The effect of aberrations on the axial response of confocal imaging systems

A confocal scanning microscope permits three-dimensional imaging of a volume specimen. The role of lens aberrations on the axial response of the system is considered both experimentally and theoretically. It is found that primary coma, for example, plays no part in the axial response from a perfect reflector. The role of detector size is also considered and compared with theory. In general, the effect of aberrations is increased as the size of the detector is increased.     

The influence of specimen refractive index,detector signal integration,and non-uniform scan speed on the imaging properties in confocal microscopy

Refraction of light in a specimen volume may cause aberrations that influence the imaging properties in confocal microscopy. In this paper the influence on three-dimensional resolution and geometry is experimentally investigated for a uniform specimen volume. It is found that the depth resolution is more severely affected than the lateral resolution. This is unfortunate, because even under ideal conditions the depth resolution is lower than the lateral resolution. Lateral image geometry is little affected by the specimen refractive index, whereas the depth scale can be considerably elongated or compressed. The influence of a finite detector integration time is also considered. This can give a noticeable, but not particularly severe effect on the image resolution in the line-scan direction. Because the integration time can be accurately controlled, a shorter integration time can be used when maximum resolution is essential, albeit at the price of a higher noise level. In scanning fluorescence microscopy a non-uniform scan speed may give large variations in bleaching over the specimen surface. Experiments illustrate how serious such non-uniform bleaching effects can be when a specimen area is repeatedly scanned, for example when recording optical serial sections.     

An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

A theory for multiphoton fluorescence imaging in high aperture scanning optical microscopes employing finite sized detectors is presented. The effect of polarisation of the fluorescent emission on the imaging properties of such microscopes is investigated. The lateral and axial resolutions are calculated for one-, two- and three-photon excitation of p-quaterphenyl for high and low aperture optical systems. Significant improvement in lateral resolution is found to be achieved by employing a confocal pinhole. This improvement increases with the order of the multiphoton process. Simultaneously, it is found that, when the size of the pinhole is reduced to achieve the best possible resolution, the signal-to-noise ratio is not degraded by more than 30%. The degree of optical sectioning achieved is found to improve dramatically with the use of confocal detection. For two- and three-photon excitation axial full width half-maximum improvement of 30% is predicted.     

Theoretical versus experimental resolution in optical microscopy

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.     

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.     

Signal strength and noise in confocal microscopy: Factors influencing selection of an optimum detector aperture

In confocal microscopy, several factors influence the selection of an optimum size and geometry of detector aperture. These include (1) strength of signal from the specimen, (2) noise level in the system, (3) optical configuration of the microscope (e.g., reflection or fluorescence), (4) time available for signal accumulation, (5) specimen thickness, and (6) amount of reduction in axial and transverse resolution (from the theoretical maximum) that can be tolerated. It is shown both theoretically and experimentally that the size of the detector aperture critically influences the type and amount of system noise that is detected along with the specimen signal. It is also demonstrated that increasing the size of the detector pinhole does not appreciably increase the signal strength from any single thin plane, but only increases the sampling depth, enhancing brightness at the cost of a reduction in axial resolution. As a result, it is shown that there is no advantage, from the standpoint of signal strength, to using a slit aperture rather than a circular detector pinhole. Finally, it is concluded that all confocal microscopes should be designed to allow the user the capability of selecting ah optimum compromise detector aperture setting based on the particular specimen properties, type of microscopy (e.g., fluorescence or reflection) and resolution required.     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.     

利用异形像元探测器的超分辨成像方法

随着光学成像全面进入光电数字成像时代,大多数成像系统的空间分辨率受限于探测器,所以提高探测器分辨率成为高分辨光电成像系统中的核心问题。而探测器的低分辨率主要是由于低采样频率和像元感光区的孔径效应而造成。最直接的解决方法就是减小像元尺寸,但会降低其他性能参数;针对最主要的限制因素——采样频率不足,目前多采用基于过采样原理的超分辨重建技术,通过提高探测器采样的频率来提高探测器的空间分辨率,但是其提升效果受到像元孔径效应的制约。为了进一步提高探测器受限的成像系统的空间分辨率,提出一种基于异形像元探测器的超分辨成像方法,将两列线阵异形像元探测器亚像元推扫实现像元细分,然后利用两列探测器所输出的灰度矩阵信息,重建出最终的高分辨图像。并分别通过理论评估和具体实验两方面验证该方法可以同时提高探测器的采样频率和截止频率,拓展带宽,从而实现高分辨率的目的。     

