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Measures of purity for 3D partially polarized fields, and in particular, the separation into circularly and linearly polarized contributions, are reexamined, and a new degree of total linear polarization introduced. Explicit expressions for the characteristic decomposition in terms of coherency matrix elements are presented, including the special case of an intrinsic coherency matrix. Parameterization of the coherency matrix in terms of ellipticity, and the directions of the ellipse normal and major axis are investigated. Phase consistency is discussed. A comprehensive collection of results regarding intrinsic polarization properties is presented.

Many of the most important resolution improvements in optical microscopy techniques are based on the reduction of scattering effects. The main benefit of polarimetry-based imaging to this end is the discrimination between scattering phenomena originating from complex systems and the experimental noise. The determination of the coherency matrix elements from the experimental Mueller matrix can take advantage of scattering measurements to obtain additional information on the structural organization of a sample. We analyze the contrast mechanisms extracted from (a) the coherency matrix elements, (b) its eigenvalues and (c) the indices of polarimetric purity at different stages of zebrafish embryos, based on previous work using Mueller matrix optical scanning microscopy. We show that the use of the coherency matrix and related decompositions leads to an improvement in the imaging contrast, without requiring any complicated algebraic operations or any a priori knowledge of the sample, in contrast to standard polarimetric methods.

Image Scanning Microscopy (ISM) [1] successfully overcomes the trade-off between resolution and signal-to-noise ratio of traditional confocal microscopes by considering the spatial distribution of the fluorescence emission light and by reassigning the detected photons accordingly (i.e., pixel-reassignment). A recent implementation [2] upgrades a confocal to an image scanning microscope by substituting the traditional single-element detector with a SPAD array detector. Notably, the SPAD array samples the fluorescence signal from the detection/probing volume both in space (such as cameras) and in time (such as single element detectors, e.g., SPADs), providing potentially significant extra information for a variety of experimental contexts. To fully exploit this advantage, we present a versatile FPGA-based time-resolved microscopy platform that parallelly acquires all the SPAD array signals with a sub-nanosecond temporal resolution, thanks to a Digital Frequency Domain architecture. In the context of stimulated emission depletion (STED) microscopy, we leverage the platform to decrease the STED power needed to achieve a target spatial resolution [3] . In particular, we show how to synergically exploit both the spatial and temporal extra information to implement a new and dedicated photon-reassignment method for STED microscopy. This method not only takes advantage of the pixel-reassignment principle but also compensates for all the different sources of background which typically reduce resolution and imaging quality in STED microscopy, i.e., incomplete depletion [4] , [5] , direct-excitation from the STED beam and out-of-focus signal. Additionally, the platform allows for fluorescence lifetime imaging and a straightforward pulsed interleaved excitation (PIE) implementation, enabling dual-color STED microscopy ( Fig. 1 ).

Mueller matrix microscopy is an advanced imaging technique providing a full characterization of the optical polarization fingerprint of a sample. The Lu-Chipman (LC) decomposition, a method based on the modeling of elementary polarimetric arrangements and matrix inversions, is the gold standard to extract each polarimetric component separately. However, this models the optical system as a small number of discrete optical elements and requires a priori knowledge of the order in which these elements occur. In stratified media or when the ordering is not known, the interpretation of the LC decomposition becomes difficult. In this work, we propose a new, to our knowledge, representation dedicated to the study of biological tissues that combines Mueller matrix microscopy with a phasor approach. We demonstrate that this method provides an easier and direct interpretation of the retardance images in any birefringent material without the use of mathematical assumptions regarding the structure of the sample and yields comparable contrast to the LC decomposition. By validating this approach through numerical simulations, we demonstrate that it is able to give access to localized structural information, resulting in a simple determination of the birefringent parameters at the microscopic level. We apply our novel, to our knowledge, method to typical biological tissues that are of interest in the field of biomedical diagnosis.

Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The resolution of two-photon image scanning microscopy with Bessel beam illumination is a factor 2.13 better than in standard two-photon fluorescence. Axial resolution and optical sectioning in two-photon or three-photon fluorescence are also improved by using the image scanning modality.

Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure similar to 1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of similar to 2 - and automatic background correction is shown to boost the image quality especially in noisy images. Most importantly, our 2PE-ISM implementation requires no calibration measurements or other input from the user, which is an important aspect in terms of day-to-day usability of the technique. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement H2020 Marie Sklodowska-Curie Actions (AdaptiveSTED, No. 794531); FondsWetenschappelijk Onderzoek (G092915, V429717N); European Research Council (Bright Eyes, No. 818699). Vicidomini, G (corresponding author), Ist Italiano Tecnol, Mol Microscopy & Spect, Genoa, Italy giuseppe.vicidomini@iit.it

We propose a straightforward implementation of two-photon image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction algorithm is shown to dramatically improve the optical resolution of two-photon imaging, in various test samples. We show how our computational ISM approach is able to adapt to changing imaging conditions, thus ensuring optimal image quality. We also show how our recently introduced blind deconvolution approaches can be integrated into the image reconstruction workflow to further improve the image quality.

Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced ISM platform – based on a new single-photon avalanche diode (SPAD) array detector – coupled with a novel blind image reconstruction method, is shown to improve the effective resolution, as well as the overall image quality of 2PE microscopy. Indeed, in stark contrast to conventional single-point detectors, SPAD array detectors give access to the images of any excited scanning region, from which it is possible to decode information about the aberrations/distortions – occurring during imaging – able to substantially improve the reconstruction. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user; it works like any familiar two-photon system, but produces higher resolution images deep into thick samples. In our view, this novel implementation is the key for making 2PE-ISM mainstream.

Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM. A single-photon detector array enables robust and versatile image scanning microscopy (ISM) on any confocal microscope. This implementation makes super-resolution FLIM possible and eases a transition from confocal microscopy to ISM.

Due to diffraction of light, the conventional fluorescent microscope is a system with a band-limited information spatial channel. Every super-resolved fluorescent microscope circumvents this limitation by avoiding the simultaneous signaling of adjacent (< diffraction limit distance) fluorophores recording them sequentially in time. Notably, the confocal microscope also agrees to this perspective. Thus, the current super-resolved systems encode extra spatial information in the temporal channel.Here we discuss if other channels (dimensions) can be used to further improve the resolution of a fluorescence microscope. In particular, we describe time-gated STED microscopy and image-scanning microscopy (ISM) as examples of exploring extra dimensions, respectively the photon arrival time and the spatial image plane dimensions.Confocal scanning microscopy (CSM) can theoretically surpass the diffraction limit by a factor of √2. Practically, this improvement is sacrificed to obtain a good signal-to-noise ratio (SNR). Image scanning microscopy (ISM) solved this limitation by substituting the single point detector with a 2D array of detector. We showed that ISM can be straightforwardly implemented by using a quadrant detector. This implementation offers resolution close to the CSM theoretical value, improves the SNR by factor of 1.5 with respect to the CSM counterpart, and may be implemented without losing the optical sectioning capability and the system versatility.Time-gated detection increases the spatial resolution of a gated continuous-wave-(CW-) STED microscope, with the drawback of reducing the SNR. Thus, in sub-optimal conditions, such as a low-photons budget regime, the SNR reduction can cancel-out the expected gain in resolution. We developed a method which does not discard photons, but instead sorts all the photons in different time-gates according to their arrival time and recombines them through a multi-image deconvolution. Our results show that the SNR of the restored image improves, thereby improving the effective resolution.

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used.

Two-photon excitation (2PE) microscopy has been adopted as the de facto standard for imaging thick or scattering biological samples at high resolution and at depths up to several hundreds of microns [1]. However, as the study of complex biological phenomena in fields as important as neuroscience demands for higher temporal resolutions over ever increasing penetration depths, 2PE faces two important challenges. First, the point scanning nature of 2PE seriously limits three-dimensional (3D) imaging speed. Second, in thick tissues or turbid media, out-of-focus background generated by 2PE scattered photons can degrade image quality and resolution by loss of signal to background (S/B) [2].Here, we present a method that enables enhanced S/B 2PE microscopy and/or volumetric imaging at high-rates. Our approach relies on coupling an acoustic optofluidic lens [3] into a standard 2PE setup. The lens enables phase modulation of the incident beam at microsecond time scales, leading to ultrafast beam shaping on a pixel by pixel basis. By using high-speed readout [4], the detected photons can be sorted according to the corresponding phase induced by the lens. When a parabolic phase profile is deployed, high-speed axial focus shifting occurs and continuous volumetric imaging is achieved. For other phase profiles, controlled aberrations can be induced just on the neighboring focal planes, and out-of-focus fluorescence can be removed through a subtractive algorithm. We demonstrate our approach by imaging a 150 um thick mouse brain slice and by comparing our results with traditional 2PE microscopy.[1] Diaspro, Wiley, 978-0-471-40920-5 (2001).[2] Helmchen et al., Nature Methods 2, 932 - 940 (2005).[3] Duocastella et al., J. Biomed. Opt. 17(5), 050505 (2012).[4] Duocastella et al., Opt. Express 22, 19293-19301 (2014).

