Multiphoton FRET Microscopy for Protein Localization in Tissue

James D. Mills; James R. Stone; David O. Okonkwo; Ammasi Periasamy; Gregory A. Helm

doi:10.1016/B978-019517720-6.50015-8

Multiphoton FRET Microscopy for Protein Localization in Tissue

James D. Mills^*, James R. Stone, David O. Okonkwo, Ammasi Periasamy, Gregory A. Helm

^*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

Abstract

This chapter discusses the multiphoton Förster resonance energy transfer (mp-FRET) microscopy and its use in localizing the proteins in tissue following traumatic brain injury (TBI).To localize proteins, two fluorophore molecules need to be attached to each protein molecule under investigation. One should have a good expression level of proteins with the selected fluorophores and use the best optics, a high quantum efficiency charge-coupled device (CCD) camera (for real-time 2p-FRET) or photomultiplier tubes (PMT) to acquire the FRET images. The efficiency of the FRET could also be improved by optimizing the concentration of the fluorophore used for protein labeling. The z-plane focus motor of the microscope to focus on the distal surface of the specimen is used to determine whether images acquired with the mp-FRET microscopy in thicker tissue specimens are appropriate for the FRET image analysis.

Original language	English
Title of host publication	Molecular Imaging
Subtitle of host publication	FRET Microscopy and Spectroscopy
Publisher	Elsevier
Pages	112-125
Number of pages	14
ISBN (Print)	9780195177206
DOIs	https://doi.org/10.1016/B978-019517720-6.50015-8
State	Published - 1 Aug 2005
Externally published	Yes

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Cite this

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title = "Multiphoton FRET Microscopy for Protein Localization in Tissue",

abstract = "This chapter discusses the multiphoton F{\"o}rster resonance energy transfer (mp-FRET) microscopy and its use in localizing the proteins in tissue following traumatic brain injury (TBI).To localize proteins, two fluorophore molecules need to be attached to each protein molecule under investigation. One should have a good expression level of proteins with the selected fluorophores and use the best optics, a high quantum efficiency charge-coupled device (CCD) camera (for real-time 2p-FRET) or photomultiplier tubes (PMT) to acquire the FRET images. The efficiency of the FRET could also be improved by optimizing the concentration of the fluorophore used for protein labeling. The z-plane focus motor of the microscope to focus on the distal surface of the specimen is used to determine whether images acquired with the mp-FRET microscopy in thicker tissue specimens are appropriate for the FRET image analysis.",

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AU - Mills, James D.

AU - Stone, James R.

AU - Okonkwo, David O.

AU - Periasamy, Ammasi

AU - Helm, Gregory A.

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AB - This chapter discusses the multiphoton Förster resonance energy transfer (mp-FRET) microscopy and its use in localizing the proteins in tissue following traumatic brain injury (TBI).To localize proteins, two fluorophore molecules need to be attached to each protein molecule under investigation. One should have a good expression level of proteins with the selected fluorophores and use the best optics, a high quantum efficiency charge-coupled device (CCD) camera (for real-time 2p-FRET) or photomultiplier tubes (PMT) to acquire the FRET images. The efficiency of the FRET could also be improved by optimizing the concentration of the fluorophore used for protein labeling. The z-plane focus motor of the microscope to focus on the distal surface of the specimen is used to determine whether images acquired with the mp-FRET microscopy in thicker tissue specimens are appropriate for the FRET image analysis.

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ER -

Multiphoton FRET Microscopy for Protein Localization in Tissue

Abstract

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