Dataset and code related to article "smFISH-on-Chip: Candida albicans biofilm growth coupled to Single-molecule Fluorescence In Situ Hybridization

This repository contains the example dataset for the protocol paper titled smFISH-on-Chip: Candida albicans biofilm growth and gene expression analysis by Single-molecule Fluorescence In Situ Hybridization. 

Abstract
Candida albicans is a major opportunistic fungal pathogen whose pathogenicity relies on extensive phenotypic plasticity and dynamic transitions between morphological and lifestyle states. Although many regulators of morphogenesis have been identified, the molecular mechanisms by which local cues are integrated remain poorly understood, partly due to limitations in preserving spatial context during gene expression analysis. Here, we present an smFISH-on-Chip protocol that couples controlled C. albicans growth in microfluidic devices with single-molecule mRNA imaging and quantification. This approach enables time-lapse tracking, maintains native spatial organization, and supports high-resolution quantification of endogenous mRNAs in intact yeast, pseudo-hyphal, and hyphal cells. We demonstrate the method’s robustness by measuring the expression and spatial localization of the cell cycle mRNA CLB2, and of the hyphal tip-localized mRNA HWP1. We benchmark smFISH-on-Chip against our established smFISH-on-Coverslip approach to compare RNA spot detection efficiency. We provide analysis pipelines for spot identification and subcellular mRNA localization analysis. Altogether, this platform provides a powerful tool for dissecting how microenvironmental cues and community architecture shape the expression of localized and non-localized mRNAs, and in turn, fungal behavior.

Content
FISH_data:
Contains raw microscopy data, processed data and analysis code.
In the folder FISH/analysis/CLB2 and FISH/analysis/HWP1 you can find ipynb files to replicate the performed analysis.

Growth_videos_and_adherence_testing
Contains images used to make Figure 2 and 3.

3D print designs:
Additionally, both .stl and .3mf files for 3D printed tools used in this protocol are published. It should be noted that prints were performed using PLA-CF from bambulab, especially for the 3D printed caps this is important as the pin that is used to close of the connector is more robust when printed with PLA-CF as opposed to standard PLA.
The 3D platforms for holding the microfluidic chips and eppendorfs were adapted from the designs of https://makerworld.com/nl/models/417346-eppendorf-tube-holder-6x3?from=search#profileId-426433.