Membrane and cell wall selective fluorescent dyes are important tools for the analysis of organelle dynamics and living fungal cells. Our protocols provide essential theoretical background and practical guidelines for the application of a selection of these dyes as truly vital stains. The key point for this is to use very low dye concentrations that do not cause cellular artifacts related to saturation or dye toxicity while still providing good signal to noise ratios for high-quality, long-term live cell imaging.
With colleagues from the Department of Nuclear Medicine of the medical university, we recently used these staining techniques to develop a novel approach for image-guided diagnosis of aapergillosis. The ability to monitor membrane and cell wall structures in real time was crucial for determining the uptake dynamics of the experimental drug compound. To begin this procedure, first obtain a prepared preculture.
Use a steril scalpel to cut a small agar block that carries non-sporulating mycelium from the colony edge of the preculture. Place the agar block at the center of a fresh, solid medium plate to inoculate the experimental culture. Incubate the experimental culture according to the developmental stage intended to be incubated.
Sample preparation is somewhat delicate and optimization of image acquisition settings is rather complex. Visual demonstration of both procedures accompanied by verbal explanation greatly facilitates replicability. To mount the samples, keep a clean 24 by 60 millimeter glass cover slip ready and add 18 microliters of liquid minimal medium or physiological salt solution onto the center.
Add two microliters of the prepared 20 micromolar dye working solution to the liquid in the center of the cover slip and mix well by pipetting up and down several times while avoiding the production of air bubbles. Using a clean scalpel, cut out a 15 by 15 millimeter sample from the periphery of the colony and place it vertically beside the medium drop on the cover slip. Use the scalpel to support the upper edge of the block and a finger to hold the rear side of the block in place.
Then, slowly lower the side carrying the mycelium onto the liquid. Mount the prepared sample onto the microscope stage. First, adjust the basic image acquisition settings to capture standing dynamics in individual hyphae as outlined in the text protocol.
For the endocytosis uptake assays, consult figure one and table one of the text protocol to identify the best excitation and emission settings for FM 143 or FM 464 that are available on the microscopy system and adjust these settings in the system accordingly. Start image recording using the previously adjusted settings and evaluate the results. Then, optimize the image acquisition settings to the spatial and temporal resolution required to capture the aspect of plasma membrane or endocytosis dynamics the experiment is focused on.
For cell wall dynamics, consult figure four and table one of the text protocol to identify the best excitation and emission settings for the applied cell wall dye and adjust the settings on the microscopy system accordingly. Start image recording using the previously adjusted settings and evaluate the results. Then, optimize the image acquisition settings to the spatial and temporal resolution required to capture the aspect of cell wall morphogenesis the experiment is focused on.
In addition to just visualizing cellular processes, live cell imaging allows extraction of quantitative information from the recorded data. In an example of the FM 464 uptake assays, fungal samples are cultivated as colonies and mounted by the inverted agar block method. The assays identified defects in the spatio-temporal organization of endocytosis in gene deletion and gene over-expressing mutants of the fungal specific protein SFP 2 of trichoderma atroviride.
Examples of FM 464 co-staining of fluorescent fusion proteins targeted to endocytic compartments is shown here. This co-staining is employed to relate the subcellular distribution of the two enhanced green fluorescent protein tagged transmembrane proteins, SFP 2 and GPR 1, to the endocytic pathway in trichoderma atroviride. An example of FM 464 co-staining for the identification of morphogenetic differences shows that this co-staining allows further relation of the subcellular localization dynamics of flourescently labeled BUD-6 polarism complex protein to end of some trafficking-dependent processes such as the formation of septa and polarized hyphal tip growth.
It also characterizes differences in the subcellular organization and hyphal architecture between wild type and mutant strains of neurospora crassa. Representative cell wall staining shows that the different interaction properties of calcofluor white, solophenyl flavine, and congo red with cell wall polymers highlight morphogenetic differences between the delta SFP 2 mutant and the wild type strain of trichoderma atroviride. Increasing the cell wall stress inflicted by elevated dye concentrations occurs more quickly and is more pronounced in the mutant compared to the wild type.
In addition, the same images allow for the quantification of morphogenetic differences regarding hyphal diameter and septal distance between both strains. Representative realtime monitoring of cell wall biosynthesis indicates that the very low calcofluor white concentration prevents saturation of the cell wall with dye molecules and allows quantitative realtime monitoring of cell wall biosynthesis. This reveals that the deposition of new cell wall material is not uniform, but responds very rapidly to localized physical stresses resulting from the relative displacement of one cell upon cell-to-cell attachment prior to dermalinfusion in neurospora crassa.
Less is more. Use as little dye as possible to preclude interference of the fluorescent marker with cellular processes. Following this procedure, flourescence recovery after photo-bleaching experiments could be performed to quantify transport and biogenesis kinetics.
The pioneering introduction of membrane and cell wall staining in filamentous fungi at the beginning of the millennium did literally revolutionize the way we look at fungi. Calcofluor white can cause eye irritations and is a classified cancerigen. Congo red is cancerigenic and teratogenic, so please wear eye and skin protection when handling these dyes.
