Conventional Wide-field Microscopy Research Articles

Folic acid functionalized gold nanoclusters for enabling targeted fluorescence imaging of human ovarian cancer cells

Photoluminescent gold nanoclusters have attracted an extensive research interest in bioimaging and therapeutics due to several distinctive advantages such as high fluorescent photostability, good dispersibility, low toxicity and large Stokes shift. However, a better understanding of the correlation between optical properties in various environments and their uptake by specific cancer cells is still needed. Herein, we developed bovine serum albumin stabilized gold nanoclusters (BSA-AuNCs) with an intrinsic tunable photoluminescence emission in the first biological window. The as-synthetized BSA-AuNCs agents consists in protein polymerized-chains dopped with AuNCs with an average size of 2–3 nm and were found to exhibit relevant properties as high photostability, temperature-dependent and excitation induced tunable red photoluminescence. The photostable BSA-AuNCs were functionalized with folic acid (FA-BSA-AuNCs) in order to achieve for the first time an active targeting of NIH:OVCAR-3 human ovarian adenocarcinoma cells, via AuNCs, towards bioimaging applications. After confirming their biocompatibility up to a concentration of 40 mg/ml, the improved cellular uptake and staining ability of FA-BSA-AuNCs compared to the BSA-AuNCs was validated by conventional wide-field epi-fluorescence microscopy, while the intracellular localization was monitored by confocal fluorescence lifetime imaging microscopy (FLIM). Considering their valuable intrinsic photoluminescent properties, the synthesized FA-BSA-AuNCs hold great promise for direct application in cellular imaging as efficient contrast agents towards early cancer diagnosis and image-guided therapy of cancer.

Deep-learning super-resolution light-sheet add-on microscopy (Deep-SLAM) for easy isotropic volumetric imaging of large biological specimens.

Isotropic 3D histological imaging of large biological specimens is highly desired but remains highly challenging to current fluorescence microscopy technique. Here we present a new method, termed deep-learning super-resolution light-sheet add-on microscopy (Deep-SLAM), to enable fast, isotropic light-sheet fluorescence imaging on a conventional wide-field microscope. After integrating a minimized add-on device that transforms an inverted microscope into a 3D light-sheet microscope, we further integrate a deep neural network (DNN) procedure to quickly restore the ambiguous z-reconstructed planes that suffer from still insufficient axial resolution of light-sheet illumination, thereby achieving isotropic 3D imaging of thick biological specimens at single-cell resolution. We apply this easy and cost-effective Deep-SLAM approach to the anatomical imaging of single neurons in a meso-scale mouse brain, demonstrating its potential for readily converting commonly-used commercialized 2D microscopes to high-throughput 3D imaging, which is previously exclusive for high-end microscopy implementations.

Efficient and cost-effective 3D cellular imaging by sub-voxel-resolving light-sheet add-on microscopy.

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving (SVR) light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a SVR algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live imaging with voxel-super-resolved capability.

Realistic, Vectorial Modeling of the Detection Point Spread Function for Single Molecule and Brightfield Microscopy

In previous work, we presented a rigorous, vectorial theory for the illumination point spread function (PSF). Here, we extend this approach to model the detection PSF, which is responsible for optical image formation in virtually all microscopy applications, such as conventional widefield microscopy, confocal scanning microscopy, fluorescence correlation spectroscopy, single molecule imaging, and super-resolution microscopy. We cover the most general case of a single dipolar scatterer or emitter, such as a single fluorescent molecule, which is imaged by a microscope objective-tube lens combination. The dipole radiation passes through stratified media composed of up to three layers (sample medium, glass coverslip, and immersion medium). The theoretical model accounts for a high numerical aperture tube lens and microscope objective (corrected for refractive indices of coverslip/immersion medium and coverslip thickness), a finite detector or detection pinhole, and an optional Babinet-Soleil compensator and polarization analyzer in the detection path. The resulting equations are readily evaluated, and multiple application examples will be discussed, such as the influence of dipole orientation and polarization detection on the detection PSF.

