TY - JOUR
T1 - Nanobody detection of standard fluorescent proteins enables multi-target DNA-PAINT with high resolution and minimal displacement errors
AU - Sograte-Idrissi, Shama
AU - Oleksiievets, Nazar
AU - Isbaner, Sebastian
AU - Eggert-Martinez, Mariana
AU - Enderlein, Jörg
AU - Tsukanov, Roman
AU - Opazo, Felipe
N1 - Publisher Copyright:
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2019/1/1
Y1 - 2019/1/1
N2 - DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
AB - DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
KW - DNA-PAINT
KW - Fluorescent proteins
KW - Microfluidics
KW - Molecular localization
KW - Multi-color imaging
KW - Nanobodies
KW - Single domain antibodies (sdAb)
KW - Super-resolution microscopy
KW - linkage error
KW - multiplexing
UR - http://www.scopus.com/inward/record.url?scp=85071638310&partnerID=8YFLogxK
U2 - 10.3390/cells8010048
DO - 10.3390/cells8010048
M3 - Article
AN - SCOPUS:85071638310
SN - 2073-4409
VL - 8
JO - Cells
JF - Cells
IS - 1
M1 - 48
ER -