Harmonic demodulation and minimum enhancement factors in field‐enhanced near‐field optical microscopy

Field‐enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near‐field information recorded in a field‐enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe–sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near‐field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near‐field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe–sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field‐enhanced scanning optical microscopy.     

关于红外探测器与红外焦平面阵列探测器性能参数描述方法的商榷

对中国国标GB/T 13584与GB/T17444关于红外探测器和红外焦平面阵列探测器噪声和相关性能参量定义方法提出了异议和见解.在国标中关于噪声的定义没有说明背景辐亮度和信号辐照度水平,因此容易导致关于噪声和相关参量的模糊观念.当信号很强时,光子噪声占主导地位,信噪比(SNR)与信号水平不成正比,此时由信噪比导出噪声等效功率(NEP)和D*时应格外小心.当信号很弱时,可以忽略光子噪声,由信噪比导出NEP和D^*就变为很简单.     

Two‐dimensional structured illumination microscopy

In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms ‘quasi‐confocal images’ can be derived from a given number of such phase‐images. Here, we present an alternative structured illumination microscopy approach, which employs two‐dimensional patterns instead of a one‐dimensional one. While in one‐dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two‐dimensional approach it is shifted at a single, pattern‐dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern‐shift and ‐rotation. Moreover, our two‐dimensional approach also yields a better signal‐to‐noise ratio in the evaluated image.     

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.     

Signal‐to‐noise ratio estimation on SEM images using cubic spline interpolation with Savitzky–Golay smoothing

A new technique based on cubic spline interpolation with Savitzky–Golay noise reduction filtering is designed to estimate signal‐to‐noise ratio of scanning electron microscopy (SEM) images. This approach is found to present better result when compared with two existing techniques: nearest neighbourhood and first‐order interpolation. When applied to evaluate the quality of SEM images, noise can be eliminated efficiently with optimal choice of scan rate from real‐time SEM images, without generating corruption or increasing scanning time.     

Alignment with sub‐pixel accuracy for images of multi‐modality microscopes using automatic calibration

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in‐depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x–y detector plane using a custom chrome‐on‐glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter‐modal and 30 intra‐modal image pairs, and the success rates are 95 and 100%, respectively, with sub‐pixel accuracy. This methodology is widely applicable to any multi‐modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.     

A novel pixellated solid‐state photon detector for enhancing the everhart–thornley detector

This article presents a pixellated solid‐state photon detector designed specifically to improve certain aspects of the existing Everhart–Thornley detector. The photon detector was constructed and fabricated in an Austriamicrosystems 0.35 µm complementary metal‐oxide‐semiconductor process technology. This integrated circuit consists of an array of high‐responsivity photodiodes coupled to corresponding low‐noise transimpedance amplifiers, a selector‐combiner circuit and a variable‐gain postamplifier. Simulated and experimental results show that the photon detector can achieve a maximum transimpedance gain of 170 dBΩ and minimum bandwidth of 3.6 MHz. It is able to detect signals with optical power as low as 10 nW and produces a minimum signal‐to‐noise ratio (SNR) of 24 dB regardless of gain configuration. The detector has been proven to be able to effectively select and combine signals from different pixels. The key advantages of this detector are smaller dimensions, higher cost effectiveness, lower voltage and power requirements and better integration. The photon detector supports pixel‐selection configurability which may improve overall SNR and also potentially generate images for different analyses. This work has contributed to the future research of system‐level integration of a pixellated solid‐state detector for secondary electron detection in the scanning electron microscope. Microsc. Res. Tech. 76:648–652, 2013. © 2013 Wiley Periodicals, Inc.     

Performance of new signal‐to‐noise ratio estimation for SEM images based on single image noise cross‐correlation

A new technique for estimation of signal‐to‐noise ratio in scanning electron microscope images is reported. The method is based on the image noise cross‐correlation estimation model recently developed. We derive the basic performance limits on a single image signal‐to‐noise ratio estimation using the Cramer–Rao inequality. The results are compared with those from existing estimation methods including the nearest neighbourhood (the simple method), the first order linear interpolator, and the autoregressive based estimator. The comparisons were made using several tests involving different images within the performance bounds. From the results obtained, the efficiency and accuracy of image noise cross‐correlation estimation technique is considerably better than the other three methods.     

