PALMsiever: a tool to turn raw data into results for single-molecule localization microscopy

During the past decade, localization microscopy (LM) has transformed into an accessible, commercially available technique for life sciences. However, data processing can be challenging to the non-specialist and care is still needed to produce meaningful results. PALMsiever has been developed to provide a user-friendly means of visualizing, filtering and analyzing LM data. It includes drift correction, clustering, intelligent line profiles, many rendering algorithms and 3D data visualization. It incorporates the main analysis and data processing modalities used by experts in the field, as well as several new features we developed, and makes them broadly accessible. It can easily be extended via plugins and is provided as free of charge open-source software. Contact: [email protected]

Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging

Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term 'wobble', results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of similar to 1 mu m. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope's pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 mu m, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample

Improved methods for sub-diffraction-limit single-molecule fluorescence measurements

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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 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

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

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

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

Observed Z-dependent lateral PSF shift (wobble).

<p>(a) A wobble-free system is denoted by the straight, light blue line (left) as compared to a system with wobble (right). The wobble effect shown in a) causes a lateral displacement of localizations as a function of axial displacement due to unwanted aberrations introduced by the microscope system. (b) Cartoon of true structures (left) and resultant warped structures (right) imaged on a system with wobble. (c) The lateral shift (or wobble), in the x and y directions as a function of the axial position (z). Dashed, green and violet lines represent data from 10 different beads over the field of view. The green and magenta solid lines represent the average value based on all 10 beads in the y and x-directions respectively. We observe a more significant shift in the y-direction, which is ~ 10 times greater than that in the x-direction. (d) Wobble curves after our correction method is applied; accuracy of correction according to error bars ≤ 6 nm. Error bars are standard deviation (S.D) values of the shift. Maximum S.D for curves in 1c: 6 nm. All data was collected on a modified Olympus IX71 inverted microscope equipped with an Olympus NA 1.40 oil immersion objective.</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