Abstract:Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely c… Show more
“…However, few examples are present in the literature that have reported how to combine optical microscopy techniques on the same instrument to perform variable-resolution imaging. Examples of correlative optical microscopies were focused on the combination of different modalities provided by nonlinear microscopy (two-photon excitation, second harmonic generation, spectral imaging) [29][30][31][32].…”
The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ. However, its use is limited to restricted regions, and consequently few cells, and frequently no more than one or two parameters. Correlative microscopy, obtained by the fusion of different optical technologies, can consequently surpass this barrier by merging results from different spatial scales. We discuss here the use of an acquisition and analysis correlative microscopy pipeline to obtain high statistical sampling, high content, and maximum spatial resolution by combining widefield, confocal, and molecular localization microscopy.
“…However, few examples are present in the literature that have reported how to combine optical microscopy techniques on the same instrument to perform variable-resolution imaging. Examples of correlative optical microscopies were focused on the combination of different modalities provided by nonlinear microscopy (two-photon excitation, second harmonic generation, spectral imaging) [29][30][31][32].…”
The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ. However, its use is limited to restricted regions, and consequently few cells, and frequently no more than one or two parameters. Correlative microscopy, obtained by the fusion of different optical technologies, can consequently surpass this barrier by merging results from different spatial scales. We discuss here the use of an acquisition and analysis correlative microscopy pipeline to obtain high statistical sampling, high content, and maximum spatial resolution by combining widefield, confocal, and molecular localization microscopy.
This publisher’s note addresses the use of an acronym in [Biomed. Opt. Express 12, 5452 (2021)10.1364/BOE.430677] that infringes on a registered trademark.
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