Live Intracellular Super-Resolution Imaging Using Site-Specific Stains

Point localization super-resolution imaging (SR) requires dyes that can cycle between fluorescent and dark states, in order for their molecular positions to be localized and create a reconstructed image. Dyes should also densely decorate biological features of interest to fully reveal structures being imaged. We tested site-specific dyes in several live-cell compatible imaging media and evaluated their performance <i>in situ</i>. We identify a number of new dyes and imaging medium-dye combinations for live staining, that densely highlight intracellular structures with excellent photophysical performance for SR

Live Intracellular Super-Resolution Imaging Using Site-Specific Stains

Point localization super-resolution imaging (SR) requires dyes that can cycle between fluorescent and dark states, in order for their molecular positions to be localized and create a reconstructed image. Dyes should also densely decorate biological features of interest to fully reveal structures being imaged. We tested site-specific dyes in several live-cell compatible imaging media and evaluated their performance <i>in situ</i>. We identify a number of new dyes and imaging medium-dye combinations for live staining, that densely highlight intracellular structures with excellent photophysical performance for SR

Live Intracellular Super-Resolution Imaging Using Site-Specific Stains

Point localization super-resolution imaging (SR) requires dyes that can cycle between fluorescent and dark states, in order for their molecular positions to be localized and create a reconstructed image. Dyes should also densely decorate biological features of interest to fully reveal structures being imaged. We tested site-specific dyes in several live-cell compatible imaging media and evaluated their performance <i>in situ</i>. We identify a number of new dyes and imaging medium-dye combinations for live staining, that densely highlight intracellular structures with excellent photophysical performance for SR

Live Intracellular Super-Resolution Imaging Using Site-Specific Stains

Point localization super-resolution imaging (SR) requires dyes that can cycle between fluorescent and dark states, in order for their molecular positions to be localized and create a reconstructed image. Dyes should also densely decorate biological features of interest to fully reveal structures being imaged. We tested site-specific dyes in several live-cell compatible imaging media and evaluated their performance <i>in situ</i>. We identify a number of new dyes and imaging medium-dye combinations for live staining, that densely highlight intracellular structures with excellent photophysical performance for SR

Wobble calibration measured on three days on the same system.

<p>Day 1 (black, x-direction: open circle, y-direction: filled circle) and day 2 (green, x-direction: open square, y-direction: filled square) show little deviation from one another. Before measurement on day 3 (magenta, x-direction: open triangle, y-direction: filled triangle), the system’s cylindrical lens was removed and replaced.</p

Wobble correction applied to an exemplar FtsZ ring in <i>Caulobacter crescentus</i>.

<p>(a) A field of view containing several bacteria (b) Zoom of the boxed region in a, containing raw localizations of the FtsZ ring, having a tilt of ~ 9.0° (c) The same FtsZ ring as in b after application of the wobble correction with a < 1° tilt. Dotted lines in b and c guide the eye to visualize the tilt of the FtsZ ring. Scale bars: 100 nm. All data, including wobble calibration, was collected on a custom-built microscope equipped with a Nikon NA 1.49 oil immersion objective lens.</p

Workflow of dual color, 3D registration.

<p>Work flow for image registration of 3D SR data. (a) At the focal plane (z0), a local weighted mean transformation T_lwmz0 is defined using a nanogrid. This allows us to map one color channel onto the other. (b) After data is acquired, it is fit to a 3D calibration curve from its channel to obtain z information and to correct the chromatic shift in z (magenta, red channel z calibration; green, far red channel z calibration), then (c) 3D data from each channel (magenta, red channel; green, far red channel) is wobble corrected. Once both the z and wobble calibrations are applied to the data from each channel, T_lwmz0 is used to map one channel onto the other. Measurements from 10 beads shown in b and c.</p

Aberrations that lack rotational symmetry produce the wobble effect.

<p>(Top row) Raw data shown at three different axial depths and corresponding wobble curves. (Middle) Phase-retrieved PSF (PR-PSF) at three axial depths and corresponding wobble curves, which are in good agreement with the raw data. (Bottom) PR-PSF with only rotationally symmetric Zernike modes. Resulting wobble calibration shows an absence of the wobble distortion.</p

Wobble distortion under different experimental conditions.

<p><b>(</b>a) Wobble curves in the presence (magenta) and absence (green) of a cylindrical lens, showing little difference in magnitude and direction. (b) Wobble curves at two different objective rotations 45° apart (0°, magenta, 45°, green), showing changes in both directions and magnitude. (c) Wobble curves with 0° (magenta) and ~ 1.5° (green) coverslip tilt. Measurements from 5–6 beads. Error bars are standard deviations of the shift value. X-directions: magenta, open circle, green, open square and y-directions: magenta, filled circle, green, filled square in unmodified (with cylindrical lens, no coverslip tilt and no objective rotation) and modified positions respectively.</p

Application of wobble correction for 2-color, 3D SR image registration.

<p>(a) A cartoon of the wobble effect in 2 independent channels is shown. Green and pink rectangles represent focal planes. Straight lines reference the wobble-free system. (b) Effect of merging channels: since the wobble differs over z, this increases the registration error <i>δ_z</i> where wobble curves differ most, which is typically at the z-limits of the imaging depth (c) the target registration error (TRE) as a function of z for a locally weighted mean (LWM) transformation defined at the focal plane (red data) and a LWM transformation defined at the focal plane with wobble correction included (black data). Error bars are standard deviation of TRE values. Measurements taken from 4 beads over the field of view.</p