Optical microangiography (OMAG) is a powerful optical angiographic tool to visualize micro-vascular flow in vivo. Despite numerous demonstrations for the past several years of the qualitative relationship between OMAG and flow, no convincing quantitative relationship has been proven. In this paper, we attempt to quantitatively correlate the OMAG signal with flow. Specifically, we develop a simplified analytical model of the complex OMAG, suggesting that the OMAG signal is a product of the number of particles in an imaging voxel and the decorrelation of OCT (optical coherence tomography) signal, determined by flow velocity, interframe time interval, and wavelength of the light source. Numerical simulation with the proposed model reveals that if the OCT amplitudes are correlated, the OMAG signal is related to a total number of particles across the imaging voxel cross-section per unit time (flux); otherwise it would be saturated but its strength is proportional to the number of particles in the imaging voxel (concentration). The relationship is validated using microfluidic flow phantoms with various preset flow metrics. This work suggests OMAG is a promising quantitative tool for the assessment of vascular flow.
We demonstrate in vivo volumetric optical microangiography at ∼200 volumes/s by the use of 1.6 MHz Fourier domain mode-locking swept source optical coherence tomography and an effective 36 kHz microelectromechanical system (MEMS) scanner. We propose an intervolume analysis strategy to contrast the dynamic blood flow signal from the static tissue background. The proposed system is demonstrated by imaging cerebral blood flow in mice in vivo. For the first time, imaging speed, sensitivity, and temporal resolution become possible for a direct four-dimensional observation of microcirculations within live body parts.
Traumatic brain injury (TBI) is a form of brain injury caused by sudden impact on brain by an external mechanical force.
Following the damage caused at the moment of injury, TBI influences pathophysiology in the brain that takes place
within the minutes or hours involving alterations in the brain tissue morphology, cerebral blood flow (CBF), and
pressure within skull, which become important contributors to morbidity after TBI. While many studies for the TBI
pathophysiology have been investigated with brain cortex, the effect of trauma on intracranial tissues has been poorly
studied. Here, we report use of high-resolution optical microangiography (OMAG) to monitor the changes in cranial
meninges beneath the skull of mouse after TBI. TBI is induced on a brain of anesthetized mouse by thinning the skull
using a soft drill where a series of drilling exert mechanical stress on the brain through the skull, resulting in mild brain
injury. Intracranial OMAG imaging of the injured mouse brain during post-TBI phase shows interesting
pathophysiological findings in the meningeal layers such as widening of subdural space as well as vasodilation of
subarachnoid vessels. These processes are acute and reversible within hours. The results indicate potential of OMAG to
explore mechanism involved following TBI on small animals in vivo.
Optical microangiography (OMAG) has been extensively utilized to study three-dimensional tissue vasculature in vivo. However, with the limited image resolution (∼10 μm) of the commonly used systems, some concerns were raised: (1) whether OMAG is capable of providing the imaging of capillary vessels that are of an average diameter of ∼6 μm; (2) if yes, whether OMAG can provide meaningful quantification of vascular density within the scanned tissue volume. Multiphoton microscopy (MPM) is capable of depth-resolved high-resolution (∼1 μm) imaging of biological tissue structures. With externally labeled plasma, the vascular network including single capillaries can be well visualized. We compare the vascular images of in vivo mouse brain acquired by both OMAG and MPM systems. We found that within the penetration depth range of the MPM system, OMAG is able to accurately visualize blood vessels including capillaries. Although the resolution of OMAG may not be able to 100% resolve two closely packed tiny capillaries in tissue, it is still capable of visualizing most of the capillaries because there are interstitial tissue spaces between them. We believe our validation results reinforce the application of OMAG in microvasculature-related studies.
Fluorescence holographic microscope (FINCHSCOPE) is a motionless fluorescence holographic imaging technique based on Fresnel incoherent correlation holography (FINCH) that shows promise in reconstructing three-dimensional fluorescence images of biological specimens with three holograms. We report a developing two-step phase-shifting method that reduces the required number of holograms from three to two. Using this method, we resolved microscopic fluorescent beads that were three-dimensionally distributed at different depths with two interferograms captured by a CCD camera. The method enables the FINCHSCOPE to work in conjunction with the frame-straddling technique and significantly enhance imaging speed.
