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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
摘要： 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.
关键词： DNA-PAINT,microfluidics,super-resolution microscopy,fluorescent proteins,molecular localization,multi-color imaging,multiplexing,single domain antibodies (sdAb),linkage error,nanobodies
更新于2025-09-19 17:15:36
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Error Assessment Model for the Inverse Kinematics Problem for Stewart Parallel Mechanisms for Accurate Aerospace Optical Linkage
摘要： In this work we develop a mathematical model to estimate the error for inverse kinematics problem for Gough-Stewart parallel mechanisms. We propose the estimation error method to include manufacture, assembly, backlash, and sensing errors. We provide the error transmission matrices for the length of each leg of the hexapod, which permits evaluation of the accuracy error in the position of each one, given a desired position and orientation of the mobile platform. We also present numerical modelling in order to estimate the accuracy of the methodology herein proposed, for specific attitude operations corresponding to performing a successful ground-LEO nanosatellite optical link. In such a case, we were able to provide the required tolerances for the actuators in order to guarantee an orientation precision requirement of the order of milliradians.
关键词： parallel mechanisms,Gough-Stewart platform,aerospace optical linkage,error assessment,inverse kinematics
更新于2025-09-04 15:30:14