Rapid and Extreme Low-light Superresolution Imaging via Artificial Intelligence

Supreresolution (SR) fluorescence microscopy breaks the optical diffraction limit and achieves unprecedented nanoscale resolution, thus producing a huge impact on biological research. Structured illumination microscopy (SIM) is one of the most broadly used SR techniques. SIM applies varying, nonuniform illumination to samples. A dedicated computational algorithm is then used to retrieve SR information from nine or fifteen sequentially acquired images, for 2D or 2D/3D reconstruction respectively. Super-resolution radial fluctuation (SRRF) is a purely computational SR approach, that can retrieve high frequency information by spatio-temporally analyzing the stochastically fluctuating signals from image time series (typically >200 frames). We utilized deep neural networks to directly reconstruct super-resolved images out of a series of images taken with either structured or conventional wide-field illumination microscopy. This substantially reduced the number of images compared to state-of-the-art methods (as few as 3 images for SIM and 5 images for SRRF). Further, leveraging skip-layer connected U-Nets, we could restore high quality, high-resolution images from raw data with over 100 times less photons. We validated our results for different cellular structures, including microtubules, mitochondria, adhesions and actin filaments. We demonstrated its applications in ultra-fast, multi-color superresolution imaging of living cells. A ready-to-use open source package is available.

Side Views in 3D Live Cell Microscopy

Commonly trans-illumination or epi-illumination techniques are used in optical microscopy, e.g. phase microscopy, fluorescence microscopy or inelastic light scattering (Raman microscopy). However, an increasing number of applications requires optical excitation perpendicular or under a certain angle to the optical axis. Methods include Mie Scattering Microscopy with angular resolution for label-free studies of cell or tissue morphology as well as Total Internal Reflection Fluorescence (TIRF) Microscopy with an evanescent electromagnetic field penetrating only about 100 nm into a sample and permitting selective studies of cell surfaces, e.g. membranes. Presently, conventional wide field or confocal microscopy are often replaced by Light Sheet Fluorescence Microscopy (LSFM) where individual planes of a specimen are illuminated from the side without any light exposure or phototoxic damage of adjacent parts of the sample. Finally, Axial Tomography permits samples to be observed from various sides with a high and isotropic resolution in all three dimensions. All these methods require experimental setups which are not integrated in conventional microscopes. Therefore, we report on some technical solutions implemented in specific modules or add-ons for these microscopes.

Efficiency-enhanced and sidelobe-suppressed super-oscillatory lenses for sub-diffraction-limit fluorescence imaging with ultralong working distance.

Super-oscillatory lens (SOL) optical microscopy, behaving as a non-invasive and universal imaging technique, as well as being a simple post-processing procedure, may provide a potential application for sub-diffraction-limit fluorescence imaging. However, the low energy concentration, high-intensity sidelobes and micrometer-scale working distance of the reported planar SOLs impose unavoidable restrictions on the ground-state applications. Here, we demonstrate step-shaped SOLs based on the multiple-phase-modulated (MPM) method to improve the focusing efficiency. Two pivotal advantages are thus generated: (i) the fabrication complexity can be effectively reduced based on several conventional optical lithography steps; (ii) the focusing efficiency is much higher than that of the random MPM ones due to the efficient manipulation of the wavefronts, bringing about a stronger light concentration to the focal spot. Additionally, the ratio of the sidelobe intensity is flexibly tuned to meet the customized requirements, and a 2 mm-working-distance MPM SOL with the sidelobe intensity highly suppressed is finally exploited. For the first time, as far as we know, a SOL-based fluorescence microscopy without the pinhole filter to map the horizontal morphology of the dispersive fluorescent particles is established. Compared with the results achieved by the conventional wide-field microscopy, the sample details beating the diffraction limit can be reconstructed by simple imaging fusion. This research demonstrates the promising applications of SOLs for low-cost, simplified and highly customized sub-diffraction-limit fluorescence imaging systems free from photobleaching and an extremely short working distance.

Pixel reassignment in image scanning microscopy: a re-evaluation

Image scanning microscopy is a technique based on confocal microscopy, in which the confocal pinhole is replaced by a detector array, and the resulting image is reconstructed, usually by the process of pixel reassignment. 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 (wide-field) microscopy. In previous studies, it has usually been assumed that pixels should be reassigned by a constant factor, to a point midway between the illumination and detection spots. Here it is shown that the peak intensity of the effective point spread function (PSF) can be further increased by 4% by a new choice of the pixel reassignment factor. For an array of two Airy units, the peak of the effective PSF is 1.90 times that of a conventional microscope, and the transverse resolution is 1.53 times better. It is confirmed that image scanning microscopy gives optical sectioning strength identical to that of a confocal microscope with a pinhole equal to the size of the detector array. However, it is shown that image scanning microscopy exhibits axial resolution superior to a confocal microscope with a pinhole the same size as the detector array. For a two-Airy-unit array, the axial resolution is 1.34 times better than in a conventional microscope for the standard reassignment factor, and 1.28 times better for the new reassignment factor. The axial resolution of a confocal microscope with a two-Airy-unit pinhole is only 1.04 times better than conventional microscopy. We also examine the signal-to-noise ratio of a point object in a uniform background (called the detectability), and show that it is 1.6 times higher than in a confocal microscope.

