Confocal microscopy is an essential imaging tool for biological systems, in solid-state physics and nano-photonics. Using confocal microscopes allows performing resonant fluorescence experiments, where the emitted light has the same wavelength as the excitation laser. Theses challenging experiments are carried out under linear cross-polarization conditions, rejecting laser light from the detector. In this work we uncover the physical mechanisms that are at the origin of the yet unexplained high polarization rejection ratio which makes these measurements possible. We show in both experiment and theory that the use of a reflecting surface (i.e. the beam-splitter and mirrors) placed between the polarizer and analyzer in combination with a confocal arrangement explains the giant cross-polarization extinction ratio of 10^8 and beyond. We map the modal transformation of the polarized optical Gaussian beam. We find an intensity 'hole' in the reflected beam under cross-polarization conditions. We interpret this as a manifestation of the Imbert-Fedorov effect, which deviates the beam depending on its polarization helicity. This implies that this topological effect is amplified here from the usually observed nanometer to the micrometer scale due to our cross-polarization dark field methods. We confirm these experimental findings for a large variety of commercially available mirrors and polarization components, allowing their practical implementation in many experiments.
Confocal imaging and phase imaging are powerful tools in life science research and industrial inspection. To coherently link the two techniques with different depth resolutions, we introduce an optical frequency comb (OFC) to microscopy. Two-dimensional (2D) image pixels of a sample were encoded onto OFC modes via 2D spectral encoding, in which OFC acted as an optical carrier with a vast number of discrete frequency channels. Then, a scan-less full-field confocal image with a depth resolution of 62.4 um was decoded from a mode-resolved OFC amplitude spectrum obtained by dual-comb spectroscopy.…
We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve hig…
We report on a method to obtain confocal imaging through multimode fibers using optical correlation. First, we measure the fiber's transmission matrix in a calibration step. This allows us to create focused spots at one end of the fiber by shaping the wavefront sent into it from the opposite end. These spots are scanned over a sample, and the light coming back from the sample via the fiber is optically correlated with the input pattern. We show that this achieves spatial selectivity in the detection. The technique is demonstrated on microbeads, a dried epithelial cell, and a cover glass.
A fluorescence correlation spectroscopy (FCS) system based on two independent measurement volumes is presented. The optical setup and data acquisition hardware are detailed, as well as a complete protocol to control the location, size and shape of the measurement volumes. A method that allows to monitor independently the excitation and collection efficiency distribution is proposed. Finally, a few examples of measurements that exploit the two spots in static and/or scanning schemes, are reported.
A novel full-field surface profilometer using diffractive-based chromatic confocal microscopy and area-scan spectral sensing is developed. A normalized cross correlation algorithm was developed to establish a spectrum-depth response curve. A measuring repeatability for a single surface depth can be controlled within 120 nanometers.
