用夏克-哈特曼探测器测量人眼波前像差

为了精确测量人眼的高低阶像差,设计并搭建了一套人眼波前像差精确测量光学系统。该系统采用夏克-哈特曼波前探测器进行波前探测,可以在不同瞳孔、不同视场和不同调焦状态下精确测量人眼的波前像差。用ZEMAX软件对系统进行模拟分析,验证了该系统的探测精度,讨论了系统的调焦性能。用该系统实验分析了人眼各阶像差的分布情况、瞳孔大小和调焦状态对人眼波前像差的影响,以及人眼波前像差的时间和空间变化特性(变化频率约3 Hz,等晕角约为1.5°)。结果表明,该系统精度高(PV1/20λ),操作方便,是人眼波像差的研究和个性化角膜手术的有力工具。     

Recording and controlling the 4D light field in a microscope using microlens arrays

By inserting a microlens array at the intermediate image plane of an optical microscope, one can record four-dimensional light fields of biological specimens in a single snapshot. Unlike a conventional photograph, light fields permit manipulation of viewpoint and focus after the snapshot has been taken, subject to the resolution of the camera and the diffraction limit of the optical system. By inserting a second microlens array and video projector into the microscope's illumination path, one can control the incident light field falling on the specimen in a similar way. In this paper, we describe a prototype system we have built that implements these ideas, and we demonstrate two applications for it: simulating exotic microscope illumination modalities and correcting for optical aberrations digitally.     

Measurement and restoration of the point spread function of fluorescence confocal microscopy

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero‐centred point spread function.     

Compensation of the shadowing error in three-dimensional imaging with a multiple detector scanning electron microscope

A method for three‐dimensional quantitative surface characterization for scanning electron microscopy is presented. The method used a quadruple scintillator detector developed by us. A surface reconstruction algorithm was performed by special software, with new algorithms for error compensation. Among these errors, detector shadowing was of particular importance. This was due to the disturbance in integration continuity when one or more detectors was screened from the flow of electrons. Several methods for the reduction of this error have been proposed and tested by us. The methods were based on software processing of complementary information, such as unshadowed detector signals, shadow depth and modified integration schemes.     

The three-dimensional point spread functions of a microscope objective in image and object space

The three-dimensional point spread function (3-D PSF) of an optical system in image space is distinguished from the 3-D PSF in object space and the relation between the two 3-D PSFs is derived. By using this relation one 3-D PSF can be easily obtained from the other. The 3-D PSFs are given in a single integral expression, which can be computed numerically. The results of this study can be used in 3-D image processing for microscopy and have been applied to the analysis of the diffusion of fluorescent molecules in a 3-D porous medium.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

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.     

Modelling of three-dimensional fluorescence images of muscle fibres: an application of the three-dimensional optical transfer function

Striated muscle fibres can be modelled by a simple geometry, which has allowed three-dimensional (3-D) images in conventional and confocal microscopes to be calculated. This model is useful for comparing different imaging methods and represents a simple example of an application of the 3-D optical transfer function (OTF) for the system. The rejection of out-of-focus blur is demonstrated, and the effects of fibre thickness and confocal pinhole size on image contrast are investigated. The effects of using a simple filter for image enhancement are studied, elucidating the characteristics of the OTF.     

Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror

We demonstrate adaptive aberration correction for depth‐induced spherical aberration in a multiphoton scanning microscope with a micromachined deformable mirror. Correction was made using a genetic learning algorithm with two‐photon fluorescence intensity feedback to determine the desired shape for an adaptive mirror. For a 40×/0.6 NA long working distance objective, the axial scanning range was increased from 150 mm to 600 mm.     

The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D data

We have measured the point-spread function (PSF) for an MRC-500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan-neofluar 63 × water immersion and Leitz plan-apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non-confocal) PSFs. Measurements of the peak width at half-maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three-dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal-to-noise ratio, and then to restore the resolution by deconvolution.     

