Abstract
ABSTRACTFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.
Highlights
Fluorescence microscopy is a standard research tool in many fields, collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence
We show that Erg11 localizes to the endoplasmic reticulum (ER) and the plasma membrane in C. albicans, while a proportion of Gcn5 localizes to the nucleus and the cytosol depending on the growth phase
Imaging of Lowly Expressed Candida albicans Proteins the background signal of C. albicans is almost 2-fold higher, indicating that fluorescence imaging of C. albicans in a similar spectral window is more challenging
Summary
Fluorescence microscopy is a standard research tool in many fields, collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. KEYWORDS Candida albicans, Erg11, Gcn5, fluorescence microscopy, photochromic fluorophores Overexpression showed localization to the nucleus during the stationary phase and in the cytosol during the exponential phase [5], imaging of Gcn5 under endogenous expression did not result in a usable signal.
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