Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors

Abstract Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural c...

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Autores principales:	Tuan A. Nguyen, Henry L. Puhl, An K. Pham, Steven S. Vogel
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2325b19a934c460e9d076cd0bad58623
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spelling	oai:doaj.org-article:2325b19a934c460e9d076cd0bad586232021-12-02T15:08:45ZAuto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors10.1038/s41598-018-25689-x2045-2322https://doaj.org/article/2325b19a934c460e9d076cd0bad586232018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-25689-xhttps://doaj.org/toc/2045-2322Abstract Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural change triggered by alterations in the bio-parameter is monitored as a change in either fluorescent intensity, or lifetime. Potentially, other photo-physical properties of fluorophores, such as fluorescence anisotropy, molecular brightness, concentration, and lateral and/or rotational diffusion could also be used. Furthermore, while it is likely that multiple photo-physical attributes of a biosensor might be altered as a function of the bio-parameter, standard measurements monitor only a single photo-physical trait. This limits how biosensors are designed, as well as the accuracy and interpretation of biosensor measurements. Here we describe the design and construction of an automated multimodal-microscope. This system can autonomously analyze 96 samples in a micro-titer dish and for each sample simultaneously measure intensity (photon count), fluorescence lifetime, time-resolved anisotropy, molecular brightness, lateral diffusion time, and concentration. We characterize the accuracy and precision of this instrument, and then demonstrate its utility by characterizing three types of genetically encoded calcium sensors as well as a negative control.Tuan A. NguyenHenry L. PuhlAn K. PhamSteven S. VogelNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-17 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tuan A. Nguyen Henry L. Puhl An K. Pham Steven S. Vogel Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
description	Abstract Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural change triggered by alterations in the bio-parameter is monitored as a change in either fluorescent intensity, or lifetime. Potentially, other photo-physical properties of fluorophores, such as fluorescence anisotropy, molecular brightness, concentration, and lateral and/or rotational diffusion could also be used. Furthermore, while it is likely that multiple photo-physical attributes of a biosensor might be altered as a function of the bio-parameter, standard measurements monitor only a single photo-physical trait. This limits how biosensors are designed, as well as the accuracy and interpretation of biosensor measurements. Here we describe the design and construction of an automated multimodal-microscope. This system can autonomously analyze 96 samples in a micro-titer dish and for each sample simultaneously measure intensity (photon count), fluorescence lifetime, time-resolved anisotropy, molecular brightness, lateral diffusion time, and concentration. We characterize the accuracy and precision of this instrument, and then demonstrate its utility by characterizing three types of genetically encoded calcium sensors as well as a negative control.
format	article
author	Tuan A. Nguyen Henry L. Puhl An K. Pham Steven S. Vogel
author_facet	Tuan A. Nguyen Henry L. Puhl An K. Pham Steven S. Vogel
author_sort	Tuan A. Nguyen
title	Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
title_short	Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
title_full	Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
title_fullStr	Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
title_full_unstemmed	Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors
title_sort	auto-fpfa: an automated microscope for characterizing genetically encoded biosensors
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/2325b19a934c460e9d076cd0bad58623
work_keys_str_mv	AT tuananguyen autofpfaanautomatedmicroscopeforcharacterizinggeneticallyencodedbiosensors AT henrylpuhl autofpfaanautomatedmicroscopeforcharacterizinggeneticallyencodedbiosensors AT ankpham autofpfaanautomatedmicroscopeforcharacterizinggeneticallyencodedbiosensors AT stevensvogel autofpfaanautomatedmicroscopeforcharacterizinggeneticallyencodedbiosensors
_version_	1718387987934347264

Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors

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