Zeiss Lattice Light Sheet 7 Point Spread Functions and example dataset

This repository contains some point spread functions (PSFs) distilled from imaging fluorescent beads, 200nm, F8811 in an agarose gel, on a Zeiss Lattice Light sheet 7 microscope. The bead images from which the PSF were distilled are contained in this repo.

Several images were acquired with varying settings of the light sheet (light sheet was always set as the 30 um x1000 nm).

These three parameters were varied:

FS: focus sheet (-100, -75, -25, 0)

FW: focus waist (40, 45, 50, 55, 60, 65, 70, 75, 80, 85)

AC: aberration control (150, 155, 160, 165, 170, 175, 180, 185, 190)

The file psf-200nm correspond to the best settings taken between the different settings. The two other files (psf-300nm, psf-400nm) correspond to resampled versions of the psf-200nm file.

The all-psfs.tif contains the deskewed psf ordered similarly as the poster uploaded in this version of the repository (ELMI-2024-poster.pdf).

A sample czi file (Hela Kyoto, raw data) is provided to test deconvolution algorithms.

Note that none of these data is deskewed.

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Here's a protocol to prepare a gel containing beads at a good enough density:

• Material:

  • Agarose 2%
  • Fluorescent beads, 200nm, F8811. Stock is 1000x

• Protocol:

  • Put the agarose to warm up at 90C.
  • Vortex the beads stock.
  • Dilute a 100 times the bead dilution in PBS (10 uL in 1mL), vortex.
  • Prepare a clean slide, and a clean coverglass (22x22mm).
  • Take 90uL of agarose, put in a 1.5mL tube, add 10uL of the diluted beads, vortex.
  • Take 50uL of this agarose solution, put it on the slide, add the coverglass on top.
  • Put this slide in the fridge and wait a few minutes.
  • Use nailpolish to seal the coverglass. Wait long enoughfor the nailpolish to dry.

You should a gel of about 100um thickness.

It should be possible to add a bit of free dye in the gel in order to help focusing the light sheet. Ideally a bit of far-red free dye, like Alexa 647.