In a traditional lensless in-line holographic microscope (LIHM), the field of view (FOV) is generally limited by the size of the imaging sensor, which is usually several millimeters wide. We propose a scanning LIHM system using a line-scan camera to achieve wide field-of-view holographic imaging. In addition, the iterative holographic reconstruction method is applied to obtain the sample image with enhanced contrast and resolution using two hologram measurements, which are acquired by slightly shifting the sample-to-sensor distance. The capability of the proposed scanning LIHM system was demonstrated by experiments with a U.S. Air Force target and a biological sample of leaf vein. The reconstructed sample image had a FOV of 2.87 cm × 4.70 cm (2048 × 3357 pixels), which was much larger than the typical FOV of several square millimeters in a traditional LIHM system.
An endoscopic probe with high resolution and multiple contrasts provides useful diagnostic information. For example, a miniature probe capable of multimodal imaging including photoacoustic imaging, optical coherence tomography (OCT), and ultrasound has been reported. However, microscale resolution is only realized in OCT modality in the probe, which may restrict the applications where high resolutions for multiple contrasts are required. Here, we present an approach to construct a miniature scanhead with 2.8 mm in diameter that achieves high-resolution multispectral photoacoustic microscopy (PAM) and OCT. The method realizes high resolution of ∼10 μm for both PAM and OCT. We experimentally demonstrate ex vivo and in vivo imaging using the scanhead.
Pixelation, blur and additional noise of imaging system limit the resolution of final images acquired. Many pixel superresolution algorithms have been applied to enhance the resolution of imaging system by merging a sequence of lowresolution holograms with different type of imaging system, for example by shifting illumination source or using wavelength scanning. Most of these pixel super-resolution imaging systems can only be implemented to single-layer sample. For multi-layer imaging system and volumetric imaging scenarios, the relative displacement of various sample at different layers will disturb each other. Herein, we report a portable, cost-effective, lensless wide-filed digital in-line holographic microscopy imaging system based on in-line hologram segmentation and pixel super-resolution algorithm, which can separate target sample from the background and improve the resolution of the sample. We demonstrated the effectiveness of our system with numerical simulation and experiment with volumetric sample. In numerical simulation, we applied a very simple two-layer sample model that samples in two layers have various moving speed and directions and also did the volumetric imaging experiment with cuvette containing algae floating in. In our system, the sensor captured a sequence of low-resolution diffraction patterns. The target sample mix with background disturbance, which will invalidate the direct pixel super-resolution technique. We applied segmentation algorithm to the reconstructed images from holograms, separating target sample from background and generating a sequence of sub-images containing only target sample with same resolution and numerical aperture as original holograms. Finally the enhanced resolution reconstructed image of target sample was obtained with pixel super-resolution algorithm, which can go beyond pixel limitation and get sub-pixel perspective microscopy. This imaging system has the advantages of wide-field, portable and lensless.
In a compact digital lensless inline holographic microscope (LIHM), where the sample-to-sensor distance is short, the imaging resolution is often limited by sensor pixel size instead of the system numerical aperture. We propose to solve this problem by applying data interpolation with an iterative holographic reconstruction method while using grating illumination in the LIHM system. In the system setup, the Talbot self-image of a Ronchi grating was used to illuminate the sample, and the inline hologram was recorded by a CMOS imaging sensor located behind the sample. The hologram was then upsampled by data interpolation before the reconstruction process. In the iterative holographic reconstruction, the sample support was defined by the bright areas of the grating illumination pattern and was used as constraint. A wide-field image can also be obtained by shifting the grating illumination pattern. Furthermore, we assumed that the sample was amplitude object, i.e., no obvious phase change was caused by the sample, which provided additional constraint to refine the interpolated data values. Besides improved resolution, the iterative holographic reconstruction also helped to reduce the twin-image background. We demonstrated the effectiveness of our method with simulation and imaging experiment by using the USAF target and polystyrene microspheres with 1 μm diameter as the sample.
KEYWORDS: Optical coherence tomography, Endoscopy, Biomedical optics, Compressed sensing, Data acquisition, 3D image processing, Mirrors, 3D acquisition, Single mode fibers, Signal detection
Conventionally endoscopic OCT images are reconstructed by uniform sampling in both axial and transverse direction of the image. To shorten the scanning time and reduce the acquisition data, we propose to sparsely sample the axial scans and use compressive sensing (CS) to reconstruct the full endoscopic OCT image with randomly chosen axial scans that are much less than conventional Nyquist criteria requirement. And we study the ratio of sparse sampling numerically and experimentally that are required to reconstruct acceptable OCT images. We demonstrate that the OCT image acquisition time can be significantly reduced because of much less acquired data.
