In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope

We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.     

Multispectral imaging using multiple-bandpass filters

We present a method for multispectral imaging. This method uses color CCD cameras with a multiple-bandpass filter, which modifies the spectral response of the cameras used and enables concurrent acquisition of multiple images at defined spectral bands. We experimentally demonstrate methodological feasibility using two color CCD cameras and a polychroic mirror to simultaneously capture eight spectral bands. We discuss how the method developed is well suited for multispectral applications of transient phenomena or for real-time measurements.     

Fast handheld two-photon fluorescence microendoscope with a 475 microm x 475 microm field of view for in vivo imaging

A fast handheld two-photon fiber-optic fluorescence endoscope for three-dimensional (3D) in vivo cellular imaging is developed. The compact handheld probe of the two-photon endoscope can simply be placed into contact with the target tissue to reveal clear 3D surface and subsurface histological images without biopsy. The new system has, to the best of our knowledge, the largest field of view (FOV) of 475 microm x 475 microm and a 3D imaging volume larger than 475 microm x 475 microm x 250 microm. A real-time two-photon fluorescence image is displayed at 0.4 mm(2)/s. The lateral and axial resolutions of the two-photon fluorescence endoscope are better than 1 and 14.5 microm, respectively.     

Multispectral laser imaging for advanced food analysis

A hardware–software apparatus for food inspection capable of realizing multispectral NIR laser imaging at four different wavelengths is herein discussed. The system was designed to operate in a through-transmission configuration to detect the presence of unwanted foreign bodies inside samples, whether packed or unpacked. A modified Lock-In technique was employed to counterbalance the significant signal intensity attenuation due to transmission across the sample and to extract the multispectral information more efficiently. The NIR laser wavelengths used to acquire the multispectral images can be varied to deal with different materials and to focus on specific aspects. In the present work the wavelengths were selected after a preliminary analysis to enhance the image contrast between foreign bodies and food in the sample, thus identifying the location and nature of the defects. Experimental results obtained from several specimens, with and without packaging, are presented and the multispectral image processing as well as the achievable spatial resolution of the system are discussed.     

Multispectral imaging of Mars: ISM results

Summary Multispectral imaging of planets opens a new class of studies of atmospheric and surface signatures. With ISM experiment on board Phobos, the first IR multispectral imaging of Mars from space was achieved. In viem of the Future Omega experiment on board Mars 94, we present here a sample of data and results (and problems) which can be achieved by studying spectral reflectance. Paper presented at the V Cosmic Physics National Conference, S. Miniato, November 27–30, 1990.     

Multispectral imaging of paintings by optical scanning

In the last few years, there has been a growing interest on multi-spectral imaging as non-destructive technique for painting diagnostics. Providing spectral and colorimetric characterization of the whole paint layer, such a technique is suitable to document the conservation state of an artwork. In this work we present a scanning system for multi-spectral imaging of paintings in the 380–800 nm spectral region, specifically developed to overcome most of the problems related to traditional detection systems based on CCD or Vidicon camera. The device is composed of a spectrometer for contact-less single-point spectral measurements, moved by two orthogonally mounted translation stages. It scans an area of 1 m² with 16 dots/mm² spatial resolution and 10 nm spectral resolution. Spectral reflection factor and tristimulus value measurements were carried out on coloured ceramic tiles and the results were compared to the corresponding certified values. We also present the first application to an ancient painting.     

Binary optics and confocal imaging

The use of two-level phase masks or spatial light modulators in confocal imaging systems is considered. It is shown how, although such masks can modify or correct the effective point-spread function of a system, their behavior for planar objects may not be as desired as a result of phase-wrapping effects. Overcoming this problem with a multilevel mask or a pair of two-level masks is discussed.     

Multispectral photoacoustic imaging of fluorochromes in small animals

Fluorochromes have become essential reporter molecules in biological research. We show that the depth-resolved distribution of fluorochromes in small animals can be imaged with 25 fmol sensitivity and 150 microm spatial resolution by means of multispectral photoacoustic imaging. The major advantage of the multispectral approach is the sensitive differentiation of chromophores and fluorochromes of interest based on self-reference measurements, as evidenced in this study by resolving a commonly used fluorochrome (Alexa Fluor 750) in mouse. The suggested method is well suited for enhancing visualization of functional and molecular information in vivo and longitudinally.     

Geometrical cross-sectional imaging by a heterodyne wavelength-scanning interference confocal microscope

We developed a heterodyne interference confocal microscope, using wavelength modulation of a laser diode to realize quick separate measurement of the refractive indices and geometrical thicknesses of multiple layers. This microscope requires only a single axial movement of the specimen. We can display the geometrical cross sections of the interfaces and the refractive indices of a three-layered object.     

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.     

Sequential optical coherence tomography and confocal imaging

We report a system capable of sequentially acquiring two en-face images of different depth resolutions. The two images are generated by use of different principles, optical coherence tomography (OCT) and confocal microscopy, and have depth resolutions, at present, of better than 20 microm and over 0.12 mm, respectively. The lower-depth-resolution image is ideal for target positioning before collection of stacks of en-face OCT images. Switching between the two types of image by flipping an opaque screen in the reference arm, coupled with self-adjusting gain operation of avalanche photodiodes in the receiver. We illustrate the usefulness of the system by imaging a leaf and an optic nerve in vivo.     

Multispectral and polarimetric imaging in the LWIR: Intersubband detectors as a versatile solution

GaAs-based intersubband infrared detectors, such as Quantum Well Infrared Photodetectors and Quantum Cascade Detectors have proven their ability to address not only conventional thermal imaging applications, but also advanced functionalities such as multispectral and polarimetric imaging. This paper illustrates this potential through the results achieved at III–V Lab in the frame of several ambitious projects, ranging from military applications to Earth observation and exo-planet detection. The advantages of these technologies at the system level are evidenced.     

Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector

We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.     

Double-pass axially resolved confocal Mueller matrix imaging polarimetry

We present experimental depth-resolved complete polarization-sensitive measurements of a stack of linear retarders and glass plates by using what is to the best of our knowledge the first combination of a confocal imaging system with a complete Mueller matrix polarimeter. The axially resolved Mueller matrices were compared with a forward simulation, with good agreement.     

Developing a contact probe for rodent fundus imaging in a confocal scanning laser ophthalmoscope

Since significant ocular differences in both anatomical structure and optical properties exist between rodents and humans, clinical imaging devices for human use are not suitable for use on rodents. In this study, we develop a contact probe with a flexible surface that can closely fit the rodent cornea for fundus imaging with a confocal scanning laser ophthalmoscope. Both Zemax simulation and in vivo fundus imaging demonstrate that this contact probe can significantly improve both the imaging quality and the operational convenience.     

Improving FRET efficiency measurement in confocal microscopy imaging

Spectral bleedthrough (SBT) ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Frster resonance energy transfer (FRET) algorithm corrects SBT ratio according to fluorescence intensity and avoids over-or under-estimation of SBT ratio. In this letter, we propose a new method to accurately measure the FRET efficiency of FRET plasmid in single living cells by combining the calculation of SBT in precision FRET algorithm with E-FRET formulae. We also use this method to measure the FRET efficiency of FRET-Bid, and find that in healthy A549 cells it is about 15%, which is verified by FRET acceptor photobleaching method.     

Dual-axis confocal microscope for high-resolution in vivo imaging

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.     

基于光照-反射成像模型和形态学操作的多谱段图像增强算法

为解决多谱段降质图像增强问题,提出了一种基于光照-反射成像模型和形态学操作的多谱段图像增强算法.首先对图像饱和度使用自适应非线性拉伸函数进行拉伸,使增强后的图像色彩更加饱和、自然;接下来利用引导滤波算法提取出图像的光照分量,提出了一种基于细节特征的加权融合策略,利用光照分布特性构造了一种自适应Gamma校正函数对光照分量进行处理,并将其与利用对比度受限的自适应直方图均衡化方法处理后的光照分量以及原始光照分量进行融合;然后在反射分量校正时,构造了一种形态学操作函数来校正反射信息;最后合并光照分量和反射分量,并与处理后的饱和度分量和色调分量一起得到增强图像.采用主客观评价指标对可见光低照度图像、水下图像、高动态范围图像、沙尘暴图像、雾天图像和热红外图像6种降质多谱段图像实验结果进行分析比较,结果表明本文算法能够有效地抑制图像噪声、增强图像细节信息、改善图像视觉效果,可应用于多种图像增强领域.     

Improvements in confocal microscopy imaging using serrated divided apertures

Confocal microscopy with serrated divided apertures is presented. Compared with using two smooth D-shaped apertures in confocal microscopy, the serrated divided apertures can suppress the out-of-focus central bright spot, thus increasing the efficiency of rejection of scattered light. Diffraction analysis shows that the serrated apertures maintain the optical sectioning strength while attenuating the background coming from far from the focal plane. In addition, the signal to background ratio (S/B) is also improved.     

3D behaviour of Frieden filters in confocal imaging

The three-dimensional (3D) focal behaviour of the super-resolving Frieden filters is investigated numerically. It is shown that, as the central bright spot is sharpened, super-giant secondary maximums are formed on the optic axis. These lobes are much higher that the well-known side-lobes inherent to spatial filtering that surround the restricted, utilisable field, whose characteristics in the meridional plane are depicted for various values of the space-bandwidth parameter and for various numbers of terms that compose the window function. The two-terms filter is found to present, for the first time to my knowledge, some axial apodizing properties. To be compatible with practical realisation, the use of this class of filters in a single- and two-photon confocally scanned system is discussed in terms of 3D super-resolution with an intentionally limited light-power loss. It is shown that these filters match particularly well with recently designed axial apodizers for the transmission-mode confocal scanning microscope and provide a 3D intensity point-spread volume reduction of variable amount as high as 37 percent. The filtering process is shown to vary significantly with the mode of operation.