Despite tremendous progress in super-resolution microscopy techniques, the imaging of non-fluorescent species with sub-diffraction resolution is a challenging task. To overcome this limit the subtraction microscopy approach has been proposed recently [1,2]. The concept is based on the acquisition and the successive mathematical subtraction of two images acquired with Gaussian and doughnut shaped focal spots, respectively.Here, we will show a novel rescaling method that avoids artifacts, maintaining the contrast and resolution enhancement. Nevertheless, Infrared absorption/saturation microscopy is also designed to be a label free super-resolution approach[3]. The main idea of InfraRed Nanoscopy (IRN) is an absorption/saturation effect similar to the conventional pump-probe technique[4], where the pump beam modifies the carrier density inside the sample, followed by the intensity changes in the transmitted probe beam. The introduction of an additional doughnut shaped pump beam can transiently saturate the induced transition in the periphery of the focal spot and enabling super-resolution. Finally we will show a novel implementation based on single wavelength 2PE-STED nanoscopy[5] by means of random access scanning, and a phasor approach for the analysis of polarization SHG images, that, providing quantitative information, can be used to extract a fingerprint of tissues.The LANIR research leading to these results has received funding from FP7/20012-2015 under grant agreement n°280804.1.Harold, D., Piche, M., De Koninck, Y.(2013) Optics express 21,13:15912-15925.2.Wang, N., Miyazaki, J., He, J., Seto, K., Kobayashi, T.(2015). Optics Communications 343,28-33.3.Silien, C., Liu, N., Hendaoui, N., Tofail, SAM., Peremans, A.(2012). Optics Express 20,29694-29704.4.Dong CY, So PT, French T, Gratton E.(1995) Biophysical Journal. 69(6):2234-2242.5. Bianchini, P., Peres, C., Oneto, M., Galiani, S., Vicidomini, G., Diaspro, A. (2015). Cell and Tissue Research 360,143-150.

If light is focused or collected with a high numerical aperture lens, as may occur in imaging and optical encryption applications, polarization should be considered in three dimensions (3D). The matrix algebra of polarization behavior in 3D is discussed. It is useful to convert between the Mueller matrix and two different Hermitian matrices, representing an optical material or system, which are in the literature. Explicit transformation matrices for converting the column vector form of these different matrices are extended to the 3D case, where they are large (81×81) but can be generated using simple rules. It is found that there is some advantage in using a generalization of the Chandrasekhar phase matrix treatment, rather than that based on Gell-Mann matrices, as the resultant matrices are of simpler form and reduce to the two-dimensional case more easily. Explicit expressions are given for 3D complex field components in terms of Chandrasekhar-Stokes parameters.

Focal Modulation Microscopy (FMM) is a single-photon excitation fluorescence microscopy technique which effectively rejects the out-of-focus fluorescence background that arises when imaging deep inside biological tissues. Here, we report on the implementation of FMM in which laser intensity modulation at the focal plane is achieved using acousto-optic modulators (AOM). The modulation speed is greatly enhanced to the MHz range and thus enables real-time image acquisition. The capability of FMM is demonstrated by imaging fluorescence labeled vasculatures in mouse brain as well as self-made tissue phantom.