Acquiring high-contrast optical images deep inside biological tissues is still a challenging problem. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. However, it suffers from low sensitivity in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images are acquired through a multimode fiber. We achieve this by digitally engi…
Planar lenses with optimized but disordered structures can focus light beyond the diffraction limit. However, these disordered structures have inevitably destroyed wide-field imaging capability, limiting their applications in microscopy. Here we introduce information entropy S to evaluate the disorder of an objective chip by using the probability of its structural deviation from standard Fresnel zone plates. Inspired by the theory of entropy change, we predict an equilibrium point S0=0.5 to balance wide-field imaging (theoretically evaluated by the Strehl ratio) and subdiffraction-limit focusi…
A recently introduced two-channel confocal microscope with correlated detection promises up to 50% improvement in transverse spatial resolution [Simon, Sergienko, Optics Express {\bf 18}, 9765 (2010)] via the use of photon correlations. Here we achieve similar results in a different manner, introducing a triple-confocal correlated microscope which exploits the correlations present in optical parametric amplifiers. It is based on tight focusing of pump radiation onto a thin sample positioned in front of a nonlinear crystal, followed by coincidence detection of signal and idler photons, each foc…
Confocal and multiphoton microscopy are effective techniques to obtain high-contrast images of 2-D sections within bulk tissue. However, scattering limits their application to depths only up to ~1 millimeter. Multimode fibers make excellent ultrathin endoscopes that can penetrate deep inside the tissue with minimal damage. Here, we present Multiview Scattering Scanning Imaging Confocal (MUSSIC) Microscopy that enables high signal-to-noise ratio (SNR) imaging through a multimode fiber, hence combining the optical sectioning and resolution gain of confocal microscopy with the minimally invasive …
Simultaneous plane illumination and detection in confocal microscopy using a mode-selective photonic lantern
Polytechnique Montréal（蒙特利尔理工学院）；Castor Optics（卡斯托光学）；Peregrine Photon（游隼光子）；Université de Montréal, Institute of Biomedical Engineering（蒙特利尔大学生物医学工程学院）
Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily impleme…
Fast, volumetric imaging over large scales has been a long-standing goal in biological microscopy. Scanning techniques such as fluorescence confocal microscopy can acquire 2D images at high resolution and high speed, but extending the acquisition to multiple planes at different depths requires an axial scanning mechanism that drastically reduces the acquisition speed. To address this challenge, we report an augmented variant of confocal microscopy where the key innovation consists to use a series of reflecting pinholes axially distributed in the detection plane, each one probing a different de…
Scanning laser confocal holographic microscopy using a spatial heterodyne detection method is presented. Spatial heterodyne detection technique employs a Mach-Zehnder interferometer with the reference beam frequency shifted by two acousto-optic modulators (AOM) relative to the object beam frequency. Different from the traditional temporal heterodyne detection technique in which hundreds temporal samples are taken at each scanning point to achieve the complex signal, the spatial heterodyne detection technique generates spatial interference fringes by use of a linear tempo-spatial relation provi…
FRET-based approaches are a unique tool for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and FLIM (Fluorescence Lifetime Imaging Microscopy) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, we demonstrate an approach to obtain super-resolved FRET imaging based on single-m…
We present a technically simple implementation of quantitative phase imaging in confocal microscopy based on synthetic optical holography with sinusoidal-phase reference waves. Using a Mirau interference objective and low-amplitude vertical sample vibration with a piezo-controlled stage, we record synthetic holograms on commercial confocal microscopes (Nikon, model: A1R; Zeiss: model: LSM-880), from which quantitative phase images are reconstructed. We demonstrate our technique by stain-free imaging of cervical (HeLa) and ovarian (ES-2) cancer cells and stem cell (mHAT9a) samples. Our techniqu…
A full-field chromatic confocal microscopy using a multispectral sensor was developed for quasi-one-shot microscopic 3D surface measurement. An innovative optical configuration employs a digital micromirror device (DMD) and a multispectral sensor is used to realize chromatic confocal microscopy with full-field area scanning. In the optical design, an area-scan type chromatic dispersive objective is specially designed to achieve measuring specification. Based on an 8x chromatic dispersive objective, the FOV for one shot measurement can be reached to 1.8mm*1.3mm which is immersive to microscopic…