Image signal‐to‐noise ratio estimation using adaptive slope nearest‐neighbourhood model

A new technique based on nearest neighbourhood method is proposed. In this paper, considering the noise as Gaussian additive white noise, new technique single‐image‐based estimator is proposed. The performance of this new technique such as adaptive slope nearest neighbourhood is compared with three of the existing method which are original nearest neighbourhood (simple method), first‐order interpolation method and shape‐preserving piecewise cubic hermite autoregressive moving average. In a few cases involving images with different brightness and edges, this adaptive slope nearest neighbourhood is found to deliver an optimum solution for signal‐to‐noise ratio estimation problems. For different values of noise variance, the adaptive slope nearest neighbourhood has highest accuracy and less percentage estimation error. Being more robust with white noise, the new proposed technique estimator has efficiency that is significantly greater than those of the three methods.     

Near-field scanning optical microscopy probes: a comparison of pulled and double-etched bent NSOM probes for fluorescence imaging of biological samples

Bent near‐field optical probes for biological applications have been fabricated using a combination of a two‐step chemical etching method and focused ion beam milling to create a well‐defined aperture. The transmission efficiencies have been evaluated as a function of laser wavelength (λ) and aperture size (D) for both large and small core fibres. The probe transmission behaviour follows a (D/λ)³ relationship. The double‐etched probes are compared to pulled probes fabricated from highly GeO₂‐doped dispersion compensating fibre and a standard single‐mode optical fibre. The transmission efficiencies of both types of pulled probes are approximately two orders of magnitude lower than double‐etched probes with similar aperture sizes. To demonstrate the utility of the various probes, their imaging performance has been evaluated for samples of polymer beads and phase‐separated phospholipid monolayers of dipalmitoylphosphatidylcholine or cholesterol/phosphatidylcholine/sphingomyelin mixtures. Both pulled and double‐etched probes are suitable for fluorescence imaging of polymer spheres. However, pulled probes are rapidly damaged at the higher input laser intensities required for fluorescence imaging of monolayer samples doped with < 1% of a fluorescent dye‐labelled lipid. The images obtained with the double‐etched probes show excellent spatial resolution and signal/noise, illustrating the potential of such probes for imaging of biological samples.     

Gated‐sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching

The spatial resolution of a stimulated emission depletion (STED) microscope is theoretically unlimited and practically determined by the signal‐to‐noise ratio. Typically, an increase of the STED beam's power leads to an improvement of the effective resolution. However, this improvement may vanish because an increased STED beam's power is often accompanied by an increased photobleaching, which worsen the effective resolution by reducing the signal strength. A way to lower the photobleaching in pulsed STED (P‐STED) implementations is to reduce the peak intensity lengthening the pulses duration (for a given average STED beam's power). This also leads to a reduction of the fluorophores quenching, thus a reduction of the effective resolution, but the time‐gated detection was proved to be successful in recovering these reductions. Here we demonstrated that a subnanosecond fiber laser beam (pulse width ∼600 ps) reduces the photobleaching with respect to a traditional stretched hundreds picosecond (∼200 ps) beam provided by a Ti:Sapphire laser, without any effective spatial resolution lost.     

Background intensity correction for terabyte‐sized time‐lapse images

Several computational challenges associated with large‐scale background image correction of terabyte‐sized fluorescent images are discussed and analysed in this paper. Dark current, flat‐field and background correction models are applied over a mosaic of hundreds of spatially overlapping fields of view (FOVs) taken over the course of several days, during which the background diminishes as cell colonies grow. The motivation of our work comes from the need to quantify the dynamics of OCT‐4 gene expression via a fluorescent reporter in human stem cell colonies. Our approach to background correction is formulated as an optimization problem over two image partitioning schemes and four analytical correction models. The optimization objective function is evaluated in terms of (1) the minimum root mean square (RMS) error remaining after image correction, (2) the maximum signal‐to‐noise ratio (SNR) reached after downsampling and (3) the minimum execution time. Based on the analyses with measured dark current noise and flat‐field images, the most optimal GFP background correction is obtained by using a data partition based on forming a set of submosaic images with a polynomial surface background model. The resulting image after correction is characterized by an RMS of about 8, and an SNR value of a 4 × 4 downsampling above 5 by Rose criterion. The new technique generates an image with half RMS value and double SNR value when compared to an approach that assumes constant background throughout the mosaic. We show that the background noise in terabyte‐sized fluorescent image mosaics can be corrected computationally with the optimized triplet (data partition, model, SNR driven downsampling) such that the total RMS value from background noise does not exceed the magnitude of the measured dark current noise. In this case, the dark current noise serves as a benchmark for the lowest noise level that an imaging system can achieve. In comparison to previous work, the past fluorescent image background correction methods have been designed for single FOV and have not been applied to terabyte‐sized images with large mosaic FOVs, low SNR and diminishing access to background information over time as cell colonies span entirely multiple FOVs. The code is available as open‐source from the following link https://isg.nist.gov/ .     

