| dc.contributor.advisor | Jakobs, Stefan Prof. Dr. | |
| dc.contributor.author | Habenstein, Florian | |
| dc.date.accessioned | 2020-09-22T14:15:59Z | |
| dc.date.available | 2021-09-01T00:50:03Z | |
| dc.date.issued | 2020-09-22 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-148A-9 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8207 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 571.4 | de |
| dc.title | Engineering of a NIR fluorescent protein for live-cell nanoscopy | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Schwarzer, Dirk Prof. Dr. | |
| dc.date.examination | 2020-09-03 | |
| dc.description.abstracteng | Since its first use as a genetically encoded fluorescent marker in 1994, GFP and its
homologues have fundamentally revolutionized live-cell fluorescence imaging and
became an essential tool for biomedical research. Many fluorescent proteins have
been engineered from GFP-like proteins with excitation and emission covering the
entirety of the visible spectrum. However, despite substantial efforts it was not possible
to reach the near infrared (NIR) spectral region beyond 650nm with a GFP
derived fluorescent protein.
The NIR spectral region between 650 to 900nm is especially suited for live-cell
deep-tissue fluorescence imaging due to reduced autofluorescence, light scattering
and absorption of tissue in this spectral region. Additionally, photo toxicity of NIR
light is reduced compared to UV/vis light.
Recently, utilization of engineered bacterial phytochrome variants opened the
NIR spectral region for fluorescence microscopy with genetically encoded fluorescent
markers. Bacterial phytochromes gain their unique optical properties by incorporating
the external chromophore and heme degradation product biliverdin which
is ubiquitous in mammalian cells. A variety of bacterial phytochrome based fluorescent
proteins has been successfully applied in various fluorescence microscopy
techniques. However, to date all engineered bacterial phytochrome variants absorbing
and emitting beyond 650nm suffer from a short fluorescence lifetime and a low
fluorescence quantum yield, limiting their potential for fluorescence microscopy.
In this work, an automated fluorescence lifetime screening microscope was built
and applied to increase the fluorescence lifetime and quantum yield of the engineered
fluorescent protein miRFP703 via directed evolution. The final protein variant
(V410) had a fluorescence lifetime of 1.1 ns and a fluorescence quantum yield of
21%. With this it is the brightest NIR fluorescent protein described to date. V410 exhibited
a good pH stability and a high extinction coefficient. In live-cell fluorescence
microscopy, V410 performed well as fusion tag for various cellular structures. With
STED microscopy, resolutions well beyond 80nm down to 40nm were measured on
endogenously tagged vimentin filaments. In consecutive confocal and STED recordings,
with 1000 and 100 consecutive frames, respectively, V410 demonstrated superior
photo stability. Utilizing the fluorescence lifetime difference between V410 and
the template miRFP703 of approximately 400 ps, two-color fluorescence lifetime confocal
and STED imaging was performed entirely in the NIR spectral region with a
single excitation beam at 660nm and a STED beam at 820 nm. | de |
| dc.contributor.coReferee | Hell, Stefan Prof. Dr. | |
| dc.subject.eng | protein-engineering | de |
| dc.subject.eng | near-infrared | de |
| dc.subject.eng | nanoscopy | de |
| dc.subject.eng | STED | de |
| dc.subject.eng | fluorescence lifetime | de |
| dc.subject.eng | fluorescence quantum yield | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-148A-9-6 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2021-09-01 | |
| dc.identifier.ppn | 1733581758 | |