Spatial resolution is one of the most important specifications of an imaging system. Recent results in quantum parameter estimation theory reveal that an arbitrarily small distance between two incoherent point sources can always be efficiently determined through the use of a spatial mode sorter. However, extending this procedure to a general object consisting of many incoherent point sources remains challenging, due to the intrinsic complexity of multi-parameter estimation problems. Here, we generalize the Richardson-Lucy (RL) deconvolution algorithm to address this challenge. We simulate its …
Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the achievable resolution both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field detection. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5S microscopy, have improved the axial resolution beyond that of the lat…
Confocal microscopy is the standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering samples. Unfortunately, a confocal microscope is quite expensive compared to traditional microscopes. In addition, the point scanning in a confocal leads to slow imaging speed and photobleaching due to the high dose of laser energy. In this paper, we demonstrate how the advances in machine learning can be exploited to "teach" a traditional wide-field microscope, one that's available in every lab, into producing 3D volumetric im…
Label-free imaging approaches seek to simplify and augment histopathologic assessment by replacing the current practice of staining by dyes to visualize tissue morphology with quantitative optical measurements. Quantitative phase imaging (QPI) operates with visible/UV light and thus provides a resolution matched to current practice. Here we introduce and demonstrate confocal QPI for label-free imaging of tissue sections and assess its utility for manual histopathologic inspection. Imaging cancerous and normal adjacent human breast and prostate, we show that tissue structural organization can b…
Intracellular structural alterations are hallmark of several disease conditions and treatment modalities. However, robust methods to quantify these changes are scarce. In view of this, we introduce a new method to quantify structural alterations in biological cells through the widely used confocal microscopy. This novel method employs optical eigenfunctions localization properties of cells and quantifies the degree of structural alterations, in terms of nano- to micron scale intracellular mass density fluctuations, in one single parameter. Such approach allows a powerful way to compare changin…
University of Bern（伯尔尼大学）；Technical University of Munich（慕尼黑工业大学）
We present a novel deep learning approach to reconstruct confocal microscopy stacks from single light field images. To perform the reconstruction, we introduce the LFMNet, a novel neural network architecture inspired by the U-Net design. It is able to reconstruct with high-accuracy a 112x112x57.6$μm^3$ volume (1287x1287x64 voxels) in 50ms given a single light field image of 1287x1287 pixels, thus dramatically reducing 720-fold the time for confocal scanning of assays at the same volumetric resolution and 64-fold the required storage. To prove the applicability in life sciences, our approach is…
We present a new flexible high speed laser scanning confocal microscope and its extension by an astigmatism particle tracking device (APTV). Many standard confocal microscopes use either a single laser beam to scan the sample at relatively low overall frame rate, or many laser beam to simultaneously scan the sample and achieve a high overall frame rate. Single-laser-beam confocal microscope often use a point detector to acquire the image. To achieve high overall frame rates, we use, next to the standard 2D probe scanning unit, a second 2D scan unit projecting the image directly on a 2D CCD-sen…
We introduce a new modality for dynamic phase imaging in confocal microscopy based on synthetic optical holography. By temporal demultiplexing of the detector signal into a series of holograms, we record time-resolved phase images directly in the time domain at a bandwidth as determined by the photo detector and digitizer. We demonstrate our method by optical imaging of transient vibrations in an atomic force microscope cantilever with 100 ns time resolution, and observe the dynamic deformation of the cantilever surface after excitation with broadband mechanical pulses. Temporal Fourier transf…
Confocal microscopy is the backbone of cellular research labs across the world but unfortunately, the imaging is restricted to a single plane. Chromatic confocal microscopy offers the possibility to image multiple planes simultaneously thus providing a manifold increase in the imaging speed whereas eliminating the need for z-axis scanning. Standard chromatic confocal systems have a limited imaging range of the order of a few hundreds of micrometers which limits their applications. In this work, using a single zinc selenide lens, we demonstrate a chromatic confocal tomography system that has an…
We present a laser scanning reflection-matrix microscopy combining the scanning of laser focus and the wide-field mapping of the electric field of the backscattered waves for eliminating higher-order aberrations even in the presence of strong multiple light scattering noise. Unlike conventional confocal laser scanning microscopy, we record the amplitude and phase maps of reflected waves from the sample not only at the confocal pinhole, but also at other non-confocal points. These additional measurements lead us to constructing a time-resolved reflection matrix, with which the sample-induced ab…