Presegmentation Procedure Generates Smooth-Sided Microfluidic Devices: Unlocking Multiangle Imaging for Everyone?

We present a simple procedure to create smooth-sided, transparent polymer-based microfluidic devices by presegmentation with hydrophobized glass slides. We study the hypothesis that the smooth side planes permit rapid multiangle imaging of microfluidic systems in contrast to the turbid side planes that result from cutting the polymer. We compare the compatibility of the entire approach with the conventional widefield microscopy, confocal and 2-photon microscopy, as well as three-dimensional (3D) rendering and discuss limitations and potential applications.

Efficient and Cost-Effective 3D Cellular Imaging by Sub-Voxel-Resolving Light-Sheet Add-On Microscopy

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a sub-voxel-resolving (SVR) algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a two-fold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live volume imaging with voxel-super-resolved capability.

Quadrature demodulation in line-scan focal modulation microscopy for imaging three-dimensional zebrafish neural structure.

Visualizing biological processes in neuroscience requires in vivo functional imaging at single-neuron resolution, high image acquisition speed and strong optical sectioning ability. However, due to light scattering of in tissue, very often conventional wide-field fluorescence microscopes are unable to resolve cells in the presence of a strong out-of-focus background. Line-scan focal modulation microscopy enables high temporal resolution and good optical sectioning ability at the same time. Here we demonstrate a quadrature demodulation method to extract the focal information with an extended frequency bandwidth and therefore higher spatial resolution. The performance of the demodulation scheme in line-scan focal modulation microscope has been evaluated by performing imaging experiments with fluorescence beads and zebrafish neural structure. Reduced background, reduced artifacts and more detailed morphological information are evident in the obtained images.

Cytological Characterization of Arabidopsis arenosa Polyploids by SIM.

Arabidopsis arenosa has recently become established as a model organism for investigating how meiosis has evolved to overcome the meiotic challenges faced by newly formed autotetraploids. Here, we describe a protocol for the preparation of spread, immunolabeled prophase I chromosomes from established A. arenosa autotetraploids for imaging with three-dimensional structured illumination microscopy (3D-SIM). This technique allows us to dissect the unique synaptic behavior in A. arenosa and identify synaptic partner switch sites that are unresolvable with conventional widefield microscopy.

Lipid droplet quantification based on iterative image processing

Lipid droplets (LDs) are ubiquitous and highly dynamic subcellular organelles required for the storage of neutral lipids. LD number and size distribution are key parameters affected not only by nutrient supply but also by lipotoxic stress and metabolic regulation. Current methods for LD quantification lack general applicability and are either based on time consuming manual evaluation or show limitations if LDs are high in numbers or closely clustered. Here, we present an ImageJ-based approach for the detection and quantification of LDs stained by neutral lipid dyes in images acquired by conventional wide-field fluorescence microscopy. The method features an adjustable preprocessing procedure that resolves LD clusters. LD identification is based on their circular edges and central fluorescence intensity maxima. Adaptation to different cell types is mediated by a set of interactive parameters. Validation was done for three different cell lines using manual evaluation of LD numbers and volume measurement by 3D rendering of confocal datasets. In an application example, we show that overexpression of the acyl-CoA synthetase, FATP4/ACSVL5, in oleate-treated COS7 cells increased the size of LDs but not their number.

Label-free bioanalysis of Leishmania infantum using refractive index tomography with partially coherent illumination.

In this work, we report the use of refractive index (RI) tomography for quantitative analysis of unstained DH82 cell line infected with Leishmania infantum. The cell RI is reconstructed by using a modality of optical diffraction tomography technique that employs partially coherent illumination, thus enabling inherent compatibility with conventional wide-field microscopes. The experimental results demonstrate that the cell dry mass concentration (DMC) obtained from the RI allows for reliable detection and quantitative characterization of the infection and its temporal evolution. The RI provides important insight for studying morphological changes, particularly membrane blebbing linked to an apoptosis (cell death) process induced by the disease. Moreover, the results evidence that infected DH82 cells exhibit a higher DMC than healthy samples. These findings open up promising perspectives for clinical diagnosis of Leishmania.

Illumination pattern design with deep learning for single-shot Fourier ptychographic microscopy.

