Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging
The measurement of Förster resonance energy transfer (FRET) in microscopes can be realized by different imaging modalities. In the present work, reference FRET constructs are developed to allow the comparison of FRET microscopy measurements using intensity, spectral, and lifetime imaging. Compliment...
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| Format: | Article |
| Language: | en_US |
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SPIE
2014
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| Online Access: | http://hdl.handle.net/1721.1/87648 https://orcid.org/0000-0003-4698-6488 |
| _version_ | 1826194440131182592 |
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| author | Pelet, Serge Previte, Michael J. R. So, Peter T. C. |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Pelet, Serge Previte, Michael J. R. So, Peter T. C. |
| author_sort | Pelet, Serge |
| collection | MIT |
| description | The measurement of Förster resonance energy transfer (FRET) in microscopes can be realized by different imaging modalities. In the present work, reference FRET constructs are developed to allow the comparison of FRET microscopy measurements using intensity, spectral, and lifetime imaging. Complimentary DNA strands are respectively labeled with Oregon Green 488 (OG488) or tetramethylrhodamine (TMR). The OG488 dye is fixed at the 5′ end of one strand, and the TMR label position is allowed to vary along the complimentary strand. Since OG488 and TMR are FRET pairs, the FRET efficiency can be determined theoretically from the distance separating the two dyes of the double-stranded DNA molecules. Microscopic images are formed by imaging microcapillaries containing various mixtures of oligonucleotides labeled with the FRET fluorophore pair, only the donor, or only acceptor. Traditional two-channel intensity measurements are compared with spectrally resolved imaging and fluorescence lifetime imaging by calculating a FRET index. The latter proves to be the best method to quantify FRET efficiency in the image. More importantly, the intensity fraction of molecules undergoing FRET can be quantitatively measured in each pixel of the image. |
| first_indexed | 2024-09-23T09:56:09Z |
| format | Article |
| id | mit-1721.1/87648 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:56:09Z |
| publishDate | 2014 |
| publisher | SPIE |
| record_format | dspace |
| spelling | mit-1721.1/876482022-09-30T17:47:55Z Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging Pelet, Serge Previte, Michael J. R. So, Peter T. C. Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Mechanical Engineering Pelet, Serge Previte, Michael J. R. So, Peter T. C. The measurement of Förster resonance energy transfer (FRET) in microscopes can be realized by different imaging modalities. In the present work, reference FRET constructs are developed to allow the comparison of FRET microscopy measurements using intensity, spectral, and lifetime imaging. Complimentary DNA strands are respectively labeled with Oregon Green 488 (OG488) or tetramethylrhodamine (TMR). The OG488 dye is fixed at the 5′ end of one strand, and the TMR label position is allowed to vary along the complimentary strand. Since OG488 and TMR are FRET pairs, the FRET efficiency can be determined theoretically from the distance separating the two dyes of the double-stranded DNA molecules. Microscopic images are formed by imaging microcapillaries containing various mixtures of oligonucleotides labeled with the FRET fluorophore pair, only the donor, or only acceptor. Traditional two-channel intensity measurements are compared with spectrally resolved imaging and fluorescence lifetime imaging by calculating a FRET index. The latter proves to be the best method to quantify FRET efficiency in the image. More importantly, the intensity fraction of molecules undergoing FRET can be quantitatively measured in each pixel of the image. National Institutes of Health (U.S.) (Grant NIHPOIHL64858) 2014-06-05T15:19:12Z 2014-06-05T15:19:12Z 2006-05 2006-02 Article http://purl.org/eprint/type/JournalArticle 10833668 1560-2281 http://hdl.handle.net/1721.1/87648 Pelet, Serge, Michael J. R. Previte, and Peter T. C. So. “Comparing the Quantification of Forster Resonance Energy Transfer Measurement Accuracies Based on Intensity, Spectral, and Lifetime Imaging.” Journal of Biomedical Optics 11, no. 3 (2006): 034017. © 2006 Society of Photo-Optical Instrumentation Engineers https://orcid.org/0000-0003-4698-6488 en_US http://dx.doi.org/10.1117/1.2203664 Journal of Biomedical Optics Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf SPIE SPIE |
| spellingShingle | Pelet, Serge Previte, Michael J. R. So, Peter T. C. Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging |
| title | Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging |
| title_full | Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging |
| title_fullStr | Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging |
| title_full_unstemmed | Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging |
| title_short | Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging |
| title_sort | comparing the quantification of forster resonance energy transfer measurement accuracies based on intensity spectral and lifetime imaging |
| url | http://hdl.handle.net/1721.1/87648 https://orcid.org/0000-0003-4698-6488 |
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