Three-dimensional biplane spectroscopic single-molecule localization microscopy

Ki Hee Song, Yang Zhang, Gaoxiang Wang, Cheng Sun, Hao F Zhang*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Spectroscopic single-molecule localization microscopy (sSMLM) captures the full emission spectra of individual molecules while simultaneously localizing their spatial locations at a precision greatly exceeding the optical diffraction limit. To achieve this, sSMLM uses a dispersive optical component to separate the emitted photons into dedicated spatial and spectral imaging channels for simultaneous acquisition. While adding a cylindrical lens in the spatial imaging channel enabled three-dimensional (3D) imaging in sSMLM, the inherent astigmatism leads to technical hurdles in spectral calibration and nonuniform lateral resolution at different depths. We found that implementing the biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows for more efficient use of the limited photon budget and provides homogeneous lateral resolution compared with the astigmatism-based method using a cylindrical lens. Here we report 3D biplane sSMLM and demonstrate its multi-color 3D imaging capability by imaging microtubules and mitochondria in fixed COS-7 cells immunostained with Alexa Fluor 647 and CF 660C dyes, respectively. We showed a lateral localization precision of 20 nm at an average photon count of 550, a spectral precision of 4 nm at an average photon count of 1250, and an axial localization resolution of 50 nm.

Original languageEnglish (US)
Pages (from-to)709-715
Number of pages7
JournalOptica
Volume6
Issue number6
DOIs
StatePublished - Jun 20 2019

Fingerprint

biplanes
Microscopic examination
microscopy
Imaging techniques
Molecules
Photons
molecules
astigmatism
Lenses
photons
Mitochondria
lenses
mitochondria
Coloring Agents
Dyes
Diffraction
Calibration
budgets
Color
acquisition

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics

Cite this

Song, Ki Hee ; Zhang, Yang ; Wang, Gaoxiang ; Sun, Cheng ; Zhang, Hao F. / Three-dimensional biplane spectroscopic single-molecule localization microscopy. In: Optica. 2019 ; Vol. 6, No. 6. pp. 709-715.
@article{2cec0ef753a14c00af13003527e3b265,
title = "Three-dimensional biplane spectroscopic single-molecule localization microscopy",
abstract = "Spectroscopic single-molecule localization microscopy (sSMLM) captures the full emission spectra of individual molecules while simultaneously localizing their spatial locations at a precision greatly exceeding the optical diffraction limit. To achieve this, sSMLM uses a dispersive optical component to separate the emitted photons into dedicated spatial and spectral imaging channels for simultaneous acquisition. While adding a cylindrical lens in the spatial imaging channel enabled three-dimensional (3D) imaging in sSMLM, the inherent astigmatism leads to technical hurdles in spectral calibration and nonuniform lateral resolution at different depths. We found that implementing the biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows for more efficient use of the limited photon budget and provides homogeneous lateral resolution compared with the astigmatism-based method using a cylindrical lens. Here we report 3D biplane sSMLM and demonstrate its multi-color 3D imaging capability by imaging microtubules and mitochondria in fixed COS-7 cells immunostained with Alexa Fluor 647 and CF 660C dyes, respectively. We showed a lateral localization precision of 20 nm at an average photon count of 550, a spectral precision of 4 nm at an average photon count of 1250, and an axial localization resolution of 50 nm.",
author = "Song, {Ki Hee} and Yang Zhang and Gaoxiang Wang and Cheng Sun and Zhang, {Hao F}",
year = "2019",
month = "6",
day = "20",
doi = "10.1364/OPTICA.6.000709",
language = "English (US)",
volume = "6",
pages = "709--715",
journal = "Optica",
issn = "2334-2536",
publisher = "OSA Publishing",
number = "6",

}

Three-dimensional biplane spectroscopic single-molecule localization microscopy. / Song, Ki Hee; Zhang, Yang; Wang, Gaoxiang; Sun, Cheng; Zhang, Hao F.

In: Optica, Vol. 6, No. 6, 20.06.2019, p. 709-715.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Three-dimensional biplane spectroscopic single-molecule localization microscopy

AU - Song, Ki Hee

AU - Zhang, Yang

AU - Wang, Gaoxiang

AU - Sun, Cheng

AU - Zhang, Hao F

PY - 2019/6/20

Y1 - 2019/6/20

N2 - Spectroscopic single-molecule localization microscopy (sSMLM) captures the full emission spectra of individual molecules while simultaneously localizing their spatial locations at a precision greatly exceeding the optical diffraction limit. To achieve this, sSMLM uses a dispersive optical component to separate the emitted photons into dedicated spatial and spectral imaging channels for simultaneous acquisition. While adding a cylindrical lens in the spatial imaging channel enabled three-dimensional (3D) imaging in sSMLM, the inherent astigmatism leads to technical hurdles in spectral calibration and nonuniform lateral resolution at different depths. We found that implementing the biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows for more efficient use of the limited photon budget and provides homogeneous lateral resolution compared with the astigmatism-based method using a cylindrical lens. Here we report 3D biplane sSMLM and demonstrate its multi-color 3D imaging capability by imaging microtubules and mitochondria in fixed COS-7 cells immunostained with Alexa Fluor 647 and CF 660C dyes, respectively. We showed a lateral localization precision of 20 nm at an average photon count of 550, a spectral precision of 4 nm at an average photon count of 1250, and an axial localization resolution of 50 nm.

AB - Spectroscopic single-molecule localization microscopy (sSMLM) captures the full emission spectra of individual molecules while simultaneously localizing their spatial locations at a precision greatly exceeding the optical diffraction limit. To achieve this, sSMLM uses a dispersive optical component to separate the emitted photons into dedicated spatial and spectral imaging channels for simultaneous acquisition. While adding a cylindrical lens in the spatial imaging channel enabled three-dimensional (3D) imaging in sSMLM, the inherent astigmatism leads to technical hurdles in spectral calibration and nonuniform lateral resolution at different depths. We found that implementing the biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows for more efficient use of the limited photon budget and provides homogeneous lateral resolution compared with the astigmatism-based method using a cylindrical lens. Here we report 3D biplane sSMLM and demonstrate its multi-color 3D imaging capability by imaging microtubules and mitochondria in fixed COS-7 cells immunostained with Alexa Fluor 647 and CF 660C dyes, respectively. We showed a lateral localization precision of 20 nm at an average photon count of 550, a spectral precision of 4 nm at an average photon count of 1250, and an axial localization resolution of 50 nm.

UR - http://www.scopus.com/inward/record.url?scp=85070781822&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85070781822&partnerID=8YFLogxK

U2 - 10.1364/OPTICA.6.000709

DO - 10.1364/OPTICA.6.000709

M3 - Article

VL - 6

SP - 709

EP - 715

JO - Optica

JF - Optica

SN - 2334-2536

IS - 6

ER -