The best reported resolution in optical failure analysis of silicon chips is 120-nm half pitch demonstrated by Semicaps Private Limited, whereas the current and future industry requirement for 10-nm node technology is 100-nm half pitch. We show the first experimental evidence for resolution of features with 100-nm half pitch buried in silicon (λ/10.6), thus fulfilling the industry requirement. These results are obtained using near-infrared reflection-mode imaging using a solid immersion lens. The key novel feature of our approach is the choice of an appropriately sized collection pinhole. Although it is usually understood that, in general, resolution is improved by using the smallest pinhole consistent with an adequate signal level, it is found that in practice for silicon chips there is an optimum pinhole size, determined by the generation of induced currents in the sample. In failure analysis of silicon chips, nondestructive imaging is important to avoid disturbing the functionality of integrated circuits. High-resolution imaging techniques like SEM or TEM require the transistors to be exposed destructively. Optical microscopy techniques may be used, but silicon is opaque in the visible spectrum, mandating the use of near-infrared light and thus poor resolution in conventional optical microscopy. We expect our result to change the way semiconductor failure analysis is performed.

We review Fourier methods used in the disciplines of electromagnetism and signal processing, with a view to reconciling differences in approach. In particular, Fourier methods well known in signal processing are applied to three-dimensional wave propagation problems. The Fourier transform of the Green function, when written explicitly in terms of a real-valued spatial frequency, consists of homogeneous and inhomogeneous components. Both parts are necessary to result in a pure out-going wave that satisfies causality. The homogeneous component consists only of propagating waves, but the inhomogeneous component contains both evanescent and propagating terms. Thus, we make a distinction between inhomogeneous waves and evanescent waves. The evanescent component is completely contained in the region of the inhomogeneous component outside the k-space sphere. Further, propagating waves in the Weyl expansion contain both homogeneous and inhomogeneous components. The connection between the Whittaker and Weyl expansions is discussed. A list of relevant spherically symmetric Fourier transforms is given.

Different methods for edge extraction have been studied in the past years. In a recent paper we have presented a rotation-invariant edge extractor based on a spiral phase filter placed in the frequency plane of a convergent correlator. In this architecture, the axial position of the output plane strongly depends on the axial position of the object. This condition limits the processing of three dimensional objects, because only a narrow axial region of the object would be correctly imaged to the output. The other axial regions of the target yield defocused results. Likewise, a rather small axial misalignment of planar scenes could produce completely inaccurate correlations. Besides, annular pupils have been widely used to regulate the depth of focus (DOF) and the transversal resolution of optical systems. In this paper we present a novel filter that combines the advantages of a spiral phase-based edge extractor and those of an axial-apodizing annular pupil. This design allows edge extraction of objects in a widened axial range. Numerical simulations and experimental results that demonstrate edge extraction with improved tolerance to defocusing are presented. Fil: Mazzaferri, Javier E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Procesado de Imágenes; Argentina Fil: Campos, Juan. Universitat Autònoma de Barcelona; España Fil: Escalera, Juan C.. Universitat Autònoma de Barcelona; España Fil: Sheppard, Colin J. R.. National University of Singapore; Singapur Fil: Ledesma, Silvia Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Procesado de Imágenes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations (p > 0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix. The authors are grateful to Peng Yanxian, Clarice Chen and Shaoping Zhong for technical support; and Dr. Ricky R Lareu for his constructive discussion. This work was supported by grants (R-397-000-025-112 and R-279-000-168-712) of the Faculty Research Committee of the Faculty of Engineering, National University of Singapore. peer-reviewed

In this paper, we show a new way to break the resolution limit and dramatically improve sensitivity to structural changes. To realize it we developed a novel label free contrast mechanism, based on the spectral encoding of spatial frequency (SESF) approach. The super-resolution SESF (srSESF) microscopy is based on reconstruction of the axial spatial frequency (period) profiles for each image point and comparison of these profiles to form super-resolution image. As a result, the information content of images is dramatically improved in comparison with conventional microscopy. Numerical simulation and experiments demonstrate significant improvement in sensitivity and resolution. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 761214. The materials presented and views expressed here are the responsibility of the author(s) only. The EU Commission takes no responsibility for any use made of the information set out. Also, this work was supported by NUI Galway, Galway University Foundation, and the National Biophotonics Imaging Platform (NBIP) Ireland and Gov. of Ireland Postdoctoral fellowship-2017 to Nandan Das. non-peer-reviewed