用于生物组织层析成像的共聚焦内窥镜

为了获取生物组织的层析图像,建立了共聚焦内窥镜成像系统,对该系统的横向和轴向分辨能力进行测量和分析,并且对其在猪皮肤组织的层析成像能力进行研究。首先根据系统工作要求,说明系统的工作原理和组成结构,然后给出系统空间分辨能力的理论计算式,最后测试系统性能,说明测量值与理论值存在偏差的主要原因。实验结果表明：建立的系统轴向分辨能力约为10 ;横向分辨能力约为1.9 ;该系统可以对猪皮肤组织进行层析成像。     

Three-dimensional imaging in confocal systems

We discuss the origin of the three-dimensional imaging characteristics of confocal optical systems. Several methods of information display are considered. The important practical question concerning the correct choice of limiting detector aperture is also considered.     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

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.     

Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light

A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.     

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.     

Three‐dimensional imaging of porous media using confocal laser scanning microscopy

In the last decade, imaging techniques capable of reconstructing three‐dimensional (3‐D) pore‐scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO₂ storage potential. CLSM has a unique capability of producing 3‐D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z‐dimension) that can be imaged in porous materials. In this study, we introduce a ‘grind and slice’ technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3‐D confocal volumetric data into pores and grains. Finally, we use the resulting 3‐D pore‐scale binarized confocal data obtained to quantitatively determine petrophysical pore‐scale properties such as total porosity, macro‐ and microporosity and single‐phase permeability using lattice Boltzmann (LB) simulations, validated by experiments.     

Confocal bi-protocol: a new strategy for isotropic 3D live cell imaging

The conventional approach for microscopic 3D cellular imaging is based on axial through-stack image series which has some significant limitations such as anisotropic resolution and axial aberration. To overcome these drawbacks, we have recently introduced an alternative approach based on micro-rotation image series. Unfortunately, this new technique suffers from a huge burden of computation that makes its use quite difficult for current applications. To address these problems we propose a new imaging strategy called bi-protocol, which consists of coupling micro-rotation acquisition and conventional z-stack acquisition. We experimentally prove bi-protocol 3D reconstruction produces similar quality to that of pure micro-rotation, but offers the advantage of reduced computation burden because it uses the z-stack volume to accelerate the registration of the micro-rotation images.     

Axial super resolution topography of focal adhesion by confocal microscopy

The protein organization within focal adhesions has been studied by state‐of‐the‐art super resolution methods because of its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation, limiting their application. In this report we present a phasor‐based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that with about 4,000 photon counts collected along a z‐scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F‐actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials. Microsc. Res. Tech., 76:1070–1078, 2013. © 2013 Wiley Periodicals, Inc.     

Axial resolution of confocal microscopes with parallel-beam detection

We present a simple theory for the evaluation of the axial resolution of a confocal scanning microscope with parallel-beam detection. The results demonstrate that, in certain cases, the collection efficiency is low compared with a conventional confocal microscope, but the axial resolution may be further improved.     

Offner双镜三反射成像光谱仪分辨率的研究

针对Offner双镜三反射成像光谱仪的消像差结构,采用几何方法推导出光谱分辨率的计算公式,分析了入射狭缝的宽度、凸面光栅分辨率、系统像差和探测器像元尺寸各个参数对光谱分辨率的影响,提出了分光系统像差的计算方法和优化设计方法,并探讨了提高光谱分辨率的方法和技术,即在优化系统像差的同时,适当减小狭缝宽度和探测器像元尺寸,有利于提高系统的光谱分辨率。该系统利用消像差优化设计同时考虑光谱分辨率的设计方法,具有十分重要的实用价值,为成像光谱仪的研制提供经验和借鉴。