Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT
| العنوان: | Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT |
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| المؤلفون: | Pablo Aurelio Gómez-García, Maria F. Garcia-Parajo, Melike Lakadamyali, E.T. Garbacik, Jason J. Otterstrom |
| المصدر: | Proceedings of the National Academy of Sciences |
| مصطلحات موضوعية: | 0301 basic medicine, Fluorophore, Color, Field of view, 02 engineering and technology, Signal, Multiplexing, Fluorescence, Frequency-division multiplexing, 03 medical and health sciences, chemistry.chemical_compound, Optics, Imaging, Three-Dimensional, Microscopy, Image Processing, Computer-Assisted, Humans, Image resolution, Fluorescent Dyes, Physics, Multidisciplinary, business.industry, DNA, Biological Sciences, 021001 nanoscience & nanotechnology, Photobleaching, Single Molecule Imaging, 030104 developmental biology, chemistry, Microscopy, Fluorescence, 0210 nano-technology, business |
| الوصف: | 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. |
| اللغة: | English |
| تدمد: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1804725115 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::78655a9a4714b004d9d44c6d54041dee |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....78655a9a4714b004d9d44c6d54041dee |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10916490 00278424 |
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| DOI: | 10.1073/pnas.1804725115 |