Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. Optical property evaluation of thin films and the surfaces of bulk materials is an important step in understanding new optical material systems and their applications. The method presented can estimate thickness, refractive index, and use absorptive properties of materials for detection. This metrology system uses evanescent field-based photoacoustics (EFPA), a field of research based upon the interaction of an evanescent field with the photoacoustic effect. This interaction and its resulting family of techniques allow the technique to probe optical properties within a few hundred nanometers of the sample surface. This optical near field allows for the highly accurate estimation of material properties on the same scale as the field itself such as refractive index and film thickness. With the use of EFPA and its sub techniques such as total internal reflection photoacoustic spectroscopy (TIRPAS) and optical tunneling photoacoustic spectroscopy (OTPAS), it is possible to evaluate a material at the nanoscale in a consolidated instrument without the need for many instruments and experiments that may be cost prohibitive.     

光学神经成像研究进展

大脑功能的成像检测在认知神经科学领域具有极其重要的意义。现代光子学技术的发展为认知脑成像提供了新的研究手段,在神经系统信息处理机制研究中发挥重要作用。文章介绍了在神经元、神经元网络、特定脑皮层功能构筑以及系统与行为等不同层次开展神经系统信息处理机制研究的各种光学成像技术,包括多光子激发荧光显微成像、内源信号光学成像、激光散斑成像和近红外光学成像等,并评述了这些有特色的光学成像技术在多层次获取和分析神经信息中的研究进展。     

Attaching biological probes to silica optical biosensors using silane coupling agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity/hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest. These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis. While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators. WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations. The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies. Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment. To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies/antigens) through surface modification. Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface. Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.     

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.     

Nonlinear optical spectroscopy of chiral molecules

We review nonlinear optical processes that are specific to chiral molecules in solution and on surfaces. In contrast to conventional natural optical activity phenomena, which depend linearly on the electric field strength of the optical field, we discuss how optical processes that are nonlinear (quadratic, cubic, and quartic) functions of the electromagnetic field strength may probe optically active centers and chiral vibrations. We show that nonlinear techniques open entirely new ways of exploring chirality in chemical and biological systems: The cubic processes give rise to nonlinear circular dichroism and nonlinear optical rotation and make it possible to observe dynamic chiral processes at ultrafast time scales. The quadratic second-harmonic and sum-frequency-generation phenomena and the quartic processes may arise entirely in the electric-dipole approximation and do not require the use of circularly polarized light to detect chirality. They provide surface selectivity and their observables can be relatively much larger than in linear optical activity. These processes also give rise to the generation of light at a new color, and in liquids this frequency conversion only occurs if the solution is optically active. We survey recent chiral nonlinear optical experiments and give examples of their application to problems of biophysical interest.     

Movies of cellular and sub-cellular motion by digital holographic microscopy

Background  

Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy.     

Imaging Carrier Transport Properties in Halide Perovskites using Time‐Resolved Optical Microscopy

Halide perovskites have remarkable properties for relatively crudely processed semiconductors, including large optical absorption coefficients and long charge carrier lifetimes. Thanks to such properties, these materials are now competing with established technologies for use in cost‐effective and efficient light‐harvesting and light‐emitting devices. Nevertheless, the fundamental understanding of the behavior of charge carriers in these materials—particularly on the nano‐ to microscale—has, on the whole, lagged behind empirical device performance. Such understanding is essential to control charge carriers, exploit new device structures, and push devices to their performance limits. Among other tools, optical microscopy and spectroscopic techniques have revealed rich information about charge carrier recombination and transport on important length scales. In this progress report, the contribution of time‐resolved optical microscopy techniques to the collective understanding of the photophysics of these materials is detailed. The ongoing technical developments in the field that are overcoming traditional experimental limitations in order to visualize transport properties over multiple time and length scales are discussed. Finally, strategies are proposed to combine optical microscopy with complementary techniques in order to obtain a holistic picture of local carrier photophysics in state‐of‐the‐art perovskite devices.     

Single molecule measurements and biological motors

Recent technological advances in lasers and optical detectors have enabled a variety of new, single molecule technologies to be developed. Using intense and highly collimated laser light sources in addition to super-sensitive cameras, the fluorescence of single fluorophores can now be imaged in aqueous solution. Also, laser optical tweezers have enabled the piconewton forces produced by pair of interacting biomolecules to be measured directly. However, for a researcher new to the field to begin to use such techniques in their own research might seem a daunting prospect. Most of the equipment that is in use is custom-built. However, most of the equipment is essence fairly simple and the aim of this article is to provide an entry point to the field for a newcomer. It focuses mainly on those practical aspects which are not particularly well covered in the literature, and aims to provide an overview of the field as a whole with references and web links to more detailed sources elsewhere. Indeed, the opportunity to publish an article such as this on the Internet affords many new opportunities (and more space!) for presenting scientific ideas and information. For example, we have illustrated the nature of optical trap data with an interactive Java simulation; provided links to relevant web sites and technical documents, and included a large number of colour figures and plots. Our group’s research focuses on molecular motors, and the bias of this article reflects this. It turns out that molecular motors have been a paradigm (or prototype) for single molecule research and the field has seen a rapid development in the techniques. It is hoped that the methods described here will be broadly applicable to other biological systems.This is an interactive contribution, which can be accessed at:     

Molecular biology in living cells by means of digital optical microscopy

The intention of this review is to introduce to microscopists some of the fundamental limits as well as the advances made in digital optical microscopy techniques, also to show their potentialities in the field of molecular cell biology. Several items within the wide field of this subject will be discussed, such as geometric and photometric features, photophysics, statistics of photon flux and detection, spatial and intensity characteristics of digital images, their manipulation and display, fluorescent probes, and measuring techniques.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optical imaging techniques for the study of malaria

Malarial infection needs to be imaged to reveal the mechanisms behind malaria pathophysiology and to provide insights to aid in the diagnosis of the disease. Recent advances in optical imaging methods are now being transferred from physics laboratories to the biological field, revolutionizing how we study malaria. To provide insight into how these imaging techniques can improve the study and treatment of malaria, we summarize recent progress on optical imaging techniques, ranging from in vitro visualization of the disease progression of malaria infected red blood cells (iRBCs) to in vivo imaging of malaria parasites in the liver.     