High resolution is always a pursuing target in the imaging field, as a new prospective technique in imaging applications, digital in-line holography has become a very active field for compactness, more information and low-cost. However, for compact system, the resolution is often limited by sensor pixel size. To overcome this problem, we propose an iterative reconstruction method with data interpolation based on the grating illumination. In our method, the Talbot self-image of a Ronchi grating is exerted in the sample plane as a priori constraint which lead to the convergence of the iteration, the iteration between the sample plane and the sensor plane can provide some extra information with interpolation in the sensor plane based on the a priori constraint, furthermore, the iteration reconstruction can also eliminates the twin-image to improve the image quality. Numerical simulation has been conducted to show the effectiveness of this method. In order to make a further verification, we have developed a lensless in-line holographic microscope with a compact and wide field-of-view design. In our setup, the sample was under the Talbot image illumination of the Ronchi grating, which was illuminated by a collimated laser beam, and holograms were recorded by a digital imaging sensor. We can shift the grating laterally to get a wide-field image. We demonstrated the resolution of our imaging system by using the USAF resolution target as a sample, and the results shown the resolution improvement of the image.
KEYWORDS: Image resolution, 3D image reconstruction, Holography, Super resolution, Digital holography, Holograms, Reconstruction algorithms, Microscopes, Sensors, Detection and tracking algorithms
We report a new holographic microscope using pixel super-resolution algorithm. In our method, a sequence of low resolution images are acquired by a complementary metal oxide semiconductor (CMOS) sensor in digital inline holography system and the resolution is limited by the sensor pixel size. Then the super-resolution algorithm is applied to the low resolution images to get the image with much higher resolution that beyond the Nyquist criteria. We perform both numerical simulation and experiments to demonstrate our method with US Air Force Target used as the sample. The sample is randomly moved in the sample plane and a set of holograms are captured by the camera in inline holographic system. We use two methods to reconstruct the sample image. In the first method, super-resolution algorithm is applied with the low resolution holograms to get the high resolution hologram. Then the high resolution hologram is reconstructed using auto-focusing algorithm to get the high resolution sample image. In the second method, the raw holograms are directly reconstructed to get a set of low resolution sample images, then the super-resolution algorithm is applied to get the high resolution sample image. We observed that the above mentioned two methods can get similar results in both numerical stimulation and experiments. We believe that the combination of pixel super-resolution algorithm and digital in-line holography can be very useful to implement a compact low-cost microscope with high resolution.
We propose to use sparsely sampled line scans with a sparsity-based reconstruction method to obtain images in a wide field of view (WFOV) multifocal scanning microscope. In the WFOV microscope, we used a holographically generated irregular focus grid to scan the sample in one dimension and then reconstructed the sample image from line scans by measuring the transmission of the foci through the sample during scanning. The line scans were randomly spaced with average spacing larger than the Nyquist sampling requirement, and the image was recovered with sparsity-based reconstruction techniques. With this scheme, the acquisition data can be significantly reduced and the restriction for equally spaced foci positions can be removed, indicating simpler experimental requirement. We built a prototype system and demonstrated the effectiveness of the reconstruction by recovering microscopic images of a U.S. Air Force target and an onion skin cell microscope slide with 40, 60, and 80% missing data with respect to the Nyquist sampling requirement.
This paper proposes an endoscopic probe for optical coherence tomography (OCT) applying on side-imaging of internal organs. The probe consists of single-mode fiber, a gradient-index (GRIN) lens, and a mirror of cylindrical wedge shape attaching to a magnetized metal piece by epoxy with a short steel wire. For OCT scanning, we use magnetic field generated by a larger magnet externally to drive the rotation of the magnetized metal. Compare with other probes, our probe design has two distinct advantages: 1) The exit beam will be unobstructed during 360 degree circumference scanning because there are no connecting wires in the scanning part. 2) In principle, the probe can be made very tiny because of the simple structure consisting only the single-mode fiber, GRIN lens, reflection mirror and the magnetized metal piece. The OCT system has axial resolution of 14µm and SNR of 98.6 dB. The probe prototype we made has an outer diameter of 1.4 mm.
