Confocal microscopy using variable-focal-length microlenses and an optical fiber bundle

The use of variable-focal-length (VFL) microlenses can provide a way to axially scan the foci across a sample by electronic control. We demonstrate an approach to coupling VFL microlenses individually to a fiber bundle as a way to create a high-throughput aperture array with a controllable aperture pattern. It would potentially be applied in real-time confocal imaging in vivo for biological specimens. The VFL microlenses that we used consist of a liquid-crystal film sandwiched between a pair of conductive substrates for which one has a hole-patterned electrode. One obtains the variation of the focal length by changing the applied voltage. The fiber bundle has been characterized by coupling with both coherent and incoherent light sources. We further demonstrate the use of a VFL microlens array in combination with the fiber bundle to build up a confocal system. The axial response of the confocal system has been measured without mechanical movement of the sample or the objective, and the FWHM is estimated to be approximately 16 microm, with asymmetric sidelobes.     

Multipoint phase calibration for improved compensation of inherent wavefront distortion in parallel aligned liquid crystal on silicon displays

The inherent distortion of a reflective parallel aligned spatial light modulator (SLM) may need compensation not only for the backplane curvature but also for other possible nonuniformities caused by thickness variations of the liquid crystal layer across the aperture. First, we build a global look-up table (LUT) of phase modulation versus the addressed gray level for the whole device aperture. Second, when a lack of spatial uniformity is observed, we define a grid of cells onto the SLM aperture and develop a multipoint calibration. The relative phase variations between neighboring cells for a uniform gray level lead us to build a multi-LUT for improved compensation. Multipoint calibration can be done using either phase-shift interferometry or Fourier diffraction pattern analysis of binary phase gratings. Experimental results show the compensation progress in diffractive optical elements displayed on two SLMs.     

Phase calibration of spatially nonuniform spatial light modulators

A new 512 x 512 pixel phase-only spatial light modulator (SLM) has been found to deviate from being flat by several wavelengths. Also, the retardation of the SLM relative to voltage varies across the device by as much as 0.25 wavelength. The birefringence of each pixel as a function of address voltage is measured from the intensity of the SLM between crossed polarizers. To these responses are added a reference spatial phase measured by phase shifting interferometry for a single address voltage. Fits to the measured data facilitate the compensation of the SLM to a root-mean-square wave-front error of 0.06 wavelength. The application of these corrections to flatten the full aperture of the SLM sharpens the focal plane spot and reduces the distortion of computer-designed diffraction patterns.     

Effect of half-stop lateral misalignment on imaging of dark-field and stereoscopic confocal microscopes

There are several ways to realize dark-field imaging in confocal microscopy. In a recent paper [J. Microsc. 181 260-268 (1996)] we suggested a simple modification of a commercial confocal microscope to incorporate dark-field imaging. This modification involved an aperture stop covering half of the entrance pupil of the objective lens. Now we investigate the lateral misalignment of the aperture stop for dark-field and stereoscopic confocal microscopes. We show the effect of lateral alignment of the half-stop on the point-spread and transfer functions and also examine the detected signal from a sloping plane reflector. Lateral and axial resolution values are given from theoretical data.     

Enhanced three-dimensional reconstruction from confocal scanning microscope images. II. Depth discrimination versus signal-to-noise ratio in partially confocal images

The enhanced depth discrimination of a confocal scanning optical microscope is produced by a pinhole aperture placed in front of the detector to reject out-of-focus light. Strictly confocal behavior is impractical because an infinitesimally small aperture would collect very little light and would result in images with a poor signal-to-noise ratio (SNR), while a finite-sized partially confocal aperture provides a better SNR but reduced depth discrimination. Reconstruction algorithms, such as the expectationmaximization algorithm for maximum likelihood, can be applied to partially confocal images in order to achieve better resolution, but because they are sensitive to noise in the data, there is a practical trade-off involved. With a small aperture, fewer iterations of the reconstruction algorithm are necessary to achieve the desired resolution, but the low a priori SNR will result in a noisy reconstruction, at least when no regularization is used. With a larger aperture the a priori SNR is larger but the resolution is lower, and more iterations of the algorithm are necessary to reach the desired resolution; at some point the a posteriori SNR is lower than the a priori value. We present a theoretical analysis of the SNR-toresolution trade-off partially confocal imaging, and we present two studies that use the expectationmaximization algorithm as a postprocessor; these studies show that a for a given task there is an optimum aperture size, departures from which result in a lower a posteriori SNR.     