Fourier ptychographic microscopy allows for the collection of images with a high space-bandwidth product at the cost of temporal resolution. In Fourier ptychographic microscopy, the light source of a conventional widefield microscope is replaced with a light-emitting diode (LED) matrix, and multiple images are collected with different LED illumination patterns. From these images, a higher-resolution image can be computationally reconstructed without sacrificing field-of-view. We use deep learning to achieve single-shot imaging without sacrificing the space-bandwidth product, reducing the acquisition time in Fourier ptychographic microscopy by a factor of 69. In our deep learning approach, a training dataset of high-resolution images is used to jointly optimize a single LED illumination pattern with the parameters of a reconstruction algorithm. Our work paves the way for high-throughput imaging in biological studies.

Photoswitching FRET to monitor protein–protein interactions

FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule's photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched "off" rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched "on" again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein-protein interactions and fluorescent protein-based biosensors.

Depth-extended, high-resolution fluorescence microscopy: whole-cell imaging with double-ring phase (DRiP) modulation.

We report a depth-extended, high-resolution fluorescence microscopy system based on interfering Bessel beams generated with double-ring phase (DRiP) modulation. The DRiP method effectively suppresses the Bessel side lobes, exhibiting a high resolution of the main lobe throughout a four- to five-fold improved depth of focus (DOF), compared to conventional wide-field microscopy. We showed both theoretically and experimentally the generation and propagation of a DRiP point-spread function (DRiP-PSF) of the imaging system. We further developed an approach for creating an axially-uniform DRiP-PSF and successfully demonstrated diffraction-limited, depth-extended imaging of cellular structures. We expect the DRiP method to contribute to the fast-developing field of non-diffracting-beam-enabled optical microscopy and be useful for various types of imaging modalities.

Partially coherent illumination engineering for enhanced refractive index tomography.

Optical diffraction tomography based on partially coherent illumination (PC-ODT) is a quantitative label-free imaging technique that allows reconstructing of the object's 3D refractive index from measured through-focus intensity images. PC illumination provides advantages such as speckle noise-free imaging and inherent compatibility with conventional wide-field microscopes. Here we experimentally demonstrate that a proper design of the PC illumination, different from the familiar bright-field one, and the use of more realistic optical transfer functions (OTFs) have crucial importance in PC-ODT to significantly increase the accuracy in 3D refractive index reconstruction. While realistic OTFs properly account for the real experimental illumination conditions, the proposed PC illumination design allows for gathering the object spatial-frequency content attenuated when bright-field illumination is used.

Parallel mapping of optical near-field interactions by molecular motor-driven quantum dots.

In the vicinity of metallic nanostructures, absorption and emission rates of optical emitters can be modulated by several orders of magnitude1,2. Control of such near-field light-matter interaction is essential for applications in biosensing3, light harvesting4 and quantum communication5,6 and requires precise mapping of optical near-field interactions, for which single-emitter probes are promising candidates7-11. However, currently available techniques are limited in terms of throughput, resolution and/or non-invasiveness. Here, we present an approach for the parallel mapping of optical near-field interactions with a resolution of <5 nm using surface-bound motor proteins to transport microtubules carrying single emitters (quantum dots). The deterministic motion of the quantum dots allows for the interpolation of their tracked positions, resulting in an increased spatial resolution and a suppression of localization artefacts. We apply this method to map the near-field distribution of nanoslits engraved into gold layers and find an excellent agreement with finite-difference time-domain simulations. Our technique can be readily applied to a variety of surfaces for scalable, nanometre-resolved and artefact-free near-field mapping using conventional wide-field microscopes.

Live-cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport.

Analysis of intracellular cholesterol transport by fluorescence microscopy requires suitable fluorescent analogues of cholesterol. Most existing cholesterol analogues contain lipophilic dyes which can compromise the sterol properties in membranes. An alternative strategy is to introduce additional double bonds into the sterol ring system resulting in intrinsic fluorescence, while at the same time keeping the cholesterol-like properties of the analogues. Existing polyene sterols, such as dehydroergosterol (DHE) or cholestatrienol (CTL), however, contain only three double bonds and suffer from low brightness, significant photobleaching and excitation/emission in the ultraviolet region. Thus, special equipment is required to image such sterols. Here, we describe synthesis, characterization and intracellular imaging of new polyene sterols containing four conjugated double bonds in the sterol ring system. We show that such analogues have red-shifted excitation and emission by ∼20 nm compared to DHE or CTL. The red shift was even more pronounced when preventing keto-enol tautomer equilibration by protecting the 3'-hydroxy group with acetate. We show that the latter analogue can be imaged on a conventional wide field microscope with a DAPI/filipin filter cube. The new polyene sterols show reduced photobleaching compared to DHE or CTL allowing for improved deconvolution microscopy of sterol containing cellular membranes.