ABSTRACT
Fluorescence microscopy in the second near-infrared optical window (NIR-II, 1000-1350 nm) has become a technique of choice for non-invasive in vivo imaging. The deep penetration of NIR light in living tissue, as well as negligible tissue autofluorescence within this optical range, offers increased resolution and contrast with even greater penetration depths. Here, we present a custom-built spinning-disc confocal laser microscope (SDCLM) that is specific to imaging in the NIR-II. The SDCLM achieves a lateral resolution of 0.5 ± 0.1 µm and an axial resolution of 0.6 ± 0.1 µm, showing a ~17% and ~45% enhancement in lateral and axial resolution, respectively, compared to the corresponding wide-field configuration. We furthermore showcase several applications that demonstrate the use of the SDCLM for in situ, spatiotemporal tracking of NIR particles and bioanalytes within both synthetic and biological systems.
ABSTRACT
In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength. Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.
Subject(s)
Embryonic Stem Cells/cytology , Microscopy, Fluorescence, Multiphoton/methods , Nanoparticles/chemistry , Regeneration/physiology , Cell Communication , Cell Differentiation/physiology , Humans , Imaging, Three-Dimensional/methods , Myocytes, Cardiac/cytology , Niobium/chemistry , Oxides/chemistry , Potassium/chemistry , Regenerative MedicineABSTRACT
Potassium niobate nonlinear nanoparticles are used for the first time to monitor the evolution of embryonic stem cells (ESC) by second harmonic microscopy. These particles feature the complete absence of photo-bleaching and unlimited excitation wavelength flexibility. The potential of this approach is made evident for tissue-regeneration studies and applications, by capturing a high-speed movie of ESC-derived cardiomyocytes autonomously beating within a cluster. Time-resolved data are analyzed to retrieve 3D information of the contraction pattern at the cellular level.
Subject(s)
Embryonic Stem Cells/cytology , Myocardium/cytology , Nanotechnology , Animals , MiceABSTRACT
We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.
Subject(s)
Image Enhancement/instrumentation , Lenses , Microscopy/instrumentation , Proteins/ultrastructure , Equipment Design , Equipment Failure Analysis , HeLa Cells , Humans , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Nonlinear optical nanocrystals have been recently introduced as a promising alternative to fluorescent probes for multiphoton microscopy. We present for the first time a complete survey of the properties of five nanomaterials (KNbO(3), LiNbO(3), BaTiO(3), KTP, and ZnO), describing their preparation and stabilization and providing quantitative estimations of their nonlinear optical response. In the light of their prospective use as biological and clinical markers, we assess their biocompatibility on human healthy and cancerous cell lines. Finally, we demonstrate the great potential for cell imaging of these inherently nonlinear probes in terms of optical contrast, wavelength flexibility, and signal photostability.