The transmission characteristics of a spherical (possibly multilayered) particle of arbitrary size under focused illumination are discussed within the generalized Lorenz-Mie theory. Expressions generalizing the total extinction and scattering cross-sections to their fractional counterparts are presented which allow for a convenient modeling of transmission signals, both on-axis and off-axis. The strong dependence of the signal on the collection angle and the complex polarizability are readily included in this minimal, yet accurate model. The precise signature of the energy redistribution and a…
Dual-comb microscopy (DCM), an interesting imaging modality based on the optical-frequency-comb (OFC) mode and image pixel one-to-one correspondence, benefits from scan-less full-field imaging and simultaneous confocal amplitude and phase imaging. However, the two fully frequency-stabilized OFC sources requirement hampers DCM practicality due to the complexity and costs. Here, a bidirectional single-cavity dual-comb fiber laser (SCDCFL) is adopted as a DCM low-complexity OFC source. Computational image correction reduces the image blur caused by the SCDCFL residual timing jitter. Nanometer-ord…
To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used [C. J. R. Sheppard, Super-resolution in confocal imaging, Optik 80, 5354 (1988)]. This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and …
Single-beam super-resolution microscopy, also known as superlinear microscopy, exploits the nonlinear response of fluorescent probes in confocal microscopy. The technique requires no complex purpose-built system, light field modulation, or beam shaping. Here, we present a strategy to enhance spatial resolution of superlinear microscopy by modulating excitation intensity during image acquisition. This modulation induces dynamic optical nonlinearity in upconversion nanoparticles (UCNPs), resulting in variations of higher spatial-frequency information in the obtained images. The high-order inform…
We report on the use of silicon nitride (SiN) photonic integrated circuits (PICs) in high-value instrumentation, namely multi-color laser engines (MLEs), a core element of cutting-edge biophotonic systems applied to confocal microscopy, fluorescent microscopy - including super-resolution stimulated emission depletion (STED) microscopy - flow cytometry, optogenetics, genetic analysis and DNA sequencing, to name just a few. These have in common the selective optical excitation of molecules - fluorophores, or, in the case of optogenetics, light-gated ion channels - with laser radiation falling wi…
Sub-nanometer 3D morphometric precision of polarisation-resolved wide-field optical extinction microscopy determines the roundness of individual gold nanospheres
Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approximately $T^{-1/2}$, where T is the transmissivity of the mask. Numerical aperture based differentiation of background from scattered light represents a general approach to increasin…
We report a new experimental technique for quantifying the angular distribution of light scattered by single plasmonic and metamaterial nanoscatterers, based on Fourier microscopy in a dark field confocal set up. This new set up is a necessary tool for quantifying the scattering properties of single plasmonic and meatamaterial building blocks, as well as small coupled clusters of such building blocks, which are expected to be the main ingredients of nano-antennas, light harvesting structures and transformation optics. We present a set of measurements on Au nanowires of different lengths and sh…
Brillouin microscopy has recently emerged as powerful technique to characterize the mechanical properties of biological tissue, cell and biomaterials. However, the potential of Brillouin microscopy is currently limited to transparent samples, because Brillouin spectrometers do not have sufficient spectral extinction to reject the predominant non-Brillouin scattered light of turbid media. To overcome this issue, we developed a spectrometer composed of a two VIPA stages and a multi-pass Fabry-Perot interferometer. The Fabry-Perot etalon acts as an ultra-narrow band-pass filter for Brillouin ligh…
Photonics detection of molecular-specific spatial structural alterations in cell nuclei due to chronic alcoholism and probiotics treatments on colon cancer via a light localization method using confocal imaging
The light scattering problem for a confocal multilayered spheroid has been solved by the extended boundary condition method (EBCM) with a corresponding spheroidal basis. The solution preserves the advantages of the approach applied previously to homogeneous and core-mantle spheroids, i.e. the separation of the radiation fields into two parts and a special choice of scalar potentials for each of the parts. The method is known to be useful in a wide range of the particle parameters. It is particularly efficient for strongly prolate and oblate spheroids. Numerical tests are described. Illustrativ…
