Fluorescence microscopy is a powerful tool for imaging biological samples with molecular specificity. In contrast, phase microscopy provides label-free measurement of the sample's refractive index (RI), which is an intrinsic optical property that quantitatively relates to cell morphology, mass, and stiffness. Conventional imaging techniques measure either the labeled fluorescence (functional) information or the label-free RI (structural) information, though it may be valuable to have both. For example, biological tissues have heterogeneous RI distributions, causing sample-induced scattering that degrades the fluorescence image quality. When both fluorescence and 3D RI are measured, one can use the RI information to digitally correct multiple-scattering effects in the fluorescence image. Here, we develop a new computational multi-modal imaging method based on epi-mode microscopy that reconstructs both 3D fluorescence and 3D RI from a single dataset. We acquire dozens of fluorescence images, each 'illuminated' by a single fluorophore, then solve an inverse problem with a multiple-scattering forward model. We experimentally demonstrate our method for epi-mode 3D RI imaging and digital correction of multiple-scattering effects in fluorescence images.
We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and by 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspe…
Adaptive optics (AO) is a powerful image correction technique with proven benefits for many life-science microscopy methods. However, the complexity of adding a reflective wavefront modulator and a wavefront sensor into already complicated microscope has made AO prohibitive for its widespread adaptation in microscopy systems. We present here the design and performance of a compact fluorescence microscope using a fully refractive optofluidic wavefront modulator yielding imaging performance on par with that of conventional deformable mirrors, both in correction fidelity and articulation. We comb…
We present a multimodal approach for measuring the three-dimensional (3D) refractive index (RI) and fluorescence distributions of live cells by combining optical diffraction tomography (ODT) and 3D structured illumination microscopy (SIM). A digital micromirror device is utilized to generate structured illumination patterns for both ODT and SIM, which enables fast and stable measurements. To verify its feasibility and applicability, the proposed method is used to measure the 3D RI distribution and 3D fluorescence image of various samples, including a cluster of fluorescent beads, and the time-…
Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet, truely quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly mea-sure the average RI in the 'footprint' region of the cell, during imaging. Our RI measurement is based on the determi…
Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near-) field and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF) is comparably less established although it achieves a similar optical sectioning as does TIRF. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission coup…
This work introduces a novel reinterpretation of structured illumination (SI) microscopy for coherent imaging that allows three-dimensional imaging of complex refractive index (RI). To do so, we show that coherent SI is mathematically equivalent to a superposition of angled illuminations. It follows that raw acquisitions for standard SI-enhanced quantitative-phase images can be processed into complex electric-field maps describing sample diffraction under angled illuminations. Standard diffraction tomography (DT) computation can then be used to reconstruct the sample 3D RI distribution at sub-…
Recent results by Hoffman et al. showed that it is possible to build a Negative Refractive Material (NIM) by interleaving InGaAs/AlInAs using current nanofabrication technologies. Their findings provide the first experimental proof of a negative refractive semiconductor long posited by transformation optics. In this paper an overview on semiconductor refractive index theory and negative refraction is presented, focusing on the results of Hoffman et al. and the future of the subject based on the theory of Transformation Optics and its importance to tailor materials electrodynamics properties.
Evanescent-wave microscopy achieves sub-diffraction axial sectioning by confining fluorescence excitation to a thin layer close to the cell/substrate interface. How thin this light sheet exactly is, however, is often unknown. Particularly in the popular objective-type total internal reflection fluorescence microscopy (TIRFM) configuration large deviations from the expected exponential intensity decay of the evanescent wave have been reported. Propagating, i.e., non-evanescent, excitation light diminishes the optical sectioning effect, reduces contrast and renders the quantification of TIRFM im…
We present simulations of an efficient high numerical aperture solid immersion metalens concept for fluorescence microscopy. The technique exploits the preferential emission of interfacial dipoles into a high refractive index substrate combined with a metalens and a conventional tube lens for imaging them. We have thus simulated dipole emission and an all-dielectric metasurface on opposite sides of a high refractive index substrate. Our calculations predict dipole collection efficiencies of up to 87 percent. The simulated beam propagation through the imaging system shows excellent performance …
Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Herein we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called Supercritical Angle Fluorescence (SAF) can be captured using a hig-NA objective and used to determine the axial position of the fluorophore with nanometer precision. We introduce a …
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…
Photothermal microscopy has recently complemented single molecule fluorescence microscopy by the detection of individual nano-objects in absorption. Photothermal techniques gain their superior sensitivity by exploiting a heat induced refractive index change around the absorbing nano-object. Numerous new applications to nanoparticles, nanorods and even single molecules have been reported all refering to the fact that photothermal microscopy is an extinction measurement on a heat induced refractive index profile. Here, we show that the actual physical mechanism generating a photothermal signal f…
In the framework of transformation optics, we show that the propagation of a locally superluminal refractive index perturbation (RIP) in a Kerr medium can be described, in the eikonal approximation, by means of a stationary metric, which we prove to be of Gordon type. Under suitable hypotheses on the RIP, we obtain a stationary but not static metric, which is characterized by an ergosphere and by a peculiar behaviour of the geodesics, which are studied numerically, also accounting for material dispersion. Finally, the equation to be satisfied by an event horizon is also displayed and briefly d…
Electrochemistry exploits local current heterogeneities at various scales ranging from the micrometer to the nanometer. The last decade has witnessed unprecedented progress in the development of a wide range of electroanalytical techniques allowing to reveal and quantify such heterogeneity through multiscale and multifonctionnal operando probing of electrochemical processes. However most of these advanced electrochemical imaging techniques, employing scanning probes, suffer from either low imaging throughput or limited imaging size. In parallel, optical microscopies, which can image a wide fie…
