DOI: 10.5061/dryad.7m0cfxq2p
title: Data from: Optical projection tomography implemented for accessibility and low cost (OPTImAL)

description: Optical projection tomography (OPT) is a 3D mesoscopic imaging modality
 that can utilise absorption or fluorescence contrast and is widely applied
 to fixed and live samples in the mm-cm scale. We present OPTImAL, an
 open-source research-grade implementation of modular OPT hardware and
 software that has been designed to be widely accessible by using low-cost
 components, including LED excitation and cooled CMOS cameras. Both the
 hardware and software are modular and flexible in their implementation,
 enabling rapid switching between sample size scales and supporting
 compressive sensing to reconstruct images from undersampled sparse OPT
 data, e.g., to facilitate rapid imaging with low
 photobleaching/phototoxicity. We also explore a simple implementation of
 focal scanning OPT to achieve higher resolution, which entails the use of
 a fan-beam geometry reconstruction method to account for variation in
 magnification.
description: This dataset consists of raw OPT (optical projection tomography)
 data, i.e. projection images of samples, recorded by an (sCMOS or cooled
 CMOS) camera as the sample is rotated. Volumetric
 images may then be reconstructed from these provided sets of projection
 data. Software is provided to do so in the associated GitHub repository.
 (https://github.com/ImperialCollegeLondon/OPTImAL) The provided datasets all comprise fluorescence images - widefield excitation illumination is incident on the sample, and the emitted fluorescence signal is recorded on the camera, with the excitation illumination blocked from reaching the camera sensor through use of filters. Select projection datasets have been deconvolved with a projection of (modelled) system 3D PSF. These are indicated in the folder name and information file, and the raw camera projection images are also provided in each case.
description: # Data from: Optical projection tomography implemented for accessibility
 and low cost (OPTImAL)
 [https://doi.org/10.5061/dryad.7m0cfxq2p](https://doi.org/10.5061/dryad.7m0cfxq2p) This repository contains OPT (optical projection tomography) projection data, which can be reconstructed to produce volumetric images using the software provided in the *OPTImAL* GitHub repository: [https://github.com/ImperialCollegeLondon/OPTImAL](https://github.com/ImperialCollegeLondon/OPTImAL). ## Data All data is compressed and stored in .7z archives, and can be extracted using 7zip, an open source archive utility, available at [https://www.7-zip.org/](https://www.7-zip.org/). This repository contains OPT projection data of 4 distinct samples, as follows: ##### fli:GFP zebrafish embryo - 4x magnification Archive name: fli_gfp_zebrafishembryo_4x.7z This folder contains a set of 400 projection images (recorded over 360 degrees) of labelled vasculature in an *in vivo* fli:GFP zebrafish embryo, recorded at 4x magnification using an Andor Zyla 5.5 sCMOS camera. Widefield excitation illumination was provided by a Cairn TriLine laser source. Figure 4 in the main *OPTImAL* paper presents a reconstruction of the full 400 projection dataset. To reconstruct the data, *OPTtools* (our open-source OPT reconstruction plugin for MATLAB, found in the *OPTImAL* GitHub repository) should be used, with FBP (filtered back projection) as the reconstruction algorithm. Figure 8 (and supplementary video 1) in the *OPTImAL* paper presents reconstructions of subsampled selections of projections from this dataset, reconstructed using the *OPTtools* plugin and the TwIST algorithm. ##### Mouse pancreas - 1x magnification Archive name: mouse_pancreas_3colour_1x.7z (archive in 3 parts, please download .001, .002 and .003 before extracting the archive) This folder contains 3 channel OPT projection data, with the separate colour channels showing CK19, pancreatic amylase and c-peptide (blue, green and red, antibody labelled with AF488 donkey antirat, AF546 donkey anti-goat and AF647 donkey anti-rabbit respectively) in fixed, cleared mouse pancreas tissue prepared using the FLASH protocol [1]. Each colour channel is saved in a separate folder ('blue_CK19_projections','green_pancreatic_amylase_projections','red_c-peptide_projections'). These projections were recorded using the CellCam Kikker cooled CMOS camera. 