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Protein‐specific, multi‐color and 3D STED imaging in cells with DNA‐labeled antibodies
摘要： Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed with exchangeable labels, which transiently bind to and off a target and thereby replenish destroyed labels by intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.
关键词： multicolor imaging,DNA-PAINT,STED microscopy,fluorescence,fluorescent probes
更新于2025-09-19 17:13:59
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Protein-specific, multi-color and 3D STED imaging in cells with DNA-labeled antibodies
摘要： Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed with exchangeable labels, which transiently bind to and off a target and thereby replenish destroyed labels by intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.
关键词： multicolor imaging,STED microscopy,fluorescent probes,DNA-PAINT,fluorescence
更新于2025-09-11 14:15:04
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Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT
摘要： Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.
关键词： STORM,frequency multiplexing,multicolor imaging,DNA-PAINT,superresolution microscopy
更新于2025-09-09 09:28:46
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Perfluorocarbon‐Based <sup>19</sup> F?MRI Nanoprobes for In?Vivo Multicolor Imaging
摘要： In vivo multicolor imaging is important for monitoring multiple biomolecular or cellular processes in biology. 19F magnetic resonance imaging (MRI) is an emerging in vivo imaging technique because it can non-invasively visualize 19F nuclei without endogenous background signals. Therefore, 19F MRI probes capable of multicolor imaging are in high demand. Herein, we report five types of perfluorocarbon-encapsulated silica nanoparticles that show 19F NMR peaks with different chemical shifts. Three of the nanoprobes, which show spectrally distinct 19F NMR peaks with sufficient sensitivity, were selected for in vivo multicolor 19F MRI. The nanoprobes exhibited 19F MRI signals with three colors in a living mouse. Our in vivo multicolor system could be utilized for evaluating the effect of surface functional groups on the hepatic uptake in a mouse. This novel multicolor imaging technology will be a practical tool for elucidating in vivo biomolecular networks by 19F MRI.
关键词： multicolor imaging,fluorine,magnetic resonance imaging,nanoparticles,imaging agents
更新于2025-09-04 15:30:14
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Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores
摘要： We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy.
关键词： PAINT,fluorogenic labels,multicolor imaging,live-cell STED microscopy,volumetric imaging,exchange-based STED microscopy
更新于2025-09-04 15:30:14
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A machine learning approach for online automated optimization of super-resolution optical microscopy
摘要： Traditional approaches for finding well-performing parameterizations of complex imaging systems, such as super-resolution microscopes rely on an extensive exploration phase over the illumination and acquisition settings, prior to the imaging task. This strategy suffers from several issues: it requires a large amount of parameter configurations to be evaluated, it leads to discrepancies between well-performing parameters in the exploration phase and imaging task, and it results in a waste of time and resources given that optimization and final imaging tasks are conducted separately. Here we show that a fully automated, machine learning-based system can conduct imaging parameter optimization toward a trade-off between several objectives, simultaneously to the imaging task. Its potential is highlighted on various imaging tasks, such as live-cell and multicolor imaging and multimodal optimization. This online optimization routine can be integrated to various imaging systems to increase accessibility, optimize performance and improve overall imaging quality.
关键词： machine learning,multicolor imaging,online automated optimization,live-cell imaging,super-resolution optical microscopy,multimodal optimization
更新于2025-09-04 15:30:14
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Comparison of MultiColor fundus imaging and colour fundus photography in the evaluation of epiretinal membrane
摘要： Purpose: To compare MultiColor fundus imaging (MC) and colour fundus photography (CFP) for the evaluation of epiretinal membrane (ERM). Methods: In this retrospective study, 192 eyes (181 patients) that underwent vitrectomy for ERM were imaged using Spectralis optical coherence tomography (for MC) and AFC-210 digital camera (for CFP) 1 week before vitrectomy. Two investigators independently determined the rate of delineability and ERM area for each modality. Results: The intergrader agreement rates for delineability were very high for both image modalities. The rate of delineability of ERM (%) was higher for MC than for CFP, for both investigators [70.8% versus 52.6% and 73.4% versus 53.6% (both p –)]. Epiretinal membrane (ERM) area measurement showed high agreement between investigators for MC (p = 0.466) but di?ered signi?cantly between the investigators for CFP (p –). The ERM area determined on MC was signi?cantly wider than that on CFP for both investigators (p – for investigator 1 and p – for investigator 2). Conclusion: The ERM area is more clearly detectable and widely demarcated in MC than in CFP images. MultiColor imaging (MC) may be more sensitive and accurate for early detection of ERM and ERM area measurement.
关键词： MultiColor imaging,spectral-domain optical coherent tomography,epiretinal membrane,colour fundus photography
更新于2025-09-04 15:30:14