheld device and distortion-free images with cellular resolution are also critically required for onsite
use in clinical applications. By integrating a custom liquid lens-based microscope and a dual-axis MEMS scanner in
a compact handheld probe, Gabor-domain optical coherence microscopy (GD-OCM) breaks the lateral resolution limit of
optical coherence tomography through depth, overcoming the tradeoff between numerical aperture and depth of focus,
enabling advances in biotechnology. Furthermore, distortion-free imaging with no post-processing is achieved with a
compact, lightweight handheld MEMS scanner that obtained a 12-fold reduction in volume and 17-fold reduction in weight
over a previous dual-mirror galvanometer-based scanner. Approaching the holy grail of medical imaging – noninvasive
real-time imaging with histologic resolution – GD-OCM demonstrates invariant resolution of 2 μm throughout a volume
of 1 x 1 x 0.6 mm3, acquired and visualized in less than 2 minutes with parallel processing on graphics processing units.
Results on the metrology of manufactured materials and imaging of human tissue with GD-OCM are presented.
Fast, robust, nondestructive 3D imaging is needed for characterization of microscopic structures in industrial and clinical applications. A custom micro-electromechanical system (MEMS)-based 2D scanner system was developed to achieve 55 kHz A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) instrument with a novel multilevel GPU architecture for high-speed imaging. GD-OCM yields high-definition volumetric imaging with dynamic depth of focusing through a bio-inspired liquid lens-based microscope design, which has no moving parts and is suitable for use in a manufacturing setting or in a medical environment. A dual-axis MEMS mirror was chosen to replace two single-axis galvanometer mirrors; as a result, the astigmatism caused by the mismatch between the optical pupil and the scanning location was eliminated and a 12x reduction in volume of the scanning system was achieved. Imaging at an invariant resolution of 2 μm was demonstrated throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. The MEMS-based scanner resulted in improved image quality, increased robustness and lighter weight of the system – all factors that are critical for on-field deployment. A custom integrated feedback system consisting of a laser diode and a position-sensing detector was developed to investigate the impact of the resonant frequency of the MEMS and the driving signal of the scanner on the movement of the mirror. Results on the metrology of manufactured materials and characterization of tissue samples with GD-OCM are presented.
We have developed a cellular resolution imaging modality, Gabor-Domain Optical Coherence Microscopy, which combines the high lateral resolution of confocal microscopy with the high sectioning capability of optical coherence tomography to image deep layers in tissues with high-contrast and volumetric resolution of 2 μm. A novelty of the custom microscope is the biomimetics that incorporates a liquid lens, as in whales’s eyes, for robust and rapid acquisition of volumetric imaging of deep layers in tissue down to 2 mm, thus overcoming the tradeoff between lateral resolution and depth of focus. The system incorporates a handheld scanning optical imaging head and fits on a movable cart that offers the flexibility in different biomedical applications and clinical settings, including ophthalmology. In the later, the microscope has successfully revealed micro-structures within the cornea and in particular the endothelial cells microenvironment, an important step in understanding the mechanisms of Fuchs’ dystrophy, a leading cause of the loss of corneal transparency. Also, the system was able to provide high definition of the edge of soft contact lenses, which is important for the fitting of the lens and the comfort of the patient. Overall, the imaging modality provides the opportunity to observe the three-dimensional features of different structures with micrometer resolution, which opens a wide range of future applications.
We study experimentally the scanning functions of galvanometer-based scanners (GSs) in order to optimize them for biomedical imaging in general, and for Optical Coherence Tomography (OCT) in particular. The main scanning parameters of the scanning process are taken into account: theoretical duty cycle (of the input signal of the GS), scan frequency (fs), and scan amplitude (θm). Triangular to sawtooth scanning regimes are thus considered. We demonstrate that when increasing the scan frequency and amplitude, the scanning function (i.e., the angular position of the galvomirror) is not able to follow anymore the input signal. Furthermore, as the theoretical duty cycle is increased, the result is unexpected: the effective duty cycle actually decreases – for high fs and θm. A saturation of the device therefore occurs. The practical limits of this phenomenon are discussed. GS users are thus provided with a multi-parameter analysis that allows them for optimizing their scanning regimes and to avoid pushing the devices to their limit – when that actually results in decreasing the quality of the images obtained, by example in OCT. Gabor Domain Optical Coherence Microscopy (GD-OCM) images are made to show effects of this phenomenon.
In biophotonics imaging, one important and quantitative task is layer-thickness estimation. In this study, we investigate
the approach of combining optical coherence tomography and a maximum-likelihood (ML) estimator for layer thickness
estimation in the context of tear film imaging. The motivation of this study is to extend our understanding of tear film
dynamics, which is the prerequisite to advance the management of Dry Eye Disease, through the simultaneous
estimation of the thickness of the tear film lipid and aqueous layers. The estimator takes into account the different
statistical processes associated with the imaging chain. We theoretically investigated the impact of key system
parameters, such as the axial point spread functions (PSF) and various sources of noise on measurement uncertainty.
