Structured illumination microscopy (SIM) has attained high spatiotemporal delineation of subcellular architecture, yet offers limited insight into chemical composition. We develop Chem-SIM, a structured-illumination fluorescence detected mid-infrared photothermal microscopy, for super-resolved chemical imaging of microorganisms and mammalian cells. Poisson maximum-likelihood demodulation and spectral normalization across wavenumber recover the weak IR-induced fluorescence intensity change under low photon budgets and convert the fluorescence intensity modulation to chemical fingerprints. Photothermal gating further rejects water backgrounds in aqueous samples, while the IR pump maintains cellular activity at near-physiological temperature. Chem-SIM preserves full vibrational fingerprints, achieves SIM-grade lateral resolution in a high-throughput camera-based format. Here, we show that this platform distinguishes stationary- from log-phase bacteria through chemical content mapping, reports deuterated fatty-acid incorporation in ovarian cancer cells, and resolves lipid-droplet dynamics in live cells, establishing a high-throughput route to super-resolved imaging of organelle chemistry, metabolism, and dynamics.
Mid-infrared photothermal microscopy is a new chemical imaging technology in which a visible beam senses the photothermal effect induced by a pulsed infrared laser. This technology provides infrared spectroscopic information at sub-micron spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependance of scattering on temperature and the image quality is vulnerable to the speckles caused by scattering. Here, we present a novel version of mid-infrared photothermal microscopy…
Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases. However, it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment. To address this challenge, we developed a computational chemical microscope integrating 3D mid-infrared photothermal imaging with fluorescence imaging, termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography (FBS-IDT). Based on a low-cost and simple optical design, FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific m…
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…
Single-molecule microscopy has become an indispensable tool for biochemical analysis. The capability of characterizing distinct properties of individual molecules without averaging has provided us with a different perspective for the existing scientific issues and phenomena. Recently, the super-resolution fluorescence microscopy techniques have overcome the optical diffraction limit by the localization of molecule positions. However, the labelling process can potentially modify the intermolecular dynamics. Based on the highly-sensitive nanomechanical photothermal microscopy reported previously…
Infrared (IR) imaging has become a viable tool for visualizing various chemical bonds in a specimen. The performance, however, is limited in terms of spatial resolution and imaging speed. Here, instead of measuring the loss of the IR beam, we utilize a pulsed visible light for a high-throughput, widefield sensing of the transient photothermal effect induced by absorption of single mid-IR pulses. To extract such transient signals, we built a virtual lock-in camera synchronized to the visible probe and IR light pulses with precisely-controlled delays, allowing sub-microsecond temporal resolution…
Leibniz Institute of Photonic Technology（莱布尼茨光子技术研究所）；Friedrich-Schiller-University Jena（耶拿弗里德里希·席勒大学）；InfectoGnostics Research Campus Jena（耶拿感染诊断研究园区）
Light absorption and scattering of plasmonic metal nanoparticles can lead to non-equilibrium charge carriers, intense electromagnetic near-fields, and heat generation, with promising applications in a vast range of fields, from chemical and physical sensing, to nanomedicine, and photocatalysis for the sustainable production of fuels and chemicals. Disentangling the relative contribution of thermal and non-thermal contributions in plasmon driven processes is however difficult. Nanoscale temperature measurements are technically challenging and macroscale experiments are often characterized by co…
Over the last decade, single-molecule optical microscopy has become the gold-standard approach to decipher complex molecular processes in cellular environments. [1-3] Single-molecule fluorescence microscopy has several advantages such as ease of application, high sensitivity, low invasiveness and versatility due the large number of available fluorescent probes. It bears however some drawbacks related to the poor photostability of organic dye molecules [4] and auto-fluorescent proteins [5-7] and and to the relatively large size of semiconductor nanoparticles in the context of live cell applicat…
Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from sub-wavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemic…
Infrared (IR) spectroscopy, a well established tool for chemical analysis of diverse materials, has significant potential in biomedical applications. While the spatial resolution of traditional IR spectroscopy is limited by the wavelength of the IR light to the few micrometres, it has been shown that nanoscale chemical analysis can be obtained by detecting IR induced local heating photothermal response via Scanning Thermal Microscopy (SThM) or local thermomechanical expansion using Atomic Force Microscopy (AFM). This paper explores the potential of a pulsed ps pulse duration high power free el…
Millimeter-scale wide-field mid-infrared photothermal imaging enabled by a broadly tunable picosecond optical parametric oscillator
Leibniz Institute of Photonic Technology（莱布尼茨光子技术研究所）；Otto Schott Institute of Materials Research, Friedrich-Schiller-University Jena（弗里德里希·席勒大学耶拿分校奥托·肖特材料研究所）；Friedrich-Schiller-University Jena（弗里德里希·席勒大学耶拿分校）
More than twenty years ago, scientists succeeded in pushing the limits of optical detection to single molecules using fluorescence. This breakthrough has revolutionized biophysical measurements, but restrictions in photophysics and labeling protocols have motivated many efforts to achieve fluorescence-free single-molecule sensitivity in biological studies. Although several interesting mechanisms using vibrational spectroscopy, photothermal detection, plasmonics or microcavities have been proposed for biosensing at the single-protein level, no method has succeeded in direct label-free detection…
Chemical element mapping is an imaging tool that provides essential information on composite materials and it is crucial for a broad range of fields ranging from fundamental science to numerous applications. Methods that exploit x-ray fluorescence are very advantageous and are widely used, but require focusing of the input beam and raster scanning of the sample. Thus the methods are slow and exhibit limited resolution due to focusing challenges. We demonstrate a new focusing free x-ray fluorescence method based ghost imaging that overcomes those limitations. We combine our procedure with compr…
Fluorescence imaging and spectroscopy remain the most powerful tools for visualization with chemical and immunological specificity of labeled biomolecules, viruses, cellular organelles, and living cells in complex biological backgrounds. However, a common drawback of fluorescence labels is that their brightness is limited by optical saturation and photobleaching. As an alternative, plasmonic metal nanoparticles are very promising as optical labels with no photobleaching and low optical saturation at realistic exciting intensities as was demonstrated in photoacoustic and photothermal sensing, i…
Single-molecule chemical reactions with individual single-walled carbon nanotubes were observed through near-infrared photoluminescence microscopy. The emission intensity within distinct submicrometer segments of single nanotubes changes in discrete steps after exposure to acid, base, or diazonium reactants. The steps are uncorrelated in space and time, and reflect the quenching of mobile excitons at localized sites of reversible or irreversible chemical attack. Analysis of step amplitudes reveals an exciton diffusional range of about 90 nanometers, independent of nanotube structure. Each exci…
We present a portable multi-channel, multi-sample UV/vis absorption and fluorescence detection device, which has no moving parts, can operate wirelessly and on batteries, interfaces with smart mobile phones or tablets, and has the sensitivity of commercial instruments costing an order of magnitude more. We use UV absorption to measure the concentration of ethylene glycol in water solutions at all levels above those deemed unsafe by the United States Food and Drug Administration; in addition we use fluorescence to measure the concentration of d-glucose. Both wavelengths can be used concurrently…
Deep Learning Domain Adaptation to Understand Physico-Chemical Processes from Fluorescence Spectroscopy Small Datasets: Application to Ageing of Olive Oil
TOELT LLC（TOELT有限责任公司）；Lucerne University of Applied Sciences and Arts（卢塞恩应用科学与艺术大学）；Zurich University of Applied Sciences（苏黎世应用科学大学）
Free-electron laser-based extended wide-field mid-infrared photothermal imaging for biomedical and microplastic analysis
