Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time. exocytosis. However, precisely which subcellular organelles become successively labeled under the tested conditions is not immediately evident and requires comparison of different FM dye variants, and co-labeling with additional organelle-specific markers. The preference of FM 1-43 for mitochondrial membranes, in comparison to FM 4-64, is one example. Cell wall selective dyes display varying specificity for the three major polymers of the fungal cell wall. CFW is thought to be a non-specific stain for β-glucans and chitin, SPF is thought to be most selective for β-1,4-glucans, and CR is thought to be highly selective for α- and β-chitins. Information on the binding specificity of PFS to fungal cell wall polysaccharides is currently not available. To what ratio which fungal cell wall polymer is most effectively labeled at a given dye concentration in the fungal species under investigation is not easily answered, and the application of detailed measurements acquired in vitro or in vivo in other organisms or other fungal species must be considered very carefully.


Introduction
Twenty years ago, the way in which hyphal morphogenesis and the underlying molecular cell biology could be visualized in filamentous fungi was revolutionized by the application of the membrane selective fluorescent Fei Mao dye FM 4-64 1 . Later, the benefit of the chitin-binding dye Calcofluor White as vital fluorescent marker of fungal cell wall dynamics was realized 2 . Since then, both dyes and variants thereof have become an inherent part of live-cell imaging analyses of organelle dynamics in fungi, and continue to provide unprecedented insights into the filamentous fungal lifestyle. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-and exocytosis and cell wall morphogenesis in filamentous fungi. Endocytosis tracking assays allow various cell biological questions related to the general study of endocytosis to be addressed 3 . For this, the localization, speed and succession of stained compartments upon FM dye addition is recorded by time lapse microscopy and quantitatively compared between the tested fungal strains 4 . Cell wall dyes delineate the outer boundary of the cell and allow tracking of morphogenetic events, including polarized hyphal tip growth 2 , hyphal branching 5 , hyphal fusion 6,7 and septum formation 8 . Furthermore, they facilitate the quantification of localized cell wall deposition and the identification of defects during cell wall biogenesis 9 . Because detailed knowledge of the biochemical and biophysical properties of any fluorescent marker is a fundamental prerequisite for its successful in vivo application, these characteristics are first summarized for the six dyes featured in this article.
Membrane selective dyes FM (Fei Mao) styryl dyes are small amphiphilic molecules that cannot pass through but reversibly associate with the outer leaflet of the lipid bilayer of biological membranes 2,14 , and evidence from BY-2 tobacco protoplasts indicates that more than 20 µM FM dye lead to plasma membrane saturation 14 . Thus, it is advisable not to exceed this limit, especially given the fact that excellent imaging has been achieved with as little as 2-5 µM 15,16 .
Notably, the spectral properties of FM dyes vary greatly depending on the particular membrane microenvironment (reviewed 14 ). Generally, excitation and emission spectra of FM dyes in pure solvent solutions (as usually provided in the product information) differ significantly from that in cellular environments and can, in most cases, not directly be consulted for selecting live-cell imaging settings. The excitation/emission maxima of FM 1-43 and FM 4-64, for instance, become blue-shifted by 37/46 nm and 43/64 nm, respectively, when bound to fungal membranes in comparison to their solutions in methanol ( Table 1).
The ground-breaking fundamentals for the use of FM 4-64 and FM 1-43 for tracking plasma membrane, endo-/exocytosis and organelle dynamics, including the Spitzenkörper and mitochondria, have already been comprehensively documented for a wide range of filamentous fungal species previously 2,4,17,18,19 . Recommended imaging settings for both FM dyes that work in various filamentous fungal species are depicted in Figure 1B. Technical limitations of the available equipment or particular cellular and experimental conditions, such as culture medium, pH or temperature, however, might require some adaptations. Fortunately, FM dyes operate over a wide spectral range, and very good imaging results are achieved by exciting FM 1-43 with 514 nm or FM 4-64 with 488 nm. Consequently, the optimal imaging settings must be determined individually for each sample type and intended application.
The considerable Stoke's shift of more than 135 nm of FM 4-64 allows excellent, simultaneous co-imaging with fluorophores emitting green light; this is frequently exploited to evaluate the intracellular localization dynamics of green fluorescent protein (GFP)-labeled fusion proteins relative to the plasma membrane and the endocytic pathway 9,20 .

Cell wall-selective dyes
Calcofluor White M2R (CFW), also marketed as Fluorescent Brightener 28, is probably the best-known fluorescent dye used to stain the cell walls of bacteria, fungi, algae, higher plants and insects. Initially used as optical whitening agