Bulletin of the American Physical Society
Four Corners Section 2023 Meeting
Friday–Saturday, October 20–21, 2023; River Woods Conference Center, Logan, Utah
Session Q01: Lustig Award Session
10:45 AM–11:45 AM,
Saturday, October 21, 2023
Riverwoods Conference Center
Room: Mahogany-Redwood
Abstract: Q01.00002 : Engineering Point Spread Functions using a Single Glass Phase Plate for 4D Super-resolution Fluorescence Microscopy*
11:05 AM–11:25 AM
Presenter:
Sanduni I Fernando
(University of Utah)
Author:
Sanduni I Fernando
(University of Utah)
Collaboration:
Sanduni I Fernando, Jason T. Martineau, Brian D. Mueller, Robert Hobson, Tien N. Vu, Erik M. Jorgensen, Jordan M. Gerton
fluorescent tags, which introduce temporal delays specially during live cell imaging, and wastes precious
photons. Alternatively, multiple fluorophore species can be simultaneously imaged and distinguished using
engineered point-spread functions (PSF) that imprint unique patterns sensitive to the spatial coordinates and
the emission wavelength of a fluorophore. Here, we insert a silicon-dioxide phase plate with only four
thickness regions at the Fourier plane of the detection path of a wide-field fluorescence microscope to
produce distinguishable PSFs (X-PSFs) at different wavelengths with sufficient 3D spatial localizability. We
localize the X-PSFs both spatially and spectrally using a Maximum Likelihood Estimation algorithm with a
faster Gaussian Cubature approximation than the standard Fourier Transforms to calculate the diffraction
integral. We demonstrate two-color and three-color super-resolution dSTORM imaging of fixed U2OS cells,
in which the peak-to-peak separation between consecutive spectra was ∼80 nm. All fluorophore species were
excited simultaneously and imaged without emission filters. The X-PSF achieves ∼21 nm lateral localization
precision, ∼17 nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a
background as high as 130 - 400 photons per pixel. The modified PSF can distinguish up to three fluorescent
probes with ∼80 nm peak-to-peak separation between consecutive spectra.
*National Science Foundation (2014862); National Institute of Neurological Disorders and Stroke (NS034307); Department of Physics and Astronomy, University of Utah; Erik M. Jorgensen is an investigator of the Howard Hughes Medical Institute.
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