Focus-grid-based wide field-of-view microscope has been developed to break the trade-off of resolution and field of view in conventional microscopy. In the wide field-of-view microscopy, the whole sample area has to be scanned with at least Nyquist-frequency sampling for image reconstruction. We propose a novel scanning mechanism using compressive sensing (CS), where the scanning of focal spots covers only a portion of the sample. Our preliminary studies show that 75% of scanning area is enough to reconstruct a decent image. Thus we can reduce the acquisition data which allow for simpler scanning mechanism and data acquisition.
Digital in-line holography (DIH) is a lensless imaging technique that can be used to build low-cost and compact imaging systems. In DIH, the in-line hologram is recorded by a CMOS or CCD sensor and later used to reconstruct the image of the sample. The imaging resolution is determined by the system numerical aperture provided that the pixel size is smaller than the required Nyquist criteria for sampling distance. In the case of short sample-to-sensor distance, pixel size is often a limiting factor for the resolution. To solve this problem, we propose to use iterative method along with data interpolation for the holographic reconstruction. Proof-of-concept numerical simulations have been done to show the effectiveness of our method. In our algorithm, the optical field is propagated back and forth between the sample plane and the sensor plane while using the measured intensity and a priori information about the sample as constraints, following Gerchberg-Saxton and Fienup’s methods. The iteration will converge and we can get both intensity and phase information of the sample. Before the iteration, the intensity data matrix measured by the sensor is interpolated to enlarge the matrix dimension and thus effectively reduce the pixel size. During the iteration, we apply the sensor plane constraints on only the measured intensity location but not the interpolated data location. In our simulation, we observed that during the iteration, the interpolated data will be changed reasonably and we can finally reconstruct the sample image with better resolution.
We propose a lensless microscopic imaging technique based on iteration algorithm with known constraint for image reconstruction in digital in-line holography. In our method, we introduce a constraint on the sample plane as known part in the lensless microscopy for iteration algorithm in order to eliminate the twin-image effect of holography and thus lead to better performance on microscopic imaging. We evaluate our method by numerical simulation and built a prototype in-line holographic imaging system and demonstrated its capability by preliminary experiments. In our proposed setup, a carefully designed photomask used to hold the sample is under illumination of a coherent light source. The in-line hologram is then recorded by a CMOS sensor. In the reconstruction, the known information of the illumination beam and the photomask is used as constraints in the iteration process. The improvement of image quality because of suppression of twin-images can be clearly seen by comparing the images obtained by direct holographic reconstruction and our iterative method.
We studied the use of Talbot pattern illumination in scanning optical microscopy (SOM). Unlike conventional illumination spots used in SOM, the focal spots in Talbot pattern are more complicated and do not have a simple Gaussian intensity distribution. To find out the resolution of SOM using Talbot pattern, we characterized the evolution of the full-width-at-half-maximum spot size of the Talbot focal spots by computer simulation. We then simulated the SOM imaging under Talbot pattern illumination using the razor blade and the U.S. Air Force target as the sample objects, and compared the results with those performed with Gaussian spots as illumination. Using several foci searching algorithms, the optimal focal distances were found to be shorter than the theoretical Talbot distances. The simulation results were consistent with the experiment results published previously. We then provide a practical guidance for searching for optimal focal distances in the SOM based on these studies.
Wide field-of-view (FOV) microscopy is useful for high-throughput applications because of the capability to obtain
large amount of information from a single image. One way to implement a wide FOV microscope is to scan the sample
with a two-dimensional focus grid. The transmission or reflection of the focal spots can then be used to reconstruct the
sample image. This scheme is effectively a parallel scanning optical microscope (SOM), where the FOV depends on the
area of the focus grid and the imaging resolution depends on the spot size of the foci. We use the Talbot image of a twodimensional
aperture grid as the focus grid and developed a wide FOV microscope. Preliminary experimental results
show the capability of our microscope to acquire wide FOV images of US air force target and MCF-7 cancer cell
samples. Fluorescence images of fluorescence beads are also acquired. Because the diffraction of incident beam by the
aperture grid contains complicated angular frequencies, the focal spots in Talbot pattern cannot be approximated as
Gaussian beams as in conventional SOM. We characterized the focal spots in Talbot pattern and studied the evolution of
the full width at half maximum (FWHM). We also simulated the SOM imaging under Talbot pattern illumination using
the razor blade as the sample objects.
