In vivo imaging of intrinsic optical signals in chicken retina with functional optical coherence tomography

Visually evoked intrinsic optical signals (IOSs) were measured in vivo for the first time to our knowledge from all retina layers of the chicken retina with a combined functional optical coherence tomography and electroretinography (ERG) system. IOS traces were recorded from a small volume in the retina with 3.5 μm axial resolution and 7 ms time resolution. Comparison of the IOS and ERG traces shows a correlation between the positive and negative IOS measured from different retinal layers and the timing of the a and b waves in the ERG recording.     

Combined confocal/en face T-scan-based ultrahigh-resolution optical coherence tomography in vivo retinal imaging

Combined confocal scanning ophthalmoscopy/en face T-scan-based ultrahigh-resolution optical coherence tomography (OCT) of the human retina in vivo is reported for the first time to our knowledge. The system uses a superluminescent diode-based broadband source, which gives an axial resolution of 3.2 microm in the retina. We demonstrate acquisition of T-scan-based OCT B-scan and simultaneous confocal/C-scan images of the human retina of large lateral size (covering a field of up to 20 degrees ) at a frame rate of 2Hz.     

In vivo imaging of mice auricle vessels using adaptive optical confocal fluorescence microscope

Facilitated with stochastic parallel gradient descent(SPGD) algorithm for wavefront sensorless correcting aberrations, an adaptive optics(AO) confocal fluorescence microscopy is developed and used to record fluorescent signals in vivo. Vessels of mice auricle at 80, 100 and 120 μm depth are obtained, and image contrast and fluorescence intensity are significantly improved with AO correction. The typical 10%–90% rise-time of the metric value measured is 5.0 s for a measured close-loop bandwidth of 0.2 Hz. Therefore, the AO confocal microscopy implemented with SPGD algorithm for robust AO corrections will be a powerful tool for study of vascular dynamics in future.     

In vivo human retinal imaging by ultrahigh-speed spectral domain optical coherence tomography

An ultrahigh-speed spectral domain optical coherence tomography (SD-OCT) system is presented that achieves acquisition rates of 29,300 depth profiles/s. The sensitivity of SD-OCT and time domain OCT (TD-OCT) are experimentally compared, demonstrating a 21.7-dB improvement of SD-OCT over TD-OCT. In vivo images of the human retina are presented, demonstrating the ability to acquire high-quality structural images with an axial resolution of 6 microm at ultrahigh speed and with an ocular exposure level of less than 600 microW.     

In vivo measurement of retinal physiology with high-speed ultrahigh-resolution optical coherence tomography

Noninvasive in vivo functional optical imaging of the intact retina is demonstrated by using high-speed, ultrahigh-resolution optical coherence tomography (OCT). Imaging was performed with 2.8 microm resolution at a rate of 24,000 axial scans per second. A white-light stimulus was applied to the dark-adapted rat retina, and the average reflectivities from different intraretinal layers were monitored as a function of time. A 10%-15% increase in the average amplitude reflectance of the photoreceptor outer segments was observed in response to the stimulus. The spatial distribution of the change in the OCT signal is consistent with an increase in backscatter from the photoreceptor outer segments. To our knowledge, this is the first in vivo demonstration of OCT functional imaging in the intact retina.     

Imaging cortical electrical stimulation in vivo: fast intrinsic optical signal versus voltage-sensitive dyes

We applied high-temporal-resolution optical imaging utilizing both the fast intrinsic optical signal (fIOS) and voltage-sensitive dyes (VSDs) to observe the spatiotemporal characteristics of rat somatosensory cortex during electrical stimulation. We find that changes in both the fIOS and VSD signals occur rapidly (<30 ms) after the stimulus is applied, suggesting that both membrane depolarization and transmembrane ion movement occur shortly after the stimulus, preceding the more gradual physiological changes in oxygen consumption revealed by the slower component of the intrinsic optical signal. We find that the VSD signal spreads through a much larger area of cortex than the fIOS.     