FINCHSCOPE is a new technology of fluorescence holographic microscopy. It has been successfully applied to recording high-resolution three-dimensional fluorescence images of biological specimens without the need for scanning. In this study, we revealed and analyzed an intrinsic phenomenon, called ghost lens effect, on spatial light modulator which is the core element enabling the incoherent correlation in the FINCHSCOPE. The ghost lens effect can degrade the imaging quality by introducing multiple spherical waves with different focal lengths into the correlation and thus increasing the noise in the recorded holograms.
Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.
We developed a novel addressable multiregional multiphoton microscope that employs a fast one-dimensional discrete-line scanning approach based on a spatial light modulator (SLM). The phase-only SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple specific discrete-lines, which are designed according to the sizes and locations of the target samples. Only the target-sample areas of are scanned
one-dimensionally, resulting in an efficient use of the laser’s power. Compared with conventional multiphoton microscopies, this technique shortens scanning time and minimizes photodamage by concentrating scanning energy and dwell time on the areas of interest. Additionally, our discrete-line-focus design eliminates the cross-talk that occurs in conventional one-dimensional line-scanning multiphoton microscopes, thus enhancing the lateral and axial resolutions of the line-scanning imaging system.
We developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain neurons and spinal cord neurons. Observation of neurons under a high-magnification microscope, which we obtained from these two cell types, showed no difference in morphology. However, when flowing in the microfluidic channel and simultaneously being laser guided, the two cell types gained quite different guidance speeds under the same experimental conditions. The results demonstrate that different cell types with the same morphology (e.g., size, shape, etc.) can be effectively distinguished from each other by measuring the difference in guidance speeds (the maximum flow speed minus the initial flow speed). This technique is expected to provide a new approach to high-throughput, label-free cell sorting with high sensitivity.
In this study, we developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain
neurons and chick embryonic spinal cord neurons. The neurons we obtained from these two cell types have no difference in
morphology as observed under a high-magnification microscope. However, when flowing in the microfluidic channel and
simultaneously being laser-guided, the two cell types gained quite different guidance velocities under the same
experimental conditions. The experimental results demonstrate that different cell types with the same morphology (e.g.,
size, shape, etc.) can be effectively distinguished from each other by measuring the difference of guidance velocities (the
maximum flow velocities minus the initial flow velocities). This technique is expected to provide a new approach to
high-throughput, label-free cell sorting with sensitivity.
Using hybrid TPEF-SHG imaging and immunocytological techniques, we studied dedifferentiation of adult
cardiomyocytes. First, the myofibrils shrank to shorten the sarcomere length. At the cell ends, the striated pattern of
myosin filaments began to dissociate; at the center of the cell, the striated pattern of alpha-actinin first faded away and
reappeared near the cell membrane during dedifferentiation. The results suggest that when freshly isolated adult
cardiomyocytes are used to model cardiac muscle, the end-to-end connection may be important to maintain their striated
myofibrillar structure and rod-shape morphology.
Through a combination of a deflective phase-only diffractive spatial light modulator (SLM) and galvo scanners, an addressable multiregional and multifocal multiphoton microscope (AM-MMM) is developed. The SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple beamlet arrays with addressable shapes and sizes that match the regions of interest on the sample. Compared with conventional multifocal multiphoton microscope (MMM), AM-MMM achieves the effective use of the laser power with an increase of imaging rate and a decrease of photodamage without sacrifice of resolution.
We developed a novel addressable scanless multifocal multiphoton microscope. This microscope works in a fast scanless
mode. Subjectively selected sample (or multiple samples located in separated areas) in a large field of view can be
imaged by illuminating only the area (or areas) where the target sample (or samples) locate(s). In this way, by precisely
designing the multiple foci according to the size and position of the area of interest, we can concentrate all the laser
energy and dwell time on that area of the sample, making full use of the available laser power and avoiding photodamage
in other areas. Since no scanning is involved, the acquisition time of a multiphoton image is decided only by the
sensitivity and readout time of the CCD camera. Moreover, the interfocal distance of the multiple foci matches the lateral
resolution of the imaging system, so that the two-photon image was recorded with high lateral resolution. However,
crosstalk (spatial interference) on out-of-focus planes occurs between adjacent points when they are too close, degrading
the resolution, especially the axial resolution of the imaging system.
Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second
harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes
during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of
existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed
mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled
onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an
important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length
during maturation.
We developed a hybrid two-photon excitation fluorescence-second harmonic generation (TPEF-SHG) imaging system with an on-stage incubator for long-term live-cell imaging. Using the imaging system, we observed the addition of new sarcomeres during myofibrillogenesis while a cardiomyocyte was spreading on the substrate. The results suggest that the TPEF-SHG imaging system with an on-stage incubator is an effective tool for investigation of dynamic myofibrillogenesis.
We propose a novel optical asymmetric cryptosystem based on a phase-truncated Fourier transform. Two decryption keys independent of each other are generated. They are referred to as universal key and special key, respectively. Each of them can be used for decryption independently in absence of the other. The universal key is applicable to decrypt any ciphertext encoded by the same encryption key, but with poor legibility. On the contrary, the special key is adequate for legible decryption, but only valid for one ciphertext corresponding to the specified plaintext. A set of simulation results show the interesting performance of two types of decryption keys.
The optical cryptosystem based on phase-shifting interferometry (PSI) is one of the most interesting optical cryptographic schemes in recent years. However, we find that the PSI technique provides an attractive method to record the ciphertext, but contributes little to the security level of the cryptosystem. From the cryptanalysis point of view, in a certain simplified case, an attacker is only required to crack two equivalent decryption keys instead of the original random phase keys and geometric key. Moreover, a chosen-plaintext attack method is proposed, in which an impulse function is chosen as a known plaintext. By using this attack, the attacker can effectively recover any plaintext from the corresponding ciphertext. The validity of the attack is verified by computer simulations.
From the perspective of optical cryptanalysis, we propose a method of chosen-plaintext attack against the optical encryption scheme based on joint transform correlator (JTC) architecture. By implementing this attack, an opponent can access the encryption key with the help of the pre-knowledge of a special chosen plaintext and the corresponding ciphertext. It indicates that the optical encryption scheme based on the JTC architecture is vulnerable to chosen-plaintext attack, even though introducing a nonlinear operation into the encryption transform. The validity of this attack is verified by numerical simulations.
In this paper, we analyze the security of the classical double random phase encoding (DRPE) technique in Fourier
domain, as well as its extended schemes in Fresnel and fractional Fourier domains. These schemes are resistant to bruteforce
attacks, for their large key spaces. However, due to the linearity property of their encryption transformations, they
are vulnerable to other attacks, such as chosen-plaintext attack and known-plaintext attack. We successfully break each
of the three encryption schemes with the help of a certain quantity of plaintext-ciphertext pairs. Each attack is validated
by computer simulations. The cryptanalysis indicates that, to minimize the risks, it is recommendable to introduce
nonlinear operations to optical encryption systems.
We report a high-power diode-double-end-pumped Q-switched Nd:YVO4 red laser through intracavity
frequency-doubling with a type-I critical phase-matched LBO crystal. At the repetition frequency of 26 kHz, the
maximum quasi-continuous-wave (QCW) output power at 671 nm was measured to be 9.2 W at the incident pump power
of 62.4 W with the corresponding optical conversion efficiency of 14.7%; the pulse width of about 106 ns; the pulse
energy of 353.8μJ; the peak power of 3.34 kW, respectively. The perfect performance indicated that the
double-end-pumped technique could effectively diminish the thermal lens effect, extend the range of the stable resonator
region and achieve a high optical conversion efficiency to obtain a high output power.
Heat sinks with impedance matching circuit have been designed and fabricated for the packaging of high-speed electroabsorption (EA) modulators. Ti/Cu/Ni/Au metallization system is adopted for the coplanar waveguide (CPW) electrodes and a 50-ohm Ta2N thin-film resistor in parallel with the EA modulator is used for impedance matching. By a matching resistance optimization, a reflection coefficient S11 better than -21 dB has been demonstrated up to 40 GHz. The heat sinks are applied successfully in the 40 GHz Modulator packaging.
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