We have developed a novel microscope technique that can achieve wide field-of-view (FOV) imaging and yet possess
resolution that is comparable to conventional microscope. The principle of wide FOV microscope system breaks the link
between resolution and FOV magnitude of traditional microscopes. Furthermore, by eliminating bulky optical elements
from its design and utilizing holographic optical elements, the wide FOV microscope system is more cost-effective. In
our system, a hologram was made to focus incoming collimated beam into a focus grid. The sample is put in the focal
plane and the transmissions of the focuses are detected by an imaging sensor. By scanning the incident angle of the
incoming beam, the focus grid will scan across the sample and the time-varying transmission can be detected. We can
then reconstruct the transmission image of the sample. The resolution of microscopic image is limited by the size of the
focus formed by the hologram. The scanning area of each focus spot is determined by the separation of the focus spots
and can be made small for fast imaging speed. We have fabricated a prototype system with a 2.4-mm FOV and 1-μm
resolution. The prototype system was used to image onion skin cells for a demonstration. The preliminary experiments
prove the feasibility of the wide FOV microscope technique, and the possibility of a wider FOV system with better resolution.
We report the narrowest to-date (21 gauge, 820-µm-diam) handheld forward-imaging optical coherence tomography (OCT) needle endoscope and demonstrate its feasibility for ophthalmic OCT inspection. The probe design is based on paired-angle-rotation scanning (PARS), which enables a linear B-scan pattern in front of the probe tip by using two counterrotating angle polished gradient-index (GRIN) lenses. Despite its small size, the probe can provide a numerical apertune (NA) of 0.22 and an experimental sensitivity of 92 dB at 0.5 frame/s. The feasibility of retinal imaging is tested on enucleated ex vivo porcine eyes, where structural features including remnant vitreous humor, retina, and choroid can be clearly distinguished. Due to its imaging quality comparable to a commercial OCT system and compatibility with the current ophthalmic surgery standard, the probe can potentially serve as a better alternative to traditional visual inspection by white light illumination during vitreoretinal surgery (e.g., vitrectomy).
Our group has reported the use of harmonically matched diffraction grating for full-field quantitative phase imaging. In
this paper, we show the improvement of this technique and the application in observing dynamics of transparent
samples. By using the grating as a beam splitter/combiner in an interferometer, we are able to obtain non-trivial phase
difference between the output ports of the grating. We have built a Mach-Zehnder interferometer using the holographic
grating with 600 and 1200 lines/mm spacing. Two CCD cameras at the output ports of the grating-based Mach-Zehnder
interferometer are used to record the full-field quadrature interferograms, which are subsequently processed to
reconstruct the phase image. Since the two quadrature interferograms are acquired at the same time, the imaging speed
of the system is limited only by the frame rate of the CCD cameras. We have demonstrated the capability of our system
by observing dynamics of transparent samples.
We report a new method for obtaining non-trivial phase difference between the output ports of an interferometer
through the use of shallow diffraction gratings. We show that as opposed to a single shallow diffraction grating-based
interferometer (which provides only trivial phase shifts, i.e., 0° or 180°), a pair of harmonically-related shallow
diffraction gratings can be used to design interferometers with non-trivial phase shifts between different output ports.
More importantly, the phase shifts can be adjusted by simply shearing one grating with respect to the other. This
approach does not change the path length relationships of the different interference beams within the interferometer,
which is an advantage for metrology and low coherence interferometry applications.
In this paper, we report the use of holographic gratings, which act as the free-space equivalent of the 3x3 fiber-optic
coupler, to perform full field phase imaging. By recording two harmonically-related gratings in the same holographic
plate, we are able to obtain nontrivial phase shift between different output ports of the gratings-based Mach-Zehnder
interferometer. The phase difference can be adjusted by changing the relative phase of the recording beams when
recording the hologram. We have built a Mach-Zehnder interferometer using harmonically-related holographic gratings
with 600 and 1200 lines/mm spacing. Two CCD cameras at the output ports of the gratings-based Mach-Zehnder
interferometer are used to record the full-field quadrature interferograms, which are subsequently processed to
reconstruct the phase image. The imaging system has ~12X magnification with ~420μmx315μm field-of-view. To
demonstrate the capability of our system, we have successfully performed phase imaging of a pure phase object and a
paramecium caudatum.
