Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation.
The combination of two-photon in vivo imaging and genetic labeling of specific cell types in the mouse brain is a powerful method to refine our understanding of brain circuitry and to dissect the contribution of specific neural classes to cortical function. The synthetic calcium indicators are the b...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2012-10-01
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| Series: | Frontiers in Molecular Neuroscience |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fnmol.2012.00096/full |
| _version_ | 1811274599452639232 |
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| author | Marco eBrondi Marco eBrondi Sebastian eSulis Sato Sebastian eSulis Sato Sebastian eSulis Sato Luigi Federico eRossi Silvia eFerrara Gian Michele eRatto Gian Michele eRatto |
| author_facet | Marco eBrondi Marco eBrondi Sebastian eSulis Sato Sebastian eSulis Sato Sebastian eSulis Sato Luigi Federico eRossi Silvia eFerrara Gian Michele eRatto Gian Michele eRatto |
| author_sort | Marco eBrondi |
| collection | DOAJ |
| description | The combination of two-photon in vivo imaging and genetic labeling of specific cell types in the mouse brain is a powerful method to refine our understanding of brain circuitry and to dissect the contribution of specific neural classes to cortical function. The synthetic calcium indicators are the best fluorescent reporters for cellular activity that are presently available but their spectral proprieties are often overlapped with those of the fluorescent proteins used for genetic labeling. Such is the case of Oregon Green BAPTA1 and EGFP, the most widely used fluorophores for targeted two photon imaging. The emission spectra of these molecules are virtually identical, precluding their separation by narrow band filters at the detector side. However, even if their one-photon excitation spectra are very similar, their two-photon excitation spectra differ significantly: here we show how it is possible to exploit this difference to separate the relative contributions of EGFP and Oregon Green to the total fluorescence signal. This approach addresses two different issues: the unbiased detection of cells expressing EGFP in a cortical volume injected with Oregon Green, and the computation of the Ca2+ insensitive fluorescence background. The latter data is essential for the quantitative comparison of the relative changes in Ca2+ concentration between different cells, containing variable concentrations of EGFP. This strategy can be easily extended to any couple of fluorophores provided that have a different two-photon excitation spectra. |
| first_indexed | 2024-04-12T23:22:03Z |
| format | Article |
| id | doaj.art-d06509d1397b48869c227a2499997f0a |
| institution | Directory Open Access Journal |
| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-04-12T23:22:03Z |
| publishDate | 2012-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-d06509d1397b48869c227a2499997f0a2022-12-22T03:12:30ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992012-10-01510.3389/fnmol.2012.0009631859Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation.Marco eBrondi0Marco eBrondi1Sebastian eSulis Sato2Sebastian eSulis Sato3Sebastian eSulis Sato4Luigi Federico eRossi5Silvia eFerrara6Gian Michele eRatto7Gian Michele eRatto8National Research CuncilNational Research CouncilNational Research CuncilScuola Normale SuperioreIITScuola Normale SuperioreNational Research CuncilNational Research CuncilScuola Normale SuperioreThe combination of two-photon in vivo imaging and genetic labeling of specific cell types in the mouse brain is a powerful method to refine our understanding of brain circuitry and to dissect the contribution of specific neural classes to cortical function. The synthetic calcium indicators are the best fluorescent reporters for cellular activity that are presently available but their spectral proprieties are often overlapped with those of the fluorescent proteins used for genetic labeling. Such is the case of Oregon Green BAPTA1 and EGFP, the most widely used fluorophores for targeted two photon imaging. The emission spectra of these molecules are virtually identical, precluding their separation by narrow band filters at the detector side. However, even if their one-photon excitation spectra are very similar, their two-photon excitation spectra differ significantly: here we show how it is possible to exploit this difference to separate the relative contributions of EGFP and Oregon Green to the total fluorescence signal. This approach addresses two different issues: the unbiased detection of cells expressing EGFP in a cortical volume injected with Oregon Green, and the computation of the Ca2+ insensitive fluorescence background. The latter data is essential for the quantitative comparison of the relative changes in Ca2+ concentration between different cells, containing variable concentrations of EGFP. This strategy can be easily extended to any couple of fluorophores provided that have a different two-photon excitation spectra.http://journal.frontiersin.org/Journal/10.3389/fnmol.2012.00096/fullspectroscopyEGFPOregon Green BAPTA 1In vivo targeted two photon microscopyscatteringin vivo functional imaging |
| spellingShingle | Marco eBrondi Marco eBrondi Sebastian eSulis Sato Sebastian eSulis Sato Sebastian eSulis Sato Luigi Federico eRossi Silvia eFerrara Gian Michele eRatto Gian Michele eRatto Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation. Frontiers in Molecular Neuroscience spectroscopy EGFP Oregon Green BAPTA 1 In vivo targeted two photon microscopy scattering in vivo functional imaging |
| title | Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation. |
| title_full | Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation. |
| title_fullStr | Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation. |
| title_full_unstemmed | Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation. |
| title_short | Finding a needle in a haystack: identification of EGFP tagged neurons during Calcium imaging by means of two-photon spectral separation. |
| title_sort | finding a needle in a haystack identification of egfp tagged neurons during calcium imaging by means of two photon spectral separation |
| topic | spectroscopy EGFP Oregon Green BAPTA 1 In vivo targeted two photon microscopy scattering in vivo functional imaging |
| url | http://journal.frontiersin.org/Journal/10.3389/fnmol.2012.00096/full |
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