Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking
Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/14/7623 |
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| author | Filippo Begarani Francesca D’Autilia Gianmarco Ferri Luca Pesce Fabio Azzarello Valentina De Lorenzi William Durso Ambra Del Grosso Marco Cecchini Francesco Cardarelli |
| author_facet | Filippo Begarani Francesca D’Autilia Gianmarco Ferri Luca Pesce Fabio Azzarello Valentina De Lorenzi William Durso Ambra Del Grosso Marco Cecchini Francesco Cardarelli |
| author_sort | Filippo Begarani |
| collection | DOAJ |
| description | Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale. |
| first_indexed | 2024-03-09T03:21:33Z |
| format | Article |
| id | doaj.art-76c78ec6e00c4ae3bd7c4cfa1d662512 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T03:21:33Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-76c78ec6e00c4ae3bd7c4cfa1d6625122023-12-03T15:09:14ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-07-012314762310.3390/ijms23147623Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital TrackingFilippo Begarani0Francesca D’Autilia1Gianmarco Ferri2Luca Pesce3Fabio Azzarello4Valentina De Lorenzi5William Durso6Ambra Del Grosso7Marco Cecchini8Francesco Cardarelli9NEST-Scuola Normale Superiore, 56127 Pisa, ItalyCenter for Nanotechnology Innovation@NEST (CNI@NEST), 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyNEST, Istituto Nanoscienze-CNR, Piazza S. Silvestro, 12, 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyNEST-Scuola Normale Superiore, 56127 Pisa, ItalyHere we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.https://www.mdpi.com/1422-0067/23/14/7623diffusioncorrelation spectroscopyfluorescenceliving cellssubcellular scalenanoscale |
| spellingShingle | Filippo Begarani Francesca D’Autilia Gianmarco Ferri Luca Pesce Fabio Azzarello Valentina De Lorenzi William Durso Ambra Del Grosso Marco Cecchini Francesco Cardarelli Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking International Journal of Molecular Sciences diffusion correlation spectroscopy fluorescence living cells subcellular scale nanoscale |
| title | Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking |
| title_full | Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking |
| title_fullStr | Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking |
| title_full_unstemmed | Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking |
| title_short | Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking |
| title_sort | measuring molecular diffusion in dynamic subcellular nanostructures by fast raster image correlation spectroscopy and 3d orbital tracking |
| topic | diffusion correlation spectroscopy fluorescence living cells subcellular scale nanoscale |
| url | https://www.mdpi.com/1422-0067/23/14/7623 |
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