A method for characterizing longitudinal chromatic aberration of microscope objectives using a confocal optical system

We describe a novel method of characterizing the longitudinal chromatic aberration of microscope objectives by recording a series of axial responses as a function of wavelength as a plane reflector is scanned through the focal region of a confocal microscope. Measurements are presented for a variety of objectives with differing degrees of correction. The use of the chromatic focal shift to measure surface profiles is also discussed.     

Simple characterisation of a deformable mirror inside a high numerical aperture microscope using phase diversity

We present a simple and versatile scheme for characterising amplitude and phase modulation by an active element, such as a deformable mirror, in the pupil plane of a high NA microscope. By placing a mirror in the vicinity of the focal plane of the objective and recording images of the reflected focal spot on a camera, we show that reliable measurements of the influence function of the mirror actuators in the pupil plane of the objective can be obtained using an iterative electric field retrieval algorithm. Compared to direct wavefront sensors, the proposed method allows characterisation for a variety of objectives with different NA and pupil sizes without modification of the setup, requires minimal space inside the microscope, and can be used with pulsed sources such as used for multiphoton microscopy. In order to validate our method, we compare our data to the results obtained with a Shack-Hartmann wavefront sensor, and show that comparable precision is achieved.     

Imaging properties of bright-field and annular-dark-field scanning confocal electron microscopy: II. point spread function analysis

The imaging properties of bright field and annular dark field scanning confocal electron microscopy (BF-SCEM and ADF-SCEM) are discussed based on their point spread functions (PSFs) in comparison with multislice simulations. Although the PSFs of BF-SCEM and ADF-SCEM show similar hourglass shapes, their numerical distributions are quite different: BF-SCEM PSF is always positive and shows a center of symmetry whereas the ADF-SCEM PSF is complex and has Hermitian symmetry. These PSF properties explain the large elongation effect in BF-SCEM for laterally extended object and almost no-elongation in ADF-SCEM, illustrating the importance of the numerical analysis of PSFs. The Hermitian symmetry of the ADF-SCEM PSF results in an interesting “edge enhancement effect” at the interface. Simulation using the PSF and the multislice method verified this effect at GaAs surfaces and InAs interfaces embedded in GaAs. This unique feature of ADF-SCEM can potentially be useful for depth sectioning. It is also pointed out that a PSF imaging model cannot be applicable for BF-SCEM of a phase object, when the system is symmetric and aberration free.     

Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: System design and calibration

A new depth encoding system (DES) is presented, which makes it possible to calculate, display, and record the z-axis position continuously during in vivo imaging using tandem scanning confocal microscopy (TSCM). In order to verify the accuracy of the DES for calculating the position of the focal plane in the cornea both in vitro and in vivo, we compared TSCM measurements of corneal thickness to measurements made using an ultrasonic pachymeter (UP, a standard clinical instrument) in both enucleated rabbit, cat, and human eyes (n = 15), and in human patients (n = 7). Very close agreement was found between the UP and TSCM measurements in enucleated eyes; the mean percent difference was 0.50 ± 2.58% (mean ± SD, not significant). A significant correlation (R=0.995, n=15, p< 0.01) was found between UP and TSCM measurements. These results verify that the theoretical equation for calculating focal depth provided by the TSCM manufacturer is accurate for corneal imaging. Similarly, close agreement was found between the in vivo UP and TSCM measurements; the mean percent difference was 1.67 ± 1.38% (not significant), confirming that z-axis drift can be minimized with proper applanation of the objective. These results confirm the accuracy of the DES for imaging of the cornea both ex vivo and in vivo. This system should be of great utility for applications where quantitation of the three-dimensional location of cellular structures is needed.     

