Point spread function (PSF) engineering by an emitter's response can code higher-spatial-frequency information of an image for microscopy to achieve super-resolution. However, complexed excitation optics or repetitive scans are needed, which explains the issues of low speed, poor stability, and operational complexity associated with the current laser scanning microscopy approaches. Here, the diverse emission responses of upconversion nanoparticles (UCNPs) are reported for super-resolution nanoscopy to improve the imaging quality and speed. The method only needs a doughnut-shaped scanning excitation beam at an appropriate power density. By collecting the four-photon emission of single UCNPs, the high-frequency information of a super-resolution image can be resolved through the doughnut-emission PSF. Meanwhile, the two-photon state of the same nanoparticle is oversaturated, so that the complementary lower-frequency information of the super-resolution image can be simultaneously collected by the Gaussian-like emission PSF. This leads to a method of Fourier-domain heterochromatic fusion, which allows the extended capability of the engineered PSFs to cover both low- and high-frequency information to yield optimized image quality. This approach achieves a spatial resolution of 40 nm, 1/24th of the excitation wavelength. This work suggests a new scope for developing nonlinear multi-color emitting probes in super-resolution nanoscopy.
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http://dx.doi.org/10.1002/adma.202008847 | DOI Listing |
Adv Mater
January 2025
Third Institute of Physics - Biophysics, Georg August University, Friedrich-Hund Platz 1, 37077, Göttingen, Germany.
In the burgeoning field of super-resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the 3D domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of 3D subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities.
View Article and Find Full Text PDFNat Methods
January 2025
Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA.
Super-resolution imaging of cell metabolism is hindered by the incompatibility of small metabolites with fluorescent dyes and the limited resolution of imaging mass spectrometry. We present ultrasensitive reweighted visible stimulated Raman scattering (URV-SRS), a label-free vibrational imaging technique for multiplexed nanoscopy of intracellular metabolites. We developed a visible SRS microscope with extensive pulse chirping to improve the detection limit to ~4,000 molecules and introduced a self-supervised multi-agent denoiser to suppress non-independent noise in SRS by over 7.
View Article and Find Full Text PDFAnal Chem
January 2025
Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, California 91010, United States.
Extracellular vesicles (EVs), membrane-encapsulated nanoparticles shed from all cells, are tightly involved in critical cellular functions. Moreover, EVs have recently emerged as exciting therapeutic modalities, delivery vectors, and biomarker sources. However, EVs are difficult to characterize, because they are typically small and heterogeneous in size, origin, and molecular content.
View Article and Find Full Text PDFCell Commun Signal
January 2025
Department of Immunology, University of Connecticut School of Medicine, Connecticut, Farmington, 06030, USA.
Background: Neutrophils are the most abundant leukocytes in human blood, and their recruitment is essential for innate immunity and inflammatory responses. The initial and critical step of neutrophil recruitment is their adhesion to vascular endothelium, which depends on G protein-coupled receptor (GPCR) triggered integrin inside-out signaling that induces β2 integrin activation and clustering on neutrophils. Kindlin-3 and talin-1 are essential regulators for the inside-out signaling induced β2 integrin activation.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
December 2024
Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen 37077, Germany.
Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue sample, superresolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments.
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