Leibniz Institute of Photonic Technology（莱布尼兹光子技术研究所）；Research Center Borstel, Leibniz Lung Center（博斯特尔研究中心，莱布尼兹肺科中心）；German Center for Infection Research (DZIF)（德国感染研究联合会）；Jena University Hospital（耶拿大学医院）；Helmholtz-Zentrum Dresden Rossendorf（德累斯顿-罗斯多夫亥姆霍兹中心）；Friedrich-Schiller-University Jena（耶拿弗里德里希·席勒大学）
Fluorescence lifetime imaging microscopy (FLIM) is an important technique to understand the chemical micro-environment in cells and tissues since it provides additional contrast compared to conventional fluorescence imaging. When two fluorophores within a diffraction limit are excited, the resulting emission leads to non-linear spatial distortion and localization effects in intensity (magnitude) and lifetime (phase) components. To address this issue, in this work, we provide a theoretical model for convolution in FLIM to describe how the resulting behavior differs from conventional fluorescenc…
In the present paper, we observed both two-photon and three-photon induced distinct photoluminescence from GO nanoparticles under fs laser excitation. Conjugated with PEG molecules, GO nanoparticles exhibited high chemical stability, and could effectively label HeLa cells. Imaged with a two-photon scanning microscope, GO nanoparticles were observed to localize in the mitochondria, endoplasmic reticulum, Golgi and lysosome of HeLa cells. Furthermore, GO nanoparticles were micro-injected into the brain of a black mouse, and in vivo two-photon luminescence imaging illustrated that GO nanoparticle…
Quantitative phase imaging (QPI) quantifies the sample-specific optical-phase-delay enabling objective studies of optically-transparent specimens such as biological samples, but lacks chemical sensitivity limiting its application to morphology-based diagnosis. We present wide-field molecular-vibrational microscopy realized in the framework of QPI utilizing mid-infrared photothermal effect. Our technique provides mid-infrared spectroscopic performance comparable to that of a conventional infrared spectrometer in the molecular fingerprint region of 1,450 - 1,600 cm-1 and realizes wide-field mole…
Molecule-surface interaction is key to many physical and chemical processes at interfaces. Here, we show that the dynamics of single molecules on a surface under ultrahigh vacuum can be resolved using fluorescence imaging. By adapting oil-immersion microscopy to a thin vacuum window, we measure the surface adsorption, translational and rotational diffusion of single perylene molecules on a fused silica surface with high spatial and temporal resolutions. Time-dependent measurements of the fluorescence signal allow us to deduce two characteristic decay time scales, which can be explained through…
Quantitative phase microscopies (QPMs) have been mainly used for applications in cell biology, for around 2 decades. In this article, we show how cross-grating phase microscopy (CGM), a high-resolution, high-sensitivity QPM, recently expanded the scope of QPMs to applications in nanophotonics. In particular, this article explains how the intensity and phase images acquired by CGM can be processed to determine all the optical properties of imaged nanoparticles, 2D-materials and metasurfaces. We also explain how CGM can be used as a temperature microscopy technique. This latter imaging modality …
Super-resolution microscopy is providing unprecedented insights into biology by resolving details much below the diffraction limit. State-of-the-art Single Molecule Localization Microscopy (SMLM) techniques for super-resolution are restricted by long acquisition and computational times, or the need of special fluorophores or chemical environments. Here, we propose a novel statistical super-resolution technique of wide-field fluorescence microscopy called MUltiple SIgnal Classification ALgorithm (MUSICAL) which has several advantages over SMLM techniques. MUSICAL provides resolution down to at …
Interferometric scattering microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimen's size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long infrared wavelengths. Here, we describe a bond…
We introduce the concept of saturated absorption competition (SAC) microscopy as a means of providing sub-diffraction spatial resolution in fluorescence imaging. Unlike the post-competition process between stimulated and spontaneous emission that is used in stimulated emission depletion (STED) microscopy, SAC microscopy breaks the diffraction limit by emphasizing a pre-competition process that occurs in the fluorescence absorption stage in a manner that shares similarities with ground-state depletion (GSD) microscopy. Moreover, unlike both STED and GSD microscopy, SAC microscopy offers a reduc…