Do SEII Electrons Really Degrade SEM Image Quality?

Generally, in scanning electron microscopy (SEM) imaging, it is desirable that a high‐resolution image be composed mainly of those secondary electrons (SEs) generated by the primary electron beam, denoted SE_I. However, in conventional SEM imaging, other, often unwanted, signal components consisting of backscattered electrons (BSEs), and their associated SEs, denoted SE_II, are present; these signal components contribute a random background signal that degrades contrast, and therefore signal‐to‐noise ratio and resolution. Ideally, the highest resolution SEM image would consist only of the SE_I component. In SEMs that use conventional pinhole lenses and their associated Everhart–Thornley detectors, the image is composed of several components, including SE_I, SE_II, and some BSE, depending on the geometry of the detector. Modern snorkel lens systems eliminate the BSEs, but not the SE_IIs. We present a microfabricated diaphragm for minimizing the unwanted SE_II signal components. We present evidence of improved imaging using a microlithographically generated pattern of Au, about 500 nm thick, that blocks most of the undesired signal components, leaving an image composed mostly of SE_Is. We refer to this structure as a “spatial backscatter diaphragm.” SCANNING 35:1‐6, 2013. © 2012 Wiley Periodicals, Inc.     

Measurement of two‐photon‐absorption spectra through nonlinear fluorescence produced by a line‐shaped excitation beam

We propose an innovative experimental approach to estimate the two‐photon absorption (TPA) spectrum of a fluorescent material. Our method develops the standard indirect fluorescence‐based method for the TPA measurement by employing a line‐shaped excitation beam, generating a line‐shaped fluorescence emission. Such a configuration, which requires a relatively high amount of optical power, permits to have a greatly increased fluorescence signal, thus avoiding the photon counterdetection devices usually used in these measurements, and allowing to employ detectors such as charge‐coupled device (CCD) cameras. The method is finally tested on a fluorescent isothiocyanate sample, whose TPA spectrum, which is measured with the proposed technique, is compared with the TPA spectra reported in the literature, confirming the validity of our experimental approach.     

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less‐than‐ideal signal‐to‐noise ratio of the lifetimes obtained: Either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10^?3 mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal‐dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

Second‐harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label‐free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two‐photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two‐photon processes like two‐photon fluorescence, it is challenging to apply the various PSF‐engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ～480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ～26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro‐nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.     

Noise-induced systematic errors in ratio imaging: serious artefacts and correction with multi-resolution denoising

Ratio imaging is playing an increasingly important role in modern cell biology. Combined with ratiometric dyes or fluorescence resonance energy transfer (FRET) biosensors, the approach allows the detection of conformational changes and molecular interactions in living cells. However, the approach is conducted increasingly under limited signal‐to‐noise ratio (SNR), where noise from multiple images can easily accumulate and lead to substantial uncertainty in ratio values. This study demonstrates that a far more serious concern is systematic errors that generate artificially high ratio values at low SNR. Thus, uneven SNR alone may lead to significant variations in ratios among different regions of a cell. Although correct average ratios may be obtained by applying conventional noise reduction filters, such as a Gaussian filter before calculating the ratio, these filters have a limited performance at low SNR and are prone to artefacts such as generating discrete domains not found in the correct ratio image. Much more reliable restoration may be achieved with multi‐resolution denoising filters that take into account the actual noise characteristics of the detector. These filters are also capable of restoring structural details and photometric accuracy, and may serve as a general tool for retrieving reliable information from low‐light live cell images.     

IVMD融合RobustICA的内燃机噪声源分离

为了准确分离识别内燃机的主要噪声源,提出了一种改进变分模态分解融合鲁棒独立分量分析的方法。首先,针对变分模态分解方法的分解数选择问题进行了算法优化,提出了基于重构信号能量比和中心频率的改进变分模态分解方法,并利用仿真信号进行了验证;其次,进行了内燃机噪声试验,利用改进变分模态分解将单通道信号分解成多个信号分量,根据信号分量与源信号的互信息主要分量识别,克服了主要噪声分量选择客观依据不足的问题;最后,通过鲁棒独立分量分析提取主要噪声分量的独立成分,并结合相干分析和时频分析进行噪声源识别。结果显示,所提出的方法能够有效进行噪声源分离,可成功识别出燃烧噪声、活塞敲击噪声和空压机噪声等内燃机主要噪声源。