ترغب بنشر مسار تعليمي؟ اضغط هنا

Fluorescence-Enhanced Mid-Infrared Photothermal Microscopy

236   0   0.0 ( 0 )
 نشر من قبل Yi Zhang
 تاريخ النشر 2021
  مجال البحث فيزياء علم الأحياء
والبحث باللغة English




اسأل ChatGPT حول البحث

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 in which thermo-sensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of speckles, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed through fingerprinting a single bacterium. Finally, the photobleaching issue is successfully addressed through the development of a wide-field fluorescence-enhanced mid-infrared photothermal microscope which allows video rate bond-selective imaging of biological specimens.



قيم البحث

اقرأ أيضاً

We demonstrate instrumentation and methods to enable fluorescence-detected photothermal infrared (F-PTIR) microscopy, then demonstrate the utility of F-PTIR to characterize the composition within phase-separated domains of model amorphous solid dispe rsions (ASDs) induced by water sorption. In F-PTIR, temperature-dependent changes in fluorescence quantum efficiency are shown to sensitively report on highly localized absorption of mid-infrared radiation. The spatial resolution with which infrared spectroscopy can be performed is dictated by fluorescence microscopy, rather than the infrared wavelength. Following proof of concept F-PTIR demonstration on model systems of polyethylene glycol (PEG) and silica gel, F-PTIR enabled the characterization of chemical composition within inhomogeneous ritonavir / polyvinylpyrrolidone-vinyl acetate (PVPVA) amorphous dispersions. Phase separation is implicated in the observation of critical behaviors in ASD dissolution kinetics, with the results of F-PTIR supporting the formation of phase-separated drug-rich domains upon water absorption in spin-cast films.
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 imagin g 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 chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over three orders of magnitude background suppression by quasi-darkfield illumination in epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 um x 70 um. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500~nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria.
Owing to its capacity for unique (bio)-chemical specificity, microscopy withmid-IR illumination holds tremendous promise for a wide range of biomedical and industrial applications. The primary limitation, however, remains detection; with current mid- IR detection technology often marrying inferior technical capabilities with prohibitive costs. This has lead to approaches that shift detection towavelengths into the visible regime, where vastly superior silicon-based cameratechnology is available. Here, we experimentally show how nonlinear interferometry with entangled light can provide a powerful tool for mid-IR microscopy, while only requiring near-infrared detection with a standard CMOS camera. In this proof-of-principle implementation, we demonstrate intensity imaging overa broad wavelength range covering 3.4-4.3um and demonstrate a spatial resolution of 35um for images containing 650 resolved elements. Moreover, we demonstrate our technique is fit for purpose, acquiring microscopic images of biological tissue samples in the mid-IR. These results open a new perspective for potential relevance of quantum imaging techniques in the life sciences.
Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for s- and p- polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient.
We theoretically investigate the plasmonic heating of graphene-based systems under the mid-infrared laser irradiation, where periodic arrays of graphene plasmonic resonators are placed on dielectric thin films. Optical resonances are sensitive to str uctural parameters and the number of graphene layers. Under mid-infrared laser irradiation, the steady-state temperature gradients are calculated. We find that graphene plasmons significantly enhance the confinement of electromagnetic fields in the system and lead to a large temperature rise compared to the case without graphene. The correlations between temperature change and the optical power, laser spot, and thermal conductivity of dielectric layer in these systems are discussed. Our numerical results are in accordance with experiments.
التعليقات
جاري جلب التعليقات جاري جلب التعليقات
سجل دخول لتتمكن من متابعة معايير البحث التي قمت باختيارها
mircosoft-partner

هل ترغب بارسال اشعارات عن اخر التحديثات في شمرا-اكاديميا