DOI: 10.5281/zenodo.15608267
title: F-actin conformations - STED-FM

description: The F-actin conformations dataset is associated with the pattern discovery task in the publication :Bilodeau, A.*, Beaupré, F.*, Chabbert, J., Bellavance, J-M, Lessard, K., Deschênes, A., Bernatchez, R., De Koninck, P., Gagné, C., Lavoie-Cardinal, F. (2025) A Self-Supervised Foundation Model for Robust and Generalizable Representation Learning in STED Microscopy. bioRxiv.The F-actin conformations dataset consists of STtimulated Emission Depletion images from several rat hippocampal neuronal cultures fixed between DIV12 and DIV15. Manual annotations of the F-actin conformations were done on the basis of morphological differences. Nanoclusters are considered as very dense small round F-actin aggregation (around 100 nm of diameter) along the dendritic shaft. Compact assemblies are segmented as F-actin dense aggregations which seem to be the basis of large but not extended protrusions from dendritic shaft. Spines are identified as protrusions that extend from the dendritic shaft. Images were acquired with a pixel size of 20 nm.The dataset contains 79 images. Images are provided with their raw intensity values. The annotations are provided as 3-channels tif images. The mask associated with an image X is given by the following: "X_mask.tif". The channels are: Nanoclusters, Compact assemblies, and Protrusions.

Methods

STED imaging. Super-resolution imaging was performed on an Abberior Expert-Line STED system (Abberior Instruments) equipped with a 100x oil immersion objective lens with a numerical aperture of 1.4 (Olympus, model UPLSAPO100XO), a motorized stage, and an auto-focus unit. The 640 nm excitation wavelength (40 MHz) and 775 nm depletion laser (40 MHz) were used for STAR635 acquisitions. The fluorescence signal was collected on an avalanche photodiode detectors (APD) through an ET685/70 (Chroma) fluorescence filter. The pinhole was set at approximatively 1 Airy unit. 

Neuronal cell culture. Extraction and culture of hippocampal neuronal cells was performed following the protocol of Nault & De Koninck 2009 and in accordance to guidelines of the animal care committee of Université Laval. Briefly, neuronal cultures from the hippocampus were obtained using neonatal Sprague-Dawley rats. P0-P1 rats were sacrificed by decapitation before dissection of the hippocampi. Cells were dissociated using enzymatic digestion and mechanical dissociation, and then seeded onto 12 mm coverslips coated with poly-D-lysine and laminin (40,000 cells/coverslip). Neurons were cultured in a growth medium composed of Neurobasal and B27 (in a 50:1 ratio), enriched with penicillin/streptomycin (25 U/mL; 25 mg/mL) and 0.5 mM L-GlutaMAX (by Invitrogen). After 5 days, Ara-C (5 mM; from Sigma-Aldrich) was added into the medium to limit the proliferation of non-neuronal cells. Cultures were fed twice a week, by replacing 50% of the growth medium with serum- and Ara-C–free medium.

Sample preparation and staining procedure. Neuronal cultures were fixed for 20 minutes in 4% PFA solution (PFA 4%, Sucrose 4%, Phosphate Buffer 100mM, Na-EGTA 2mM). Permeabilization and blocking of aspecific binding sites were done simultaneously with a solution of Triton X-100 and 2% goat serum in PBS 20 mM, for 30 min. Labeling of F-actin was performed by 2h incubation of phalloidin-STAR635 (Sigma Aldrich, cat. 30972-20, 1:50 dilution) diluted in blocking solution. Stained coverslips were mounted in Mowiol-DABCO for imaging.