Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.     

Polyelectrolyte surface interface for single-molecule fluorescence studies of DNA polymerase

We report the use of polyelectrolyte multilayers in a stable robust surface chemistry for specific anchoring of DNA to glass. The nonspecific binding of fluorescently tagged nucleotides is suppressed down to the single-molecule level, and DNA polymerase is active on the anchored DNA template. This surface-chemistry platform can be used for single-molecule studies of DNA and DNA polymerase and may be more broadly applicable for other situations in which it is important to have specific biomolecular surface chemistry with extremely low nonspecific binding.     

Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515–535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.     

Frequency division multiplexed multichannel high-speed fluorescence confocal microscope

In this article, we report a new type of fluorescence confocal microscope: frequency division multiplexed multichannel fluorescence confocal microscope, in which we encode the spatial location information into the frequency domain. In this microscope, the exciting laser beam is first split into multiple beams and each beam is modulated at a different frequency. These multiple beams are focused at different locations of the target to form multiple focal points, which further generate multiple fluorescent emission spots. The fluorescent emissions from different focal points are also modulated at different frequencies, because the exciting beams are modulated at different frequencies (or difference carrier frequency). Then, all the fluorescent emissions (modulated at different frequencies) are collected together and detected by a highly sensitive, large-dynamic-range photomultiplier tube. By demodulating the detected signal (i.e., via the Fourier transform), we can distinguish the fluorescent light emitted from the different locations by the corresponding carrier frequencies. The major advantage of this unique fluorescence confocal microscope is that it not only has a high sensitivity because of the use of photomultiplier tube but also can get multiple-point data simultaneously, which is crucial to study the dynamic behavior of many biological process. As an initial step, to verify the feasibility of the proposed multichannel confocal microscope, we have developed a two-channel confocal fluorescence microscope and applied it to study the dynamic behavior of the changes of the calcium ion concentration during the single cardiac myocyte contraction. Our preliminary experimental results demonstrated that we could indeed realize multichannel confocal fluorescence microscopy by utilizing the frequency division multiplexed microscope, which could become an effective tool to study the dynamic behavior of many biological processes.     

激光扫描共聚焦显微镜在医学研究中的应用

激光扫描共聚焦显微镜（Confocal laser scanning microscope,CLSM）具有高分辨率、高灵敏度、三维重建、动态分析等优点,使图像更为精确清晰和数字化。该仪器现已广泛应用于细胞生物学、生理学、病理学、遗传学和药理学等研究领域中。本文简述了激光扫描共聚焦显微镜的结构、工作原理并归纳了其在医学各领域研究中的应用。     

A custom confocal and two-photon digital laser scanning microscope

We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD ( approximately 5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is approximately 5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a x63 1.4 NA oil-immersion objective, the resolution of the confocal system is 0.25 microm laterally and 0.52 microm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 microm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).     

How the confocal laser scanning microscope entered biological research

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.     

激光共焦显微成像的最新进展及其在生命科学研究中的应用

激光扫描共聚焦显微镜近年来得到了迅速发展,是近代最先进的细胞生物医学分析仪器之一。通过它可以对观察样品进行无创断层扫描和成像,在生物学和医学研究诊断的各个方面都得到了广泛的应用。本文主要介绍了激光扫描共焦显微镜的基本原理和发展状况,并着重介绍了在共焦荧光显微镜中采用薄荧光层和切片成像特性图来表征成像状态的功能。这种方法一般用于表征共聚焦和多光子显微镜的成像特性,是比较显微镜切片成像条件、成像质量等相关性能的重要依据。     

Design for a fast fluorescence laser scanning microscope

The design of a fast fluorescence laser scanning microscope is described and illustrated, with discussion of the design consideration of the principal components, including the optical elements. The system, now under construction at the Optical Sciences Center of the University of Arizona, is expected to provide very-high-speed scanning, at a high spatial sampling density, of large object areas while retaining a flexibility of applications. The projected scanning rate approximates the rate achieved by flow cytometry; the projected rates of information generation should be orders of magnitude higher.     

Analysis of DNA interactions using single-molecule force spectroscopy

Protein–DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein–DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide– and protein–DNA interactions are given.     

Use of UV excitation in confocal laser scanning fluorescence microscopy

By making only minor modifications, we adapted a conventional confocal beam-scanning laser microscope for the recording of UV-excited fluorescence. The major, and most expensive, change is that we coupled an external UV argon ion laser, providing the wavelengths 334, 351 and 364 nm, to the microscope scanner. We also replaced some optical components to obtain improved transmission and reflection properties in the UV. Only easily obtainable and inexpensive off-the-shelf components were used. The most serious problem encountered was the chromatic aberration of the microscope objective when using both UV and visible wavelengths. This is of no consequence in conventional microscopy where good imaging properties are important only in the visible region. In confocal microscopy on the other hand, good imaging properties are necessary for both the exciting and fluorescent light. Rather than having new optics designed, we tried with simple means to reduce the effects of the chromatic aberration to a tolerable level. This was done by mechanical adjustments in the ray-path. In addition we also tested two mirror objectives, which are inherently free from chromatic aberrations. However, such objectives have rather limited numerical apertures and are not of the immersion type. Their value in biomedical applications is therefore limited.The objective most frequently used in our experiments was a 63/1.25 oil-immersion fluorite. Without any compensation this objective had a depth resolution in UV-excited confocal fluorescence that was an order of magnitude worse than when using visible-light excitation. The useful field of view was also very small due to lateral chromatic aberration. By simple means we managed to improve the depth resolution by a factor of 4.4, and at the same time increase the useful field of view substantially. Still, the depth resolution was worse than what is obtained using visible light excitation. We think this is due to the fact that after compensation the objective is working with an incorrect tube length.Using the modified instrument, we recorded specimens labelled with AMCA and Fluoro-Gold, obtaining 1.5 μm thick optical sections.     

激光共聚焦扫描显微镜在抗菌机理研究中的应用

激光共聚焦扫描显微镜采用激光光源、共聚焦技术和点扫描技术,使其分辨力较传统光学显微镜大为提高。与其他的生物学技术相配合,可定性、定量、定位地检测组织细胞内的多种生化成分。它具有的活细胞动态监测、断层扫描及三维图像重建等功能,使其在抗菌机理研究、尤其是抗生物膜研究中得到大量的应用。本文就激光共聚焦扫描显微镜在抗菌剂作用位点,抗菌剂对微生物细胞膜,微生物生理代谢,以及微生物生物膜形成与结构的影响等研究中的应用做一综述。     

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.     

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes

OBJECTIVE: To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. STUDY DESIGN: Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. RESULTS: Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. CONCLUSION: Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.     

Laminar flow cells for single-molecule studies of DNA-protein interactions

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.     

Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was approximately 1000 bases/s and also demonstrated high processivity.     

Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy.     

激光共聚焦显微镜在微痕定量分析中的应用综述

微痕分析是石器、骨器、牙齿等工艺、功能研究的常用方法,但无论是高倍还是低倍等常用方法,受限于体式光学显微镜、扫描电镜等观察方法,微痕分析通常很难进行原位定量分析,因此微痕分析的定量化是目前学术界遇到的主要问题。近年来随着激光共聚焦显微镜（LSCM）在微痕分析中的应用,微痕定量化在西方学术界得到了新的实践和进展。本文主要介绍激光共聚焦显微镜的原理及其在微痕分析中的应用案例,以期促进微痕分析在中国的进一步发展。