Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

Conventional, confocal and two-photon fluorescence microscopy investigations of polymer-supported oxygen sensors

Luminescence‐based, polymer‐supported oxygen sensors, particularly those based on platinum group complexes, continue to be of analytical importance. Commercial applications range from the macroscopic (e.g. aerodynamic investigations in wind tunnels, monitoring of oxygen concentration during fermentation, and measurement of biological oxygen demand) to the microscopic (e.g. imaging of oxygen in blood, tissue, cells and other biological samples). Problems hindering the design of improved oxygen sensors include non‐linear Stern–Volmer calibration plots and the multi‐exponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule in the polymer support matrix. Conventional, confocal and two‐photon fluorescence microscopy have proven to be invaluable tools with which the microscale heterogeneity and response of luminescence‐based oxygen sensors can be investigated and compared to the macroscopic response. Results obtained for three ruthenium(II) α‐diimine complexes in polydimethylsiloxane polymer supports indicate the presence of unquenched microcrystals within the polymer matrix that probably degrade oxygen quenching sensitivity and linearity of the Stern–Volmer quenching plot. Two‐photon fluorescence microscopy proved most useful for imaging microcrystals within sensor films, and conventional microscopy allowed direct comparison between microscopic and macroscopic sensor response. The implications of the results in the rational design and mass production of luminescence‐based oxygen sensors are significant.     

Extension of imaging depth in two-photon fluorescence microscopy using a long-wavelength high-pulse-energy femtosecond laser source

We experimentally demonstrate, for the first time to the best of our knowledge, two-photon fluorescence imaging with a femtosecond optical parametric amplifier. In particular, we systematically compare the imaging depths of two-photon fluorescence microscopes based on three different excitation sources, including a femtosecond oscillator, a femtosecond regenerative amplifier and the optical parametric amplifier. The results show that the optical parametric amplifier can greatly extend the penetration depth by approximately 227% as compared with that obtained with the femtosecond oscillator due to effective suppression of scattering at longer wavelength and enhanced excitation efficiency enabled by higher pulse energy.     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

Studies of the microstructure of polymer-modified bitumen emulsions using confocal laser scanning microscopy

Polymer-modified bitumen emulsions present a safer and more environmentally friendly binder for enhancing the properties of roads. Cationic bitumen emulsion binders containing polymer latex were investigated using confocal laser scanning microscopy. The latex was incorporated into the bitumen emulsion by using four different addition methods and all emulsions were processed with a conventional colloid mill. The emulsion binder films were studied after evaporation of the emulsion aqueous phase. We show how the microstructure and distribution of the polymer varies within the bitumen binder depending on latex addition method, and that the microstructure of the binder remains intact when exposed to elevated temperature. It was found that a distinctly fine dispersion of polymer results when the polymer is blended into the bitumen before the emulsifying process (a monophase emulsion). In contrast, bi-phase emulsion binders produced by either post-adding the latex to the bitumen emulsion, or by adding the latex into the emulsifier solution phase before processing, or by comilling the latex with the bitumen, water and emulsifier all resulted in a network formation of bitumen particles surrounded by a continuous polymer film. The use of emulsified binders appears to result in a more evenly distributed polymer network compared to the use of hot polymer-modified binders, and they therefore have greater potential for consistent binder cohesion strength, stone retention and therefore improved pavement performance.     

Microfractography of granitic rocks under confocal scanning laser microscopy

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three-dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures, and the rock-forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.     

单点式位移平台激光共聚焦扫描荧光显微镜

为了获得细胞图像,利用Visual Studio C#开发了移动位移平台的控制程序,使用位移平台单点扫描的方式设计激光共聚焦扫描显微镜(laser confocal scanning microscope,LCSM)。为了获得高分辨率的位移,位移由精度可以达到1nm的压电陶瓷驱动器驱动。设计了梳状和矩形两种扫描路径,通过程序设计位移补偿的方法弥补了机械运动的偏差。利用算术平均值的数字滤波方法处理数据采集卡采集的数据以减小随机噪声的影响。实验结果证明,利用C#程序控制的单点式平台扫描LCSM具有较好地测量效果。     

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy

Objective: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods: Cardiac H9c2 cells were exposed to H₂O₂ (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H₂DCFDA). Results: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H₂O₂ compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H₂DCFDA. Conclusions: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.     

A new confocal scanning beam laser MACROscope using a telecentric,f-theta laser scan lens

A new confocal scanning beam system (MACROscope) that images very large-area specimens is described. The MACROscope uses a telecentric, f-theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small-area, high-speed, high-resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large-area, high-speed, good-resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected-light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.     

Single-lens theta microscopy — a new implementation of confocal theta microscopy

A new microscopical technique based on the principle of confocal theta microscopy (Stelzer, E.H.K., Lindek, S. & Pick, R. (1996) Konfokales Mikroskop . German Patent Office DE 43 26 473 (filed 6.8.1993, granted 6.12.1996)) is described. It uses a single objective lens in combination with a mirror unit to achieve the theta configuration that leads to axial and volume resolution improvements. In this paper we present technical details of possible microscopical set-ups, and we discuss different versions of mirror units.     

Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope

A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 μm with a vertical resolution of 2 μm. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-μm vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.     

Development of laser scanning microscopy using a near ultraviolet laser

We have developed a legitimate fluorescence con-focal scanning microscope (CLSM) using a near ultraviolet (UV) laser. This system has almost no chromatic aberration from the near UV region to the visible region (350–600 nm), and the objectives are designed as water-immersion type. Therefore this system provides the high-quality fluorescence image excited by the near UV laser, and high-quality image of deep points in a sample.     

Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy

Resilin is a rubber-like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin-dominated structures, which were visualized successfully using wide-field fluorescence microscopy (WFM) and a 'classical' UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilin-dominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods.     

Three‐dimensional visualization of dermal skin structure using confocal laser scanning microscopy

The properties and performance of collagen‐based materials may be affected by the collagen fibre bundle pattern, orientation and weave. The aim of this study was to develop and apply methods to visualize the dermis using confocal laser scanning microscopy from thin tissue sections stained with haematoxylin and eosin. The data was processed to allow three‐dimensional (3‐D) visualization on a PC and using a 3‐D immersive technology system. The 3‐D visualization of the confocal microscope image stacks allowed the evaluation of the collagen macromolecular structure including the collagen fibre bundles. The methods developed provide a novel way of viewing complex organic structures with further potential applications in the medical field.