3 colour reconstructions are presented in Figure 5 of the *OPTImAL* paper (and supplementary video 2), and the data can be reconstructed using the *OPTtools* reconstruction plugin, using the FBP reconstruction algorithm. Each colour channel should be reconstructed separately, and then combined in ImageJ or Icy as desired. The folder 'zyla_projections' contains the same 3 colour channel data of the same sample, however the projection data was recorded using the Andor Zyla 5.5 sCMOS camera. Reconstructions from this dataset are presented in Supplementary Figure 3 of the *OPTImAL* paper. ##### MMTV-PyMT mouse mammary gland - 1x magnification Archive name: mouse_mammary_gland_fixedlens_1x.7z (archive in 3 parts) This folder contains conventional OPT projections of labelled mammary epithelial cells in a MMTV-PyMT mouse mammary gland, fixed and cleared using the FLASH protocol [1]. The projection images were recorded with a 1x telecentric lens, with the depth of field set to cover the half of the sample closest to the camera. This folder contains multiple projection datasets of the sample, recorded using different cameras, illumination sources, and with different camera integration times, as indicated by the folder names and in the information.txt file. Reconstructions from these datasets are presented in Figures 1, 6 and 7 of the *OPTImAL* paper. The data can be reconstructed using the *OPTtools* MATLAB plugin, using the FBP reconstruction algorithm. The folder also contains a deconvolved set of projections. This deconvolution process was performed using scripts from [3], and a modelled system 3D PSF, generated using PSF Generator, an open-source java program (available as a MATLAB plugin) available at [3]. Deconvolution scripts for OPT projection images are provided in the *OPTImAL* GitHub repository. A reconstruction of this deconvolved set of projections is presented in Figure 14 of the *OPTImAL* paper, and the deconvovled projections may be reconstructed using the *OPTtools* software, just as the raw projection images are. ##### MMTV-PyMT mouse mammary gland - 0.75x magnification focal scanning OPT projection data Archive name: mouse_mammary_gland_focal_scanning_0point75x.7z.001 This folder contains focal scanning OPT data of the same MMTV-PyMT mouse mammary gland sample presented in the previous section. The data was recorded using a 0.75x telecentric lens, which contained a tunable liquid lens element, such that the focal plane could be axially scanned through the sample during acquisition. 'Slice 2' presents focal scanning projection data from a small section of the sample. LED illumination was restricted to illuminate only ~ 600 pixel rows in the centre of the field of view, which corresponded with the field of view which approximated a fan-beam projection geometry (as discussed in the *OPTImAL* paper). In order to image the entire sample in this way, the sample was physically translated, and repeatedly imaged in 8 sections. Only a single section is presented here, as is shown in Figure 14 of the *OPTImAL* paper. 'Section_away_from_fanlike_plane' presents focal scanning data of the same section of the sample, however in this case the entire sample was illuminated, and the field of view was centred on a different part of the sample. This section was therefore positioned below the central plane, and the projection geometry was no longer well described by a fan-beam. Accordingly, reconstruction of this projection data assuming a fan-beam projection geometry results in artefacts, as demonstrated in Supplementary Figure 8. Both focal scanning datasets may be reconstructed following the focal scanning reconstruction script provided in the *OPTImAL* GitHub repository. This script extensively utilises functions from the open-source ASTRA toolbox [4] and FPSOPT [2], and utilises the PSF Generator to generate modelled system PSFs [3]. ##### 1 micron fluorescent beads suspended in agarose Archive name: focal_scanning_1micron_fluorescent_beads.7z (archive in 2 parts) This folder contains a set of 800 focal scanning OPT projections of 1 micron fluorescent beads suspended in a cylinder of agarose, recorded on the CellCam Kikker CMOS camera, using the 0.75x telecentric lens containing a tunable liquid lens. Reconstructions of these fluorescent beads are shown in Figures 12 and 13, as well as supplementary figures 5 and 7 of the *OPTImAL* paper. This focal scanning dataset can also be reconstructed using the provided focal scanning MATLAB scipt provided in the *OPTImAL* GitHub repository. ###### References \[1] Messal, H. et al, Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH, Nature Protocols 16 (2021)239–262; [https://doi.org/10.1038/s41596-020-00414-z](https://doi.org/10.1038/s41596-020-00414-z) \[2] Chan, K. G., & Liebling, M. (2017). Direct inversion algorithm for focal plane scanning optical projection tomography. Biomedical Optics Express, 8(11), 5349. [https://doi.org/10.1364/BOE.8.005349](https://doi.org/10.1364/BOE.8.005349) \[3] H. Kirshner and D. Sage, PSF Generator, 2019. [Online]. Available: [http://bigwww](http://bigwww). epfl.ch/algorithms/psfgenerator/ \[4] ASTRA Toolbox. [Online]. Available: [https://astra-toolbox.com/](https://astra-toolbox.com/)