Design of a high-numerical-aperture miniature microscope objective for an endoscopic fiber confocal reflectance microscope

An endoscopic confocal microscope requires a high-performance, miniaturized microscope objective. We present the design of a miniature water-immersion microscope objective that is approximately 10 times smaller in length than a typical commercial objective. The miniature objective is 7 mm in outer diameter and 21 mm in length (from object to image). It is used in a fiber confocal reflectance microscope. The miniature microscope objective has a numerical aperture of 1.0, a field of view of 250 microm, and a working distance of 450 microm. It delivers diffraction-limited performance at lambda = 1064 nm. Micrometer-level resolution has been experimentally demonstrated.     

Binary zone-plate array for a parallel joint transform correlator applied to face recognition

Taking advantage of small aberrations, high efficiency, and compactness, we developed a new, to our knowledge, design procedure for a binary zone-plate array (BZPA) and applied it to a parallel joint transform correlator for the recognition of the human face. Pairs of reference and unknown images of faces are displayed on a liquid-crystal spatial light modulator (SLM), Fourier transformed by the BZPA, intensity recorded on an optically addressable SLM, and inversely Fourier transformed to obtain correlation signals. Consideration of the bandwidth allows the relations among the channel number, the numerical aperture of the zone plates, and the pattern size to be determined. Experimentally a five-channel parallel correlator was implemented and tested successfully with a 100-person database. The design and the fabrication of a 20-channel BZPA for phonetic character recognition are also included.     

Efficient implementation of a spatial light modulator as a diffractive optical microlens array in a digital Shack-Hartmann wavefront sensor

A traditional Shack-Hartmann wavefront sensor (SHWS) uses a physical microlens array to sample the incoming wavefront into a number of segments and to measure the phase profile over the cross section of a given light beam. We customized a digital SHWS by encoding a spatial light modulator (SLM) with a diffractive optical lens (DOL) pattern to function as a diffractive optical microlens array. This SHWS can offer great flexibility for various applications. Through fast-Fourier-transform (FFT) analysis and experimental investigation, we studied three sampling methods to generate the digitized DOL pattern, and we compared the results. By analyzing the diffraction efficiency of the DOL and the microstructure of the SLM, we proposed three important strategies for the proper implementation of DOLs and DOL arrays with a SLM. Experiments demonstrated that these design rules were necessary and sufficient for generating an efficient DOL and DOL array with a SLM.     

Approximate phase conjugation with a retroreflecting array in microscopy

Some experiments on image recovery in microscopy are described. The aperture stop of a microscope objective is partially obscured, and a retroreflecting array is used as an approximate phase conjugator to return the beam through the unobstructed part of the aperture. An application is made to the measurement of the absolute reflectance of small samples by use of a variant of Strong's method.     

Confocal theta fluorescence microscopy with annular apertures

In a confocal theta fluorescence microscope, two objective lenses with circular apertures are used, one to illuminate the sample and the other to detect the emitted light at an angle to the illumination axis. We show that annular illumination and detection apertures lead to a reduction in the extent of the point-spread function. A spatial resolution improved by more than 50% can be achieved with a central obstruction blocking the inner 80% of the diameter. For the limit of a very narrow annular aperture and a numerical aperture of 0.75, the volume at half-maximum of the point-spread function is reduced from 15to5 aL. Amixed setup with anannular illumination aperture and a circular detection aperture is also considered.     

Fluorescence correlation microscopy with real-time alignment readout

In confocal fluorescence correlation microscopy (FCM) it is important to ensure that the correlation measurement is actually performed at the chosen location of the three-dimensional image of the specimen. We present a confocal FCM design that provides an automatic real-time readout of the location in the confocal microscopic image, which is aligned with the detector of the fluorescence correlation spectrometer. The design accomplishes this without using any special positioning device. The design is based on an apertured fluorescence detector placed close to the back aperture of the objective lens and can be easily incorporated into virtually any confocal microscope. We demonstrate the method by performing FCM measurements of a dye diffusing on a cell membrane.     

基于DMD的并行共焦图像处理方法研究

在并行共焦测量中,用于构造点光源阵列的光分束器件普遍存在参数固化、对不同测量对象适应性较差的缺点。将数字微镜器件（DMD）作为光分束器件应用于并行共焦测量,可以控制DMD构造柔性化点光源阵列,应对不同的测量场合。推导了DMD的空间光调制模型,规避了“泰伯效应”对并行共焦测量的影响,设计了可抑制光点横向漂移的远心光路,构建了基于DMD的并行共焦测量装置,对比了两种不同的并行共焦图像处理方法。实验结果表明,在没有设置高数值孔径物镜组的情况下,测量装置的纵向分辨率受到一定影响,但所设计的图像处理程序能够快速、准确地获取被测物面的形貌特征。     

Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes

Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined. The results verify earlier calculations [Appl. Opt. 33, 603 (1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.     

