Microlens array recording of localized retinal responses

We designed a rapid functional imager for the parallel recording of localized intrinsic optical signals (IOSs). This imager used a microlens array (MLA)-based illuminator to deliver visible stimulus light and near-infrared (NIR) recording light simultaneously. The parfocal configuration of the stimulus and recording light illumination enabled confocal recording of the stimulus-evoked IOSs. Because the MLA stimulation/recording spots were widely separated on the retina, and only the photoreceptors within the MLA stimulation/recording spots were stimulated, the potential IOS cross talk effect among neighboring retinal areas was minimized. Our experiments revealed robust IOS activities tightly correlated with localized retinal responses.     

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.     

Confocal calcium imaging with ultraviolet laser- scanning microscopy and indo-1

We developed a newly designed ultraviolet laser-scanning confocal microscopy (UV-LSCM) system and applied it for quantitative confocal imaging of intracellular calcium concentration ([Ca²⁺]_i) with the dual-emission wavelength indicator indo-1. The resolution and contrast of the biological sample images obtained using the current UV system were found to be comparable to those obtained with the conventional visible LSM optics and hence the UV-transmittable optics in our LSM system employing an achromatic objective lens provides appreciable confocality in the UV range. When indo-1 is used with this UV-LSCM system, dual- imaging ratiometric measurement of [Ca²⁺]_i can be easily performed without requiring time-consuming geometrical decalibration of the two simultaneously obtained images. The resulting confocal images allow quantitative analysis of [Ca²⁺]_i in rapidly contractile cardiac cells at a high temporal resolution in line-scan and fast frame-scan modes. The combined use of the UV-LSCM and dual- emission ratiometric indicator is now practical and we anticipate its widespread application in physiological and pathological studies in living cells.     

Optimized electron–optical system of a static mass-spectrometer for simultaneous isotopic and chemical analysis

A new approach to control the linear dimensions of analytical electrophysical systems is suggested. This approach uses the lens properties of electron–optical elements with a curvilinear axis. It is shown that such an approach can be effectively applied, in particular, to synthesize ion–optical systems (IOSs) for static magnetic mass spectrometers and can be implemented owing to off-axis fundamental points, the “poles” of an electron–optical system, introduced earlier by one of the authors. The capabilities of the new approach are demonstrated with the synthesis of the IOS of a static mass spectrometer dedicated for isotopic and chemical analysis with an increased resolution. A new IOS not only provides desired high ion–optical parameters at decreased dimensions of the mass spectrometer as a whole but also makes it possible to loosen requirements for the manufacturing accuracy of IOS main elements.     

Holoscopy--holographic optical coherence tomography

Scanning optical coherence tomography (OCT) is limited in sensitivity and resolution by the restricted focal depth of the confocal detection scheme. Holoscopy, a combination of holography and Fourier-domain full-field OCT, is proposed as a way to detect photons from all depths of a sample volume simultaneously with uniform sensitivity and lateral resolution, even at high NAs. By using the scalar diffraction theory, as frequently applied in digital holographic imaging, we fully reconstruct the object field with depth-invariant imaging quality. In vivo imaging of human skin is demonstrated with an image quality comparable to conventionally scanned OCT.     

光谱编码成像系统的数据压缩技术

利用时间拉伸显微成像系统观察并记录非重复动态随机现象,在其超高成像速度和高空间分辨率下必定会产生大量的数据。一种基于差分检测和游程编码的数据压缩方法,可以有效地解决时间拉伸成像系统的数据存储问题。差分检测可以消除连续相同的信号,只检测出相邻信号的差异,从而提高游程编码算法的有效性。实验中,采用扫描频率为77.76 MHz的时间拉伸显微成像对分辨率板、人红细胞和人乳腺癌细胞线性扫描成像。实验结果表明,数据压缩比可以达到8.47,对比分析发现经过差分检测方法可以获得更高的压缩比。另外,通过计算重建后的图像与原图的结构相似性(SSIM)值发现,经过数据压缩后高质量的图像可以被重建。     