用近场光学显微镜观察红细胞的自发荧光

传统的自体荧光检测技术均是对大量细胞或组织进行检测,而近场光学显微技术由于具有较高分辨率和能够同时获取样品的外部形貌和光学信息等特点,有望成为一种研究单个细胞自体发光机理、疾病诊断和检测单个细胞自体荧光光谱的新技术。本文通过应用近场光学显微镜观察不同形状红细胞的外部形貌和光学信息,来初步探讨近场光学显微技术在这方面的应用前景。     

Imaging in flow.

Imaging in flow has been valuable in investigating discrepancies in flow cell measurements due to cell orientation and flow dynamics. This paper discusses optical consideration in flow imaging, slit and full field imaging systems and various cell motion arresting techniques from the standpoint of image plane exposure and suitable detector choices. It concludes with an explanation of the slit-imaging techniques employed in a multidimensional slit-scan flow system and slit-scan correlation system.     

Optogenetics: a new enlightenment age for zebrafish neurobiology

Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of \optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.     

New ways to admire zebrafish: progress in functional genomics research methodology

The main challenge of the post-genomic era is to functionally characterize genes identified by the genome sequencing projects. Model organisms, including zebrafish, are indispensable for this demanding task. Zebrafish has been successfully incorporated into large-scale genetic screens due to the optical clarity of the embryos and their accessibility to various experimental techniques throughout development. The attractiveness of the zebrafish as a model organism is enhanced by the availability of continuously improving genomic tools and methodologies for functional characterization of the gene. This article will highlight the current techniques used in the field, with the focus on transgenesis.     

Determination of absolute configuration--an overview related to this special issue

Rapid progress in asymmetric synthesis stimulated a further development of methods and techniques for the determination of absolute configuration of chiral molecules. In recent years the direct methods, i.e. X-ray diffraction analysis, circular dichroism (vibrational and electronic), Raman optical activity, optical rotation measurements, as well as indirect methods for relative configuration assignment with the use of NMR spectroscopy or enzymatic transformations, are receiving increasing attention not only by specialists in the field but also by synthetic and structural chemists alike. This paper provides a short overview of the methods currently used, as well as references to contributions collected in this Thematic Issue of Chirality.     

Enhanced operation of femtosecond lasers and applications in cell transfection

In this work we present a review and discussion on the enhancement of femtosecond (fs) lasers for use within biophotonics with a particular focus on their use in optical transfection techniques. We describe the broad range of source options now available for the generation of femtosecond pulses before briefly reviewing the application of fs laser in optical transfection studies. We show that major performance enhancements may be obtained by optimising the spatial and temporal performance of the laser source before considering possible future directions in this field. In relation to optical transfection we describe how such laser sources initiate a multiphoton process to permeate the cell membrane in a transient fashion. We look at aspects of this technique including the ability to combine transfection with optical trapping. For future implementation of such transfection we explore the role of new sources and “nondiffracting” light fields. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular.In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.     

基于稳定同位素的SPAC水碳拆分及耦合研究进展

土壤-植被-大气连续体(SPAC)是陆地水文学、生态学和全球变化领域的重要研究对象,其水碳循环过程及耦合机制是前沿性问题.稳定同位素技术示踪、整合和指示的特征有助于评估分析生态系统固碳和耗水情况.本文在简述稳定同位素应用原理和技术的基础上,重点阐释了基于稳定同位素光学技术的SPAC系统水碳交换研究进展,包括:在净碳通量中拆分光合与呼吸量,在蒸散通量中拆分蒸腾与蒸发量,以及在系统尺度上的水碳耦合研究.新兴的技术和方法实现了生态系统尺度上长期高频的同位素观测,但在测量精准度、生态系统呼吸拆分、非稳态模型适应性、尺度转换和水碳耦合机制等方面存在挑战.本文探讨了现有主要研究成果、局限性以及未来研究展望,以期对稳定同位素生态学领域的新研究和技术发展有所帮助.     

The dynamic nature of mollusc egg surface architecture and its relation to the microtubule network

Dynamic changes in the surface architecture pattern of embryos of the slipper limpet (Crepidula fornicata, Mollusca) were found in this study to correlate with the dynamic activity and pattern of the underlying mitotic spindle microtubule network, revealed by fluorescent labelling and confocal imaging techniques. Examination of a series of optical sections indicate that this network appears to be spatially co-ordinated together as a whole throughout the embryo. The microtubule pattern also associated with abnormal multipolar spindles resulting from an applied static magnetic field, indicating that the pattern may be generated by a natural endogenous field source. The patterning characteristics of the surface and microtubule network together provide further morphological evidence for a primary morphogenetic or developmental field system which organises the primary body axis and co-ordinates the pattern of cleavage.