TY - JOUR
T1 - POLCAM
T2 - instant molecular orientation microscopy for the life sciences
AU - Bruggeman, Ezra
AU - Zhang, Oumeng
AU - Needham, Lisa Maria
AU - Körbel, Markus
AU - Daly, Sam
AU - Cheetham, Matthew
AU - Peters, Ruby
AU - Wu, Tingting
AU - Klymchenko, Andrey S.
AU - Davis, Simon J.
AU - Paluch, Ewa K.
AU - Klenerman, David
AU - Lew, Matthew D.
AU - O’Holleran, Kevin
AU - Lee, Steven F.
N1 - Publisher Copyright:
© The Author(s) 2024.
PY - 2024/10
Y1 - 2024/10
N2 - Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design and developed a fast algorithm based on Stokes parameter estimation that can operate over 1,000-fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.
AB - Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design and developed a fast algorithm based on Stokes parameter estimation that can operate over 1,000-fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.
UR - http://www.scopus.com/inward/record.url?scp=85205821874&partnerID=8YFLogxK
U2 - 10.1038/s41592-024-02382-8
DO - 10.1038/s41592-024-02382-8
M3 - Article
C2 - 39375574
AN - SCOPUS:85205821874
SN - 1548-7091
VL - 21
SP - 1873
EP - 1883
JO - Nature Methods
JF - Nature Methods
IS - 10
ER -