Confocal pattern period in multiple-aperture confocal imaging systems with coherent illumination

We have built a programmable multiple-aperture confocal imaging system that uses a spatial light modulator (a Texas Instruments digital micromirror device). Excellent axial resolution and confocal imaging quality have been experimentally demonstrated with this system, even when the distance between adjacent ON pixels is four times the size of the pixel aperture. By contrast, typical pinhole-based systems (e.g., a Nipkow disk) employ an adjacent aperture distance-to-size ratio of 10x . We have achieved improvement over such systems by 6.25x in both light-utilization efficiency and confocal image rate without adding more components and extra processing time.     

基于电动可调焦透镜的大范围快速光片显微成像

搭建了一种基于电动可调焦透镜(electrically tunable lens)的大范围快速光片荧光显微成像系统.通过引入电动可调焦透镜与一维振镜以实现成像物平面和光片位置的快速移动,再结合高速s CMOS完成快速光片荧光显微成像.另外实验中通过改善光路与提升动态成像质量,实现了大范围扫描并减少了伪像.最终对成像性能进行测试,本系统的纵向分辨率和横向分辨率分别达到约5.5μm和约0.7μm,单幅图像稳定成像的速度约为275 frames/s,成像深度可超过138μm,能满足对具有一定尺寸的生物样本进行实时清晰成像的需求.     

Fast EPR imaging at 300 MHz using spinning magnetic field gradients

Electron paramagnetic resonance imaging (EPRI) technology has rapidly progressed in the last decade enabling many important applications in the fields of biology and medicine. At frequencies of 300-1200 MHz a range of in vivo applications have been performed. However, the requisite imaging time duration to acquire a given number of projections, limits the use of this technique in many in vivo applications where relatively rapid kinetics occur. Therefore, there has been a great need to develop approaches to accelerate EPRI data acquisition. We report the development of a fast low-frequency EPRI technique using spinning magnetic field gradients (SMFG). Utilizing a 300 MHz CW (continuous wave) EPRI system, SMFG enabled over 10-fold accelerated acquisition of image projections. 2D images with over 200 projections could be acquired in less than 3s and with 20s acquisitions good image quality was obtained on large aqueous free radical samples. This technique should be particularly useful for in vivo studies of free radicals and their metabolism.     

Circulation times of hepatocellular carcinoma cells by in vivo flow cytometry

Hepatocellular carcinoma （HCC） may metastasize to many organs. The survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. We have developed the ＂in vivo microscopy＂ to study the mechanisms that govern liver tumor cells spreading through the microenvironment in vivo. A recently developed ＂in vivo flow cytometer＂ combined with real-time confocal fluorescence imaging is used to assess spreading and the circulation kinetics of liver tumor cells. We measure the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which are from the same origin and obtained by repetitive screenings in mice. More than 60% of the HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, less than 40% of the HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.     

Circular polarization intrinsic optical signal recording of stimulus-evoked neural activity

Linear polarization intrinsic optical signal (LP-IOS) measurement can provide sensitive detection of neural activities in stimulus-activated neural tissues. However, the LP-IOS magnitude and signal-to-noise ratio (SNR) are highly correlated with the nerve orientation relative to the polarization plane of the incident light. Because of the complexity of orientation dependency, LP-IOS optimization and outcome interpretation are time consuming and complicated. In this study, we demonstrate the feasibility of circular polarization intrinsic optical signal (CP-IOS) measurement. Our theoretical modeling and experimental investigation indicate that CP-IOS magnitude and SNR are independent from the nerve orientation. Therefore, CP-IOS promises a practical method for polarization IOS imaging of complex neural systems.     