Simulations show that an OCT system with a 1 μm axial PSF (FWHM) allows unbiased estimates down to nanometers
with nanometer precision. In implementation, we built a customized Fourier domain OCT system that operates in the
600 to 1000 nm spectral window and achieves 0.93 micron axial PSF in corneal epithelium. We then validated the
theoretical framework with physical phantoms made of custom optical coatings, with layer thicknesses from tens of
nanometers to microns. Results demonstrate unbiased nanometer-class thickness estimates in three different physical
phantoms.
Gabor-domain optical coherence microscopy (GD-OCM) is a volumetric high-resolution technique capable of acquiring three-dimensional (3-D) skin images with histological resolution. Real-time image processing is needed to enable GD-OCM imaging in a clinical setting. We present a parallelized and scalable multi-graphics processing unit (GPU) computing framework for real-time GD-OCM image processing. A parallelized control mechanism was developed to individually assign computation tasks to each of the GPUs. For each GPU, the optimal number of amplitude-scans (A-scans) to be processed in parallel was selected to maximize GPU memory usage and core throughput. We investigated five computing architectures for computational speed-up in processing 1000×1000 A-scans. The proposed parallelized multi-GPU computing framework enables processing at a computational speed faster than the GD-OCM image acquisition, thereby facilitating high-speed GD-OCM imaging in a clinical setting. Using two parallelized GPUs, the image processing of a 1×1×0.6 mm 3 skin sample was performed in about 13 s, and the performance was benchmarked at 6.5 s with four GPUs. This work thus demonstrates that 3-D GD-OCM data may be displayed in real-time to the examiner using parallelized GPU processing.
This paper proposes a theoretical and experimental analysis of the saturation effect in digital Fresnel holography and
generalizes the linear image formulation to the case of the non linear pixel saturation.
The method of digital holography wavefront reconstruction with adjustable magnification is introduced. Since there is
strong zero-order diffraction interference on the reconstruction plane, three different methods are proposed to eliminate
zero-order diffraction interference, and compared through experiment. The experimental results demonstrated that the
mean subtracted from a hologram is an effective method.
This letter presents a method for real-time three-color digital holographic interferometry based on Foveon CCD. The
control mode of CCD is optimized and color aliasing is limited consumedly. The influence on color hologram becomes
very little. On the whole, color aliasing is eliminated especially in the phase difference detecting. Moreover, the optical
setup is considerably simplified, since the reference beams are combined into a unique beam. Experimental results
confirm the suitability of the proposed method.
We present a simple set-up for digital color holography in which the reference beam has a unique way and the recording
uses a stacked photodiode sensor. A dedicated algorithm allows the color object to be reconstructed along each channel.
Experimental results confirm the proposed approach.
New double FFT convolution algorithms based on the use of spatial spectrum scanning or numerical spherical
reconstruction wave allow the full complex amplitude of large objects to be reconstructed. Experimental results in color
holography and contact less metrology validate the proposed methods.
This paper presents work and results performed with LAUM collaboration in digital three-color holographic
interferometry applied to Fluid Mechanics. In this method, three different wavelengths are used as luminous light source
of the interferometer and the optical setup generates three micro interferences fringes which constitute three spatial
carrier frequencies. When these images are recorded with a color sensor, the resolution of reconstructed hologram
depends on the pixel size and pixel number of the sensor used for recording and also, the shape and the overlapping of
three filters of color sensor influence strongly the three reconstructed images. This problem can be directly visualized in
2D Fourier planes on red, green and blue channels. To better understand this problem and to avoid parasitic images
generated at the reconstruction, three different sensors have been tested : a CCD sensor equipped with a Bayer filter, a
Foveon sensor and a 3CCD sensor. The best results have been obtained with the last one. In the recording principle,
interference micro fringes produced by the superimposition of three reference waves and three measurement waves can
be simultaneously recorded on the three spectral bands (red, green, and blue). Phase and amplitude images are computed
using 2D Fourier transform in delayed time. Spectral filtering is applied on each Fourier plane in order to eliminate the
parasitic diffraction orders. Then, phase differences are obtained by subtracting the reference phase to the probe phase.
Several optical setups were tested and the best configuration allows the visualization of field about 70mm and increases
the sensitivity since the measurement wave crosses twice the test section. Interferences induced by the wake flow have
been recorded and intensities have been computed from the phase differences. Finally, one shows that fringes obtained
with this process are those found with real-time color holographic interferometry using classical holographic plates.
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