The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.     

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z‐stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal‐to‐background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.     

面向微纳材料的激光扫描二维成像系统

在对微纳材料光学特性表征中,需要获得分辨率更高的波长和强度的荧光图像。普通的显微镜无法满足测试的要求,因此将普通的成像显微镜、光谱仪以及纳米移动台组成激光扫描显微镜成像系统,并利用LabVIEW开发了一套完整的集二维扫描采集与信号图像处理一体的系统上位机软件。扫描采集过程使用了低通滤波等数字信号处理方法消除光谱仪信号噪声的影响。利用本系统测量硒化镉纳米带、单层二硫化钼得到了荧光强度图像以及荧光峰值波长图像,能分辨出最小波长为0.03 nm的荧光。将采集长度与实际长度进行比较并分析荧光强度差异,取得了较好的效果。     

Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology

Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

Chromatic two-photon excitation fluorescence imaging

We report on a chromatic axial scanning method for two-photon excitation fluorescence imaging. Effective axial scanning is achieved by incorporating a Fresnel lens in the system, which has large chromatic aberration and can therefore focus the excitation beam to different axial positions depending on its wavelength. We experimentally demonstrated this technique and used it to image the cross-section of fluorescent microspheres.     

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.     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.     

Image scanning fluorescence emission difference microscopy based on a detector array

We propose a novel imaging method that enables the enhancement of three‐dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.     

Dispersion, aberration and deconvolution in multi-wavelength fluorescence images

The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi-wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image.     

Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy

Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging.     

Offner成像光谱仪的消像差技术

介绍了基于同心结构的Offner成像光谱仪光学系统的工作原理、结构特点及优势,综述了Offner型成像光谱仪在国内外的研究进展。较详细地介绍了国内外目前常用的三种消像差方式,即通过改变光学元件装调结构实现消像差,基于单光栅像差理论的解析方法进行消像差以及通过设计凸面全息光栅来提高光谱图像的分辨率。文中总结了Offner成像光谱仪存在的关键问题及发展趋势,强调该系统需要解决的问题的是消除系统像差,提高系统光谱分辨率、空间分辨率和对弱信号的探测能力,其发展方向为更高的分辨率和探测能力以及系统的小型化和轻量化。基于对Offner成像光谱仪的研究,文中提出了一种从凸面全息光栅与光谱仪一体化设计的角度进行消像差的思路。     

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330-337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ～8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ～4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).     

The effects of refractive index heterogeneity within kidney tissue on multiphoton fluorescence excitation microscopy

Although multiphoton fluorescence excitation microscopy has improved the depth at which useful fluorescence images can be collected in biological tissues, the reach of multiphoton fluorescence excitation microscopy is nonetheless limited by tissue scattering and spherical aberration. Scattering can be reduced in fixed samples by mounting in a medium whose refractive index closely matches that of the fixed material. Using optical 'clearing', the effects of refractive index heterogeneity on signal attenuation with depth are investigated. Quantitative measurements show that by mounting kidney tissue in a high refractive index medium, less than 50% of signal attenuates in 100 μm of depth.     

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.     

Image analysis for denoising full-field frequency-domain fluorescence lifetime images

Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super-resolution imaging).     

High-resolution imaging with an aberration-corrected transmission electron microscope

Recently an electromagnetic hexapole system for the correction of the spherical aberration of the objective lens of a 200 kV transmission electron microscope has been constructed by Haider and coworkers. By appropriately exciting the hexapole elements it is possible to adjust specific values of the spherical aberration coefficient ranging from the value of the original uncorrected instrument over zero even to negative values. In the first part of the paper the consequences of the tunable spherical aberration are investigated. New imaging modes are available: By adjustment of an optimum value for the spherical-aberration coefficient, the point resolution of phase-contrast imaging can be extended to the information limit. Phase-contrast imaging can be improved by a reduced level of contrast delocalisation. For zero aberration contrast delocalisation does not occur. In this case high-resolution investigations are carried out under amplitude-contrast conditions, where the local image intensity of crystalline objects is controlled by electron diffraction channelling. The defocus and spherical aberration values related to the new imaging modes are given. In the second part novel applications of the instrument to semiconductor heterostructures and ceramic grain boundaries are examined.     

Optimized temporal response in multichannel two-photon fluorescence lifetime microscopy using a photonic crystal fibre

The integration of fibre optics into an imaging system for the convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Although it is extremely robust and cost effective, fibre integration requires special consideration in a time‐domain fluorescence lifetime imaging schema where multipath propagation in the fibre causes significant spread in photon transit times. In this study, we investigated the effect of the length of a multimode collection fibre on the temporal performance of a multichannel fluorescence lifetime microscope and demonstrated the effectiveness of a photonic crystal fibre as a means of optimizing the collection and delivery of emitted fluorescence in terms of temporal resolution. The findings are pertinent to all studies that employ a multimode optical fibre to collect and deliver an emitted fluorescence signal from a sample to a remote detector for measurement of the characteristic fluorescence lifetime.     

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.