Observing secretory granules with a multiangle evanescent wave microscope

In total internal reflection fluorescence microscopy (TIRFM), fluorophores near a surface can be excited with evanescent waves, which decay exponentially with distance from the interface. Penetration depths of evanescent waves from 60 nm to 300 nm were generated by varying the angle of incidence of a laser beam. With a novel telecentric multiangle evanescent wave microscope, we monitored and investigated both single secretory granules and pools of granules in bovine chromaffin cells. By measuring the fluorescence intensity as a function of penetration depth, it is possible through a Laplace transform to obtain the fluorophore distribution as a function of axial position. We discuss the extent to which it is possible to determine distances and diameters of granules with this microscopy technique by modeling the fluorescent volumes of spheres in evanescent fields. The anisotropic near-field detection of fluorophores and the influence of the detection point-spread function are considered. The diameters of isolated granules between 70 nm and 300 nm have been reconstructed, which is clearly beyond the resolution limit of a confocal microscope. Furthermore, the paper demonstrates how evanescent waves propagate along surfaces and scatter at objects with a higher refractive index. TIRFM will have a limited applicability for quantitative measurements when the parameters used to define evanescent waves are not optimally selected.     

Expanding Two-Photon Intravital Microscopy to the Infrared by Means of Optical Parametric Oscillator

Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.     

Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching

It has not been possible to measure diffusion deep in solid tissues such as tumors because of the limited light penetration of conventional optical techniques. Here we report a microfiberoptic epifluorescence photobleaching (MFEP) method in which photobleaching is done by laser epi-illumination through a multimode fiberoptic whose micron-sized tip can be introduced deep into tissues. We applied MFEP to measure the diffusion of fluorescent macromolecules in tumors in living mice, at depths well beyond those accessible by surface optical measurements. Macromolecule diffusion was slowed about twofold within 200 microm of the surface of a solid tumor, but was slowed greater than tenfold beyond 500 microm. Our results reveal a remarkable and previously unrecognized slowing of diffusion deep in tumors, which correlated with the differing tissue architectures of tumor periphery versus core, and with altered tumor vasculature produced by aquaporin-1 deletion. MFEP should have wide applications for measuring diffusion in organs, solid tumors and other light-inaccessible tissue masses.     

Optical coherence tomography for ultrahigh resolution in vivo imaging

Optical coherence tomography (OCT) is an emerging biomedical optical imaging technique that performs high-resolution, cross-sectional tomographic imaging of microstructure in biological systems. OCT can achieve image resolutions of 1-15 microm, one to two orders of magnitude finer than standard ultrasound. The image penetration depth of OCT is determined by the optical scattering and is up to 2-3 mm in tissue. OCT functions as a type of 'optical biopsy' to provide cross-sectional images of tissue structure on the micron scale. It is a promising imaging technology because it can provide images of tissue in situ and in real time, without the need for excision and processing of specimens.     

Full-field OCT

Optical coherence tomography (OCT) is an emerging technique for imaging of biological media with micrometer-scale resolution, whose most significant impact concerns ophthalmology. Since its introduction in the early 1990's, OCT has known a lot of improvements and sophistications. Full-field OCT is our original approach of OCT, based on white-light interference microscopy. Tomographic images are obtained by combination of interferometric images recorded in parallel by a detector array such as a CCD camera. Whereas conventional OCT produces B-mode (axially-oriented) images like ultrasound imaging, full-field OCT acquires tomographic images in the en face (transverse) orientation. Full-field OCT is an alternative method to conventional OCT to provide ultrahigh resolution images (approximately 1 microm), using a simple halogen lamp instead of a complex laser-based source. Various studies have been carried, demonstrating the performances of this technology for three-dimensional imaging of ex vivo specimens. Full-field OCT can be used for non-invasive histological studies without sample preparation. In vivo imaging is still difficult because of the object motions. A lot of efforts are currently devoted to overcome this limitation. Ultra-fast full-field OCT was recently demonstrated with unprecedented image acquisition speed, but the detection sensitivity has still to be improved. Other research directions include the increase of the imaging penetration depth in highly scattering biological tissues such as skin, and the exploitation of new contrasts such as optical birefringence to provide additional information on the tissue morphology and composition.     

无损光学法测量人胃粘膜/粘膜下层组织的光衰减特性

研究了人正常胃粘膜及粘膜下层组织对640 nm,690 nm,740 nm,790 nm,840 nm和890 nm波长的钛宝石激光的光衰减特性以及光学穿透深度,实验采用激光斜入射式空间分辨反射光和CCD探测器以及非线性拟合确定组织光学特性。结果表明:人正常胃粘膜及粘膜下层组织对六个波长的激光的有效衰减系数和光学穿透深度都是随着激光波长的变化而变化的。其有效衰减系数的最大值在640 nm,其值为1.12 mm-1,最小值在790 nm,其值为0.901 mm-1,最大差异在790 nm和890 nm之间,其值为19.9%,最小差异在690 nm和740nm之间,其值为2.83%。其光学穿透深度的最大值在790 nm,其值为1.11 mm,最小值在640 nm,其值为0.890 mm,最大差异在640 nm和790 nm之间,其值为24.7%,最小差异在690 nm和740 nm之间,其值为2.97%。     

Dual-wavelength phosphorimetry for determination of cortical and subcortical microvascular oxygenation in rat kidney.

This study presents a dual-wavelength phosphorimeter developed to measure microvascular PO2 (microPO2) in different depths in tissue and demonstrates its use in rat kidney. The used phosphorescent dye is Oxyphor G2 with excitation bands at 440 and 632 nm. The broad spectral gap between the excitation bands combined with a relatively low light absorption of 632 nm light by tissue results in a marked difference in penetration depths of both excitation wavelengths. In rat kidney, we determine the catchments depth of the 440-nm excitation to be 700 microm, whereas the catchments depth of 632 nm is as much as 4 mm. Therefore, the measurements differentiate between cortex and outer medulla, respectively. In vitro, no difference in PO2 readings between both channels was found. On the rat kidney in vivo, the measured cortical microPO2 was on average 20 Torr higher than the medullary microPO2 over a wide PO2 range induced by variations in inspired oxygen fraction. Examples provided from endotoxemia and resuscitation show differences in responses of mean cortical and medullary PO2 readings as well as in the shape of the PO2 histograms. It can be concluded that oxygen-dependent quenching of phosphorescence of Oxyphor G2 allows quantitative measurement of microPO2 noninvasively in two different depths in vivo. Oxygen levels measured by this technique in the rat renal cortex and outer medulla are consistent with previously published values detected by Clark-type oxygen electrodes. Dual-wavelength phosphorimetry is excellently suited for monitoring microPO2 changes in two different anatomical layers under pathophysiological conditions with the characteristics of providing oxygen histograms from two depths and having a penetration depth of several millimeters.     

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

基因表达产物蛋白质的亚细胞定位是解析基因生物学功能的重要证据之一。近年来出现的超分辨率光学成像技术已成功应用于人类和动物细胞中,预示着显微成像技术继激光共聚焦技术后的又一重要进步。由于植物细胞的特殊性和成像技术的研发取向,超分辨率光学成像技术在植物细胞蛋白质亚细胞定位的应用尚未见报道。该研究利用Delta Vision OMX显微镜技术,克服了叶绿体基粒中叶绿素自发荧光与融合蛋白荧光不易区分的缺陷,解决了受分辨率局限无法将植物细胞中蛋白质在亚细胞器内可视化精确定位的技术难题,成功地将植物蔗糖合成酶Zm SUS-SH1定位在烟草表皮细胞叶绿体基粒周围。该研究同时建立了一套基于撕片制片法的简便OMX显微镜制片方法,并针对OMX显微成像技术在植物细胞中蛋白质亚细胞定位的应用进行了讨论。     

Prestress as an optimal biomechanical parameter for needle penetration

Drug delivery requires precise intradermal and subcutaneous injections of formulations to clinically relevant penetration depths. However, penetration depth is confounded by skin deflection, which occurs prior to and during penetration as the skin surface deforms axially with the needle, and which varies profoundly due to differing intrinsic mechanical (e.g. viscoelastic) tissue properties, disease state, aging, and ethnicity. Herein, an ex vivo model was utilized to study factors that affect skin deflection and the efficacy of injection, including prestress applied at the tissue surface, needle gauge, velocity, and actuation depth. The application of prestress minimized skin deflection during needle penetration and allowed for needle actuation to the targeted penetration depths with minimum variability. The force required to achieve target penetration depths was found to increase with prestress and decrease with needle gauge. Our findings emphasize the need for prestress applied to the skin surface to minimize variation in skin properties and administer formulations for intradermal and subcutaneous treatments with maximum precision.     

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Non-Scanning Fiber-Optic Near-Infrared Beam Led to Two-Photon Optogenetic Stimulation In-Vivo

Stimulation of specific neurons expressing opsins in a targeted region to manipulate brain function has proved to be a powerful tool in neuroscience. However, the use of visible light for optogenetic stimulation is invasive due to low penetration depth and tissue damage owing to larger absorption and scattering. Here, we report, for the first time, in-depth non-scanning fiber-optic two-photon optogenetic stimulation (FO-TPOS) of neurons in-vivo in transgenic mouse models. In order to optimize the deep-brain stimulation strategy, we characterized two-photon activation efficacy at different near-infrared laser parameters. The significantly-enhanced in-depth stimulation efficiency of FO-TPOS as compared to conventional single-photon beam was demonstrated both by experiments and Monte Carlo simulation. The non-scanning FO-TPOS technology will lead to better understanding of the in-vivo neural circuitry because this technology permits more precise and less invasive anatomical delivery of stimulation.     

Making blind robots see: the synergy between fluorescent dyes and imaging devices in automated proteomics

Proteomics investigations endeavor to provide a global understanding of gene product synthesis rate, degradation rate, functional competence, posttranslational modification, subcellular distribution and physical interactions with other cell components. Protein expression encompasses an enormous dynamic range. Since rare proteins cannot be amplified by any type of PCR method, sensitive detection is critical to proteome projects. Fluorescence methods deliver streamlined detection protocols, superior detection sensitivity, broad linear dynamic range and excellent compatibility with modern microchemical identification methods such as mass spectrometry. Two general approaches to fluorescence detection of proteins are currently practiced: the covalent derivatization of proteins with fluorophores or noncovalent interaction of fluorophores either via the SDS micelle or through direct electrostatic interaction with proteins. One approach for quantifying fluorescence is to use a photomultiplier tube detector combined with a laser light scanner. In addition, fluorescence imaging is performed using a charge-coupled device camera combined with a UV light or xenon arc source. Fluorescent dyes with bimodal excitation spectra may be broadly implemented on a wide range of analytical imaging devices, permitting their widespread application to proteomics studies and incorporation into semiautomated analysis environments.     

Scanning Super-Resolution Imaging in Enclosed Environment by Laser Tweezer Controlled Superlens

Super-resolution imaging using microspheres has attracted tremendous scientific attention recently because it has managed to overcome the diffraction limit and allowed direct optical imaging of structures below 100 nm without the aid of fluorescent microscopy. To allow imaging of specific areas on the surface of samples, the migration of the microspheres to specific locations on two-dimensional planes should be controlled to be as precise as possible. The common approach involves the attachment of microspheres on the tip of a probe. However, this technology requires additional space for the probe and could not work in an enclosed environment, e.g., in a microfluidic enclosure, thereby reducing the range of potential applications for microlens-based super-resolution imaging. Herein, we explore the use of laser trapping to manipulate microspheres to achieve super-resolution imaging in an enclosed microfluidic environment. We have demonstrated that polystyrene microsphere lenses could be manipulated to move along designated routes to image features that are smaller than the optical diffraction limit. For example, a silver nanowire with a diameter of 90 nm could be identified and imaged. In addition, a mosaic image could be constructed by fusing a sequence of images of a sample in an enclosed environment. Moreover, we have shown that it is possible to image Escherichia coli bacteria attached on the surface of an enclosed microfluidic device with this method. This technology is expected to provide additional super-resolution imaging opportunities in enclosed environments, including microfluidic, lab-on-a-chip, and organ-on-a-chip devices.     

In vivo quantitative microvasculature phenotype imaging of healthy and malignant tissues using a fiber-optic confocal laser microprobe

Real-time in vivo imaging of the microvasculature may help both earlier clinical detection of disease and the understanding of tumor-host interaction at various stages of progression. In vivo confocal and multiphoton microscopy is often hampered by bulky optics setup and has limited access to internal organs. A fiber-optic setup avoids these limitations and offers great user maneuverability. We report here the in vivo validation of a fiber-optic confocal fluorescence microprobe imaging system. In addition, we developed an automated fractal-based image analysis to characterize microvascular morphology based on vessel diameter distribution, density, volume fraction, and fractal dimension from real-time data. The system is optimized for use in the far-red and near-infrared region. The flexible 1.5-mm-diameter fiber-optic bundle and microprobe enable great user maneuverability, with a field of view of 423 x 423 microm and a tissue penetration of up to 15 microm. Lateral and axial resolutions are 3.5 and 15 microm. We show that it is possible to obtain high temporal and spatial resolution images of virtually any abdominal viscera in situ using a far-red blood pool imaging probe. Using an orthotopic model of pancreatic ductal adenocarcinoma, we characterized the tumor surface capillary and demonstrated that the imaging system and analysis can quantitatively differentiate between the normal and tumor surface capillary. This clinically approved fiber-optic system, together with the fractal-based image analysis, can potentially be applied to characterize other tumors in vivo and may be a valuable tool to facilitate their clinical evaluation.     

Multiphoton fluorescence microscopy with GRIN objective aberration correction by low order adaptive optics

Graded Index (GRIN) rod microlenses are increasingly employed in the assembly of optical probes for microendoscopy applications. Confocal, two-photon and optical coherence tomography (OCT) based on GRIN optical probes permit in-vivo imaging with penetration depths into tissue up to the centimeter range. However, insertion of the probe can be complicated by the need of several alignment and focusing mechanisms along the optical path. Furthermore, resolution values are generally not limited by diffraction, but rather by optical aberrations within the endoscope probe and feeding optics. Here we describe a multiphoton confocal fluorescence imaging system equipped with a compact objective that incorporates a GRIN probe and requires no adjustment mechanisms. We minimized the effects of aberrations with optical compensation provided by a low-order electrostatic membrane mirror (EMM) inserted in the optical path of the confocal architecture, resulting in greatly enhanced image quality.     

Polarization-modulated second harmonic generation in collagen

Collagen possesses a strong second-order nonlinear susceptibility, a nonlinear optical property characterized by second harmonic generation in the presence of intense laser beams. We present a new technique involving polarization modulation of an ultra-short pulse laser beam that can simultaneously determine collagen fiber orientation and a parameter related to the second-order nonlinear susceptibility. We demonstrate the ability to discriminate among different patterns of fibrillar orientation, as exemplified by tendon, fascia, cornea, and successive lamellar rings in an intervertebral disc. Fiber orientation can be measured as a function of depth with an axial resolution of approximately 10 microm. The parameter related to the second-order nonlinear susceptibility is sensitive to fiber disorganization, oblique incidence of the beam on the sample, and birefringence of the tissue. This parameter represents an aggregate measure of tissue optical properties that could potentially be used for optical imaging in vivo.     

Molecular analysis of complex tissues is facilitated by laser capture microdissection

Every tissue contains heterogeneous cell populations. Laser capture microdissection (LCM) facilitates cell isolation from complex tissues followed by molecular analysis. LCM entails placing a transparent film over a tissue section or a cytological sample, visualizing the cells microscopically, and selectively adhering the cells of interest to the film with a focused pulse from an infrared laser. The film with the procured cells is then removed from the original sample and placed directly into DNA, RNA, or protein-extraction buffer for processing. LCM has revolutionized molecular analysis of complex tissues because it combines the topographic precision of microscopy with the power of molecular genetics, genomics, and proteomics. However, the success of molecular analysis still depends on the experimental design and requires the understanding of each technical step involved in specimen preparation. This review attempts to rationalize and demystify the choice of various technical options in upstream tissue processing supporting global analytical strategies.     

Quantitative bioluminescence imaging of tumor-targeting bacteria in living animals

We describe a protocol for imaging bacterial luciferase (Lux)-expressing bacteria in small living animals. In this protocol, light emitted by Lux-expressing bacteria is detected and monitored by a cooled charge-coupled device detector. When these bacteria are administered to animals, it provides a potentially valuable approach to generate sensitive whole-body images with extremely low background. This imaging technology should enable the real-time monitoring of bacterial migration into both primary and metastatic tumors in several different mouse tumor models at a strong quantification power.