Volumetric real-time imaging using a CMUT ring array

A ring array provides a very suitable geometry for forward-looking volumetric intracardiac and intravascular ultrasound imaging. We fabricated an annular 64-element capacitive micromachined ultrasonic transducer (CMUT) array featuring a 10-MHz operating frequency and a 1.27-mm outer radius. A custom software suite was developed to run on a PC-based imaging system for real-time imaging using this device. This paper presents simulated and experimental imaging results for the described CMUT ring array. Three different imaging methods--flash, classic phased array (CPA), and synthetic phased array (SPA)--were used in the study. For SPA imaging, two techniques to improve the image quality--Hadamard coding and aperture weighting--were also applied. The results show that SPA with Hadamard coding and aperture weighting is a good option for ring-array imaging. Compared with CPA, it achieves better image resolution and comparable signal-to-noise ratio at a much faster image acquisition rate. Using this method, a fast frame rate of up to 463 volumes per second is achievable if limited only by the ultrasound time of flight; with the described system we reconstructed three cross-sectional images in real-time at 10 frames per second, which was limited by the computation time in synthetic beamforming.     

Infrared microspectroscopy using prism-based spectrographs and focal plane array detection

Several prism-based spectrographs employing a mercury cadmium telluride (MCT) focal plane array detector have been interfaced to an infrared microscope. In the combined system, the area-defining aperture of the microscope also served as the entrance slit to the spectrograph. This investigation considered the fundamental limits of diffraction for both the spectrograph and microscope in order to determine both the spatial and spectral resolution of the system as a whole. Experimental results for spectral resolution, spectral range, and peak-to-peak noise have been presented. Finally, the dynamic capabilities of one spectrograph/microscope combination were investigated.     

Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts

We study the use of coherent counterpropagating interfering waves to increase threefold to sevenfold the optical bandwidth and the resolution of fluorescence microscopy along the optic axis. Systematic comparison of the point-spread function and the optical transfer function (OTF) for the standing-wave microscope (SWM), the incoherent illumination interference image interference microscope (I5M), and the 4Pi confocal microscope reveals essential differences among their resolution capabilities. It is shown that the OTF's of these microscopes differ strongly in contiguity and amplitude within the enlarged range of transferred frequencies, and therefore they also differ in their ability to provide data from which interference artifacts can be removed. We demonstrate that for practical aperture angles the production of an interference pattern is insufficient for improving the axial resolution by the expected factor of 3-7. Conditions of the OTF for unambiguous improvement of axial resolution of arbitrary objects are fulfilled not at all in the SWM, partially in the I5M, and fully in the two-photon 4Pi confocal microscope.     

Experimental generation of Mathieu-Gauss beams with a phase-only spatial light modulator

We present a novel method for the efficient generation of even, odd, and helical Mathieu-Gauss beams of arbitrary order and ellipticity by means of a phase-only spatial light modulator (SLM). Our method consists of displaying the phase of the desired beam in the SLM; the reconstructed field is obtained on-axis following a spatial filtering process with an annular aperture. The propagation invariance and topological properties of the generated beams are investigated numerically and experimentally.     

Robust determination of the anisotropic polarizability of nanoparticles using coherent confocal microscopy

A coherent confocal microscope is proposed as a means to fully characterize the elastic scattering properties of a nanoparticle as a function of wavelength. Using a high numerical aperture lens, two-dimensional scanning, and a simple vector-beam shaper, the rank-2 polarizability tensor is estimated from a single confocal image. A method for computationally efficient data processing is described, and numerical simulations show that this algorithm is robust to noise and uncertainty in the focal plane position. The proposed method is a generalization of techniques that provide an estimate of a limited set of scattering parameters, such as a single orientation angle for rodlike particles. The measurement of the polarizability obviates the need for a priori assumptions about the nanoparticle.     

Implementation of multilens micro-optical systems with large numerical aperture by stacking of microlenses

In the field of micro-optics there is a demand for objectives with large numerical aperture (NA). One example is optical storage in which a NA greater than 0.5 is required. For planar microlenses the NA is determined by means of the maximal index difference and the degree of exchange and reaches typical values of 0.13-0.2. Thus stacking is needed to build high NA objectives from planar microlenses. An additional benefit of stacking lenses is the possibility to correct for different types of aberrations. We realized two stacked systems: an array of micro-objectives with a NA of 0.45 from three microlens arrays and a confocal sensor head from four microlens arrays and one pinhole array mask.     

Low-coherence fiber-optic sensor with a large numerical aperture for topographic measurements

We describe a compact fiber-optic setup for three-dimensional topometry. The measuring principle is based on low-coherence interferometry in combination with a confocal microscope. Integrating an optical arrangement based on compact disk optics together with fiber optics into one sensor yields a compact unit. The sensor has a numerical aperture of 0.45 and is suitable for measuring large surface slopes with high spatial resolution. Further, a depth measuring range of more than 1 mm can be achieved. The capabilities of our setup for three-dimensional measurements are demonstrated.