Simultaneous optical coherence tomography--Indocyanine Green dye fluorescence imaging system for investigations of the eye's fundus

We have developed a dual-channel optical coherence tomography-Indocyanine Green dye (OCT-ICG) fluorescence system based on a previously reported ophthalmic OCT confocal imaging system. The confocal channel is tuned to the fluorescence wavelength range of the ICG, and light from the same optical source is used to generate the OCT image and to excite the ICG fluorescence. The system enables the clinician to visualize simultaneously en face OCT slices and corresponding ICG angiograms of the ocular fundus, displayed side by side. C-scan (constant depth) and B-scan (cross section) images are collected by a fast en face scan (T scan). The pixel-to-pixel correspondence between the OCT and angiography images allows the user to capture OCT B scans precisely at selected points on the ICG confocal images.     

Characterization of ultrasonic imaging systems using transfer functions

The transfer functions for focussed and defocussed, coherent and confocal optical imaging systems have been applied to the equivalent ultrasonic imaging systems. The transfer functions with varying degrees of defocus were calculated to show the defocus effects for ultrasonic imaging systems. Assuming that the acoustic waves are reflected perfectly on the surface of the step edge, the theoretical line-scans for small amplitude signals across a step edge, with various degrees of defocus, were generated. The first derivative of the line-scan for a step edge is shown theoretically to yield the same impulse response as that calculated using the inverse Fourier transform applied to the original transfer function. These results show how the real and imaginary parts of the transfer functions contribute to ultrasonic image formation. A method for the experimental determination of the impulse response, and the transfer functions for the characterization of an ultrasonic imaging system, such as an acoustic microscope, is provided.     

低场MRI系统中线扫描扩散张量成像方法的研究

磁共振扩散张量成像(DTI)是在扩散加权成像(DWI)基础上发展起来的一种新型技术,可以无创伤显示脑白质纤维,诊断脑白质病变. 但是由于各种原因,DTI一般只在超导高场磁共振成像(MRI)仪器上进行,这就限制了这一重要诊断手段临床应用的广泛性. 本文在低场磁共振成像系统上应用线扫描实现了扩散张量成像,并测量了健康志愿者大脑内主要解剖结构的表观扩散系数(ADC)和各项异性分数(FA),得到的数据与高场仪器上的相关数据比较是吻合的. 因此临床上使用在低场强上得到的DTI图像评价脑白质是可行的,而且通常在临床上这也是足够的.     

Spectrally encoded confocal microscopy

An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique.     

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy

Optical sectioning techniques offer the ability to acquire three‐dimensional information from various organ tissues by discriminating between the desired in‐focus and out‐of‐focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.     

Multispectral imaging with a confocal microendoscope

The concept of a multispectral confocal microscope for in vivo imaging is introduced. To demonstrate the concept we modified a slit-scan fluorescence confocal microendoscope incorporating a fiber-optic catheter for in vivo imaging to record multispectral images. The system was designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. Preliminary experiments were carried out in phantoms and cell cultures to demonstrate the potential of the technique.     

Quasi-localization and quasi-mobility edge for light atoms mixed with heavy ones

A mixture of light and heavy atoms is considered. We study the kinetics of the light atoms, scattered by the heavy ones, the latter undergoing slow diffusive motion. In three-dimensional space we claim the existence of a crossover region (in energy), which separates the states of the light atoms with fast diffusion and the states with slow diffusion; the latter is determined by the dephasing time. For the two dimensional case we have a transition between weak localization, observed when the dephasing length is less than the localization length (calculated for static scatterers), and strong localization observed in the opposite case.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

基于线阵TDI-CCD器件的扫描成像系统设计

提出了由步进电机控制振镜运转,基于线阵TDI-CCD相机的扫描成像设计方案.通过研究线阵TDI-CCD器件的结构与工作原理,得到对振镜扫描速率和相机行扫描速率进行同步的方法,实现了空间位置配准.采用EC-11相机进行成像实验,扫描成像系统输出了可辨别的图像.