On the complex three-dimensional amplitude point spread function of lenses and microscope objectives: theoretical aspects, simulations and measurements by digital holography

The point spread function is widely used to characterize the three‐dimensional imaging capabilities of an optical system. Usually, attention is paid only to the intensity point spread function, whereas the phase point spread function is most often neglected because the phase information is not retrieved in noninterferometric imaging systems. However, phase point spread functions are needed to evaluate phase‐sensitive imaging systems and we believe that phase data can play an essential role in the full aberrations' characterization. In this paper, standard diffraction models have been used for the computation of the complex amplitude point spread function. In particular, the Debye vectorial model has been used to compute the amplitude point spread function of ×63/0.85 and ×100/1.3 microscope objectives, exemplifying the phase point spread function specific for each polarization component of the electromagnetic field. The effect of aberrations on the phase point spread function is then analyzed for a microscope objective used under nondesigned conditions, by developing the Gibson model ( Gibson & Lanni, 1991 ), modified to compute the three‐dimensional amplitude point spread function in amplitude and phase. The results have revealed a novel anomalous phase behaviour in the presence of spherical aberration, providing access to the quantification of the aberrations. This work mainly proposes a method to measure the complex three‐dimensional amplitude point spread function of an optical imaging system. The approach consists in measuring and interpreting the amplitude point spread function by evaluating in amplitude and phase the image of a single emitting point, a 60‐nm‐diameter tip of a Near Field Scanning Optical Microscopy fibre, with an original digital holographic experimental setup. A single hologram gives access to the transverse amplitude point spread function. The three‐dimensional amplitude point spread function is obtained by performing an axial scan of the Near Field Scanning Optical Microscopy fibre. The phase measurements accuracy is equivalent to λ/60 when the measurement is performed in air. The method capability is demonstrated on an Achroplan ×20 microscope objective with 0.4 numerical aperture. A more complete study on a ×100 microscope objective with 1.3 numerical aperture is also presented, in which measurements performed with our setup are compared with the prediction of an analytical aberrations model.     

Measurement of potential distribution function on object surface by using an electron microscope in the mirror operation mode

The quantitative theory of image contrast in an electron microscope in the mirror operation mode is given in this paper. This theory permits us to calculate the potential distribution on the object surface from the current density distribution on the microscope screen. The potential distribution results in image formation on the screen. Local electric fields existing on the object surface lead to a perturbation of electron trajectories above the object and to a redistribution of the current density on the screen, causing image contrast. Using the quantitative correlation between these fields and the function of current density distribution on the screen, it is possible to calculate the magnitude of these microfields as well. As illustration, a measured potential distribution on an object surface with spiral structures of adsorbates was analysed. These structures are formed during reaction of CO oxidation on Pt(110). The value of the measured contact potential difference comprised a few hundredths of volt.     

Calibration of the automated z-axis of a microscope using focus functions

Applications in automated microscopy and three-dimensional microscopy require careful calibration of the microscope system. This paper presents methods for calibration of the motorized z -axis (focus or optical axis) of an automated microscope. Apart from the automated microscope the procedures require a CCD camera and a test slide containing a simple bar pattern. The calibration embraces the following characteristics of the z -axis: (a) measuring the motor step size in nanometres; (b) measuring the mechani-cal backlash in the focus mechanism of the microscope and (c) measuring the reproducibility and the stability of the focus position over time. The measurements employ focus functions to determine the z -position of the microscope stage.     

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.     

Measurement of absolute tracer concentrations in tissue sections by using digital imaging fluorescence microscopy. Application to the study of plasma protein uptake by the arterial wall

Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to other studies employing DIFM. Nonlinearities were found to arise from offsets in the video digitizers, from background fluorescence and stray light within the microscope and from the transfer characteristics of the intensified CCD camera. Camera gain controls showed complex behaviour. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in response were caused by the geometry of the microscope optics and by the image intensifier. However, the ratios between areas were not affected by light intensity or camera gain settings. Measured intensities were independent of the depthwise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not concentration dependent. Methods used to correct these effects and obtain a uniform, linear and constant relationship between concentration and grey level are described. Using the system and appropriate corrections, in vivo uptake of sulphorhodamine-B-labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean uptake of tracer and its local variation were quantitatively similar to those previously obtained with nonmicroscopic methods.     

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new,efficient upconverting fluorophore

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sap-phire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.