In vivo whole-body imaging of optical agent dynamics using full angle fluorescence diffuse optical tomography

Dynamic fluorescence diffuse optical tomography （FDOT） is important in drug deliver research. In this letter, we first image the metabolic processes of micelles indocyanine green throughout the whole body of a nude mouse using the full-angle FDOT system with line illumination （L-FDOT）. The resolution of L-FDOT is evaluated using phantom experiment. Next, in vivo dynamic tomographic images （100 frames; approximately 170 min） of mouse liver and abdomen are shown and cross-validated by planar fluorescence reflectance imaging in vitro. Results provide evidence on applicability of the tomographic image wholebody biological activities in vivo on minute timescale （approximately 1.7 min） using L-FDOT.     

Contrast improvement of confocal retinal imaging by use of phase-correcting plates

We have developed a custom scanning laser ophthalmoscope that uses phase plates produced by photolithography to improve the contrast of human retinal images. We combined the scanning engine from a commercial real-time confocal microscope with custom optics to provide medium magnification imaging of the human retina (3 degrees field of view). Defocus and astigmatism were corrected with conventional trial lenses. Higher-order aberrations of the eye were corrected with a phase plate. A 633-nm laser was used for illuminating the retina. Inserting the phase plate into the optical system increased the contrast of a sample retinal vessel by 26%. Additionally, a number of small features of the retina, which were not visible with standard commercial imaging systems, became visible. There results illustrate that, with the rapid development of custom fabrication techniques for refractive corrections, improved diagnostic imaging with little added complexity to existing ophthalmic imaging systems may be realistic.     

Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.     

Linear self-referenced complex-field characterization of fast optical signals using photonic differentiation

This paper reviews recent work on a new group of linear and self-referenced techniques for full (amplitude and phase) characterization of fast optical signals based upon the concept of photonic differentiation, generally referred to as ‘phase reconstruction using optical ultrafast differentiation’ (PROUD). These techniques are particularly well adapted for applications in the context of fiber-optics telecommunications. PROUD methods can be implemented using simple and practical optical fiber-based setups and they rely on a direct, non-iterative phase recovery numerical algorithm. They can be used over a very wide range of pulse time durations, from the sub-picosecond to the nanosecond regime, and they can provide measurements in a single shot and in real time with power sensitivities down to the microwatt level. Previously reported PROUD methods are treated here under a unified, single framework, facilitating their analysis and comparison.     

激光差动共焦显微成像中的轴向快速定焦方法

提出了一种基于光强信号实时判断的快速定焦方法,在对样品进行横向扫描的同时,驱动测量物镜轴向移动,对前焦探测器和后焦探测器采集到的光强信号进行实时处理,通过实时判断测量面与样品的位置关系迅速找到焦面位置,可实现快速、准确的轴向定焦,极大地提升了定焦速度。理论分析和初步的实验表明,该方法在一副图像的采集周期内能够实现轴向定焦,在同等定焦精度条件下,与清晰度定焦方法相比,可节省90℅以上的定焦时间。     

In vivo bidirectional color Doppler flow imaging of picoliter blood volumes using optical coherence tomography

We describe a novel optical system for bidirectional color Doppler imaging of flow in biological tissues with micrometer-scale resolution and demonstrate its use for in vivo imaging of blood flow in an animal model. Our technique, color Doppler optical coherence tomography (CDOCT), performs spatially localized optical Doppler velocimetry by use of scanning low-coherence interferometry. CDOCT is an extension of optical coherence tomography (OCT), employing coherent signal-acquisition electronics and joint time-frequency analysis algorithms to perform flow imaging simultaneous with conventional OCT imaging. Cross-sectional maps of blood flow velocity with <50-microm spatial resolution and <0.6-mm/s velocity precision were obtained through intact skin in living hamster subdermal tissue. This technology has several potential medical applications.     

In vitro birefringence imaging with spectral domain polarization-sensitive optical coherence tomography

Spectral domain polarization-sensitive optical coherence tomography （SDPS-OCT） is a depth-resolved polarization-sensitive interferometry which integrates polarization optics into spectral domain optical co- herence tomography （SD-OCT）. This configuration can obtain birefringence information of samples and improve the imaging speed. In this paper, horizontally polarized light is used to replace natural light of the source. Then, right-rotated circularly polarized light is the incident sample light. To obtain two orthogonal components of the polarized interferogram, the reflected light of the reference arm is set to be 45° linearly polarized light. These two components are acquired by two spectrometers synchronously. The system was employed to achieve 12.8μm axial resolution and 4.36μm transverse resolution. We have imaged in vitro chicken tendon and muscle tissues with these svstem.     

High-speed, high-resolution optical coherence tomography retinal imaging with a frequency-swept laser at 850 nm

High-speed, high-resolution optical coherence tomography (OCT) imaging of the human retina is demonstrated using a frequency-swept laser at 850 nm. A compact external cavity semiconductor laser design, optimized for swept-source ophthalmic OCT, is described. The laser enables an effective 16 kHz sweep rate with >10 mm coherence length and a tuning range of approximately 35 nm full width at half-maximum, yielding an axial resolution of <7 micro m in tissue.     

In vivo imaging of the human rod photoreceptor mosaic

Although single cone receptors have been imaged in vivo, to our knowledge there has been no observation of rods in the living normal eye. Using an adaptive optics ophthalmoscope and post processing, evidence of a rod mosaic was observed at 5° and 10° eccentricities in the horizontal temporal retina. For four normal human subjects, small structures were observed between the larger cones and were observed repeatedly at the same locations on different days, and with varying wavelengths. Image analysis gave spacings that agree well with rod measurements from histological data.     

In vivo visualization of displacement-distribution-derived parameters in q-space imaging

OBJECTIVE: This study aimed to explore the potential of in vivo q-space imaging in the differentiation between different cerebral water components. MATERIALS AND METHODS: Diffusion-weighted imaging was performed in six directions with 32 equally spaced q values and a maximum b value of 6600 s/mm(2). The shape of the signal-attenuation curve and the displacement propagator were examined and compared with a normal distribution using the kurtosis parameter. Maps displaying kurtosis, fast and slow components of the apparent diffusion coefficients, fractional anisotropy and directional diffusion were calculated. The displacement propagator was further described by the full width at half and at tenth maximum and by the probability density of zero displacement P(0). Three healthy volunteers and three patients with previously diagnosed multiple sclerosis (MS) were examined. RESULTS: Simulations indicated that the kurtosis of a signal-attenuation curve can determine if more than one water component is present and that care must be taken to select an appropriate threshold. It was possible to distinguish MS plaques in both signal and diffusional kurtosis maps, and in one patient, plaques of different degree of demyelinization showed different behavior. DISCUSSION: Our results indicate that in vivo q-space analysis is a potential tool for the assessment of different cerebral water components, and it might extend the diagnostic interpretation of data from diffusion magnetic resonance imaging.     

In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure

We developed dual-modality microscope integrating photoacoustic microscopy (PAM) and fluorescence confocal microscopy (FCM) to noninvasively image hemoglobin oxygen saturation (sO?) and oxygen partial pressure (pO?) in vivo in single blood vessels with high spatial resolution. While PAM measures sO? by imaging hemoglobin optical absorption at two wavelengths, FCM quantifies pO? using phosphorescence quenching. The variations of sO? and pO? values in multiple orders of vessel branches under hyperoxic (100% oxygen) and normoxic (21% oxygen) conditions correlate well with the oxygen-hemoglobin dissociation curve. In addition, the total concentration of hemoglobin is imaged by PAM at an isosbestic wavelength.     

All-optical demultiplexing of 4-ASK optical signals with four-wave mixing optical gates

A new all-optical technique to demultiplex quaternary-amplitude-shift keying (4-ASK) signals into two on-off keying (OOK) signals is proposed. We show that such quaternary-to-binary conversion may be performed with two types of optical gates (OGs); a first one that implements an S-shaped optical power transfer function (TF), which is also the characteristic TF of optical pulse reshaping devices, and a second type that implements a U-shaped TF. Furthermore, we describe a heuristic approach to design such OGs by using the fiber four-wave mixing effect in highly non-linear dispersion-shifted fibers. The performance of the technique is evaluated by simulating the demultiplexing of 4-ASK signals with these OGs. Results indicate that the proposed devices are able to provide binary signals with acceptable BERs even after the 4-ASK signals are propagated through a cascade of eight fiber links of 50 km followed by optical amplifiers with a 4.5-dB noise figure.     

In vivo video rate multiphoton microscopy imaging of human skin

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.