Vitrectomy (removal of the vitreous humor) is an ophthalmic surgery required as a precursor to several posterior chamber procedures. Vitrectomy is commonly performed using an endoscopic vitreous cutter and fiber based light delivery for observation through a surgical microscope. Cross-sectional visualization of the retina and remnant vitreous layers during surgery using an external optical coherence tomography (OCT) scanner is impractical due to deformation in the shape of the eye and the cornea. We present a forward imaging probe with 820 &mgr;m outer diameter (21 gauge needle) for cross-sectional endoscopic OCT imaging during ophthalmic surgeries. The Paired-Angle-Rotating Scanner (PARS) OCT probe is based on angle polished gradient index (GRIN) lenses which are rotated about the optical axis. The scan pattern is determined by the angle between the GRIN lenses and the relative angular velocity. Endoscopic placement of the PARS-OCT probe tip near the retinal surface permits use of a longer wavelength light, in particular 1310 nm, which would otherwise suffer significant attenuation traversing the vitreous humor. The prototype endoscopic PARS-OCT probe is coupled to a commercially available 1310 nm swept laser source, and uses commercial software for data acquisition, processing, and display of retinal images in real time at an A-scan rate of 16 kHz. We present an analysis of aberrations due to off axis use of GRIN lenses and measure the scan pattern of the PARS probe. Images acquired on an ex vivo porcine retina are presented, motivating development of the endoscopic PARS-OCT probe for clinical evaluation.
We review the current state of research in endoscopic optical coherence tomography (OCT). We first survey the range of available endoscopic optical imaging techniques. We then discuss the various OCT-based endoscopic methods that have thus far been developed. We compare the different endoscopic OCT methods in terms of their scan performance. Next, we examine the application range of endoscopic OCT methods. In particular, we look at the reported utility of the methods in digestive, intravascular, respiratory, urinary and reproductive systems. We highlight two additional applications—biopsy procedures and neurosurgery—where sufficiently compact OCT-based endoscopes can have significant clinical impacts.
The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (~190 kJ/cm2) is then used to permanently photobleach the ICG molecules—resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, B,cum, defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that B,cum decreases with fluence as well as with increasing dye concentration. The quantity B,cum is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.
Use of indocyanine green (ICG), an FDA-approved dye, in a pump-probe scheme for optical coherence tomography (OCT) is reported. Aqueous solutions of ICG are not stable, i.e., the dye degrades over time especially in the presence of light. Addition of protein such as bovine serum albumin (BSA) stabilizes the ICG; however, when exposed to high intensity illumination, the dye still degrades. Moreover, the photodegradation is permanent and occurs swiftly if the illumination band corresponds to the ICG absorption peak. The permanence of the photobleached state illustrates that ICG photobleaching phenomenon has great potential to achieve contrast in OCT. ICG solutions with 50 micromolar concentration were prepared in water, 1% BSA, and 0.8% agarose to study the dynamics of the dye for different illumination intensity levels. In addition, different molar concentrations of ICG in water were studied for fixed illumination intensity. In each case, probability of photobleaching, defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground-state molecules, is evaluated to characterize the photobleaching phenomenon in ICG. We also demonstrate ICG-based pump-probe MCOCT imaging by mapping the distribution of ICG in a stage 54 Xenopus laevis.
We propose a novel forward-imaging OCT needle probe. The probe is based on the use of two angled GRIN lenses that can freely rotate with respect to each other. The probe is capable of scanning a forward cone volume ahead of the probe tip. Different scanning modes, such as the conventional OCT B-scan mode, spiral mode and starburst B-scan mode, can be obtained by adjusting the angular scan velocities of the two GRIN lenses. We develop a prototype probe and demonstrate its capability to acquire OCT images. In this paper we give the characteristics of the prototype probe and display images of different part of tadpole acquired by the probe. The longitudinal resolution, lateral resolution and the signal-to-noise ratio of the system are 10 μm, 10 μm and 93 dB, respectively.
Optical Coherence Tomography is a new technique mainly used in biomedical imaging. Here we present a Particle-Fixed Monte Carlo (PFMC) simulation for OCT signal. In PFMC model the scattering particles of the sample are assumed to be temporarily fixed randomly in simulation process of the backscattering light. The new model, beyond the convention Monte Carlo simulation, explains very well the exponential decay signal at the interface of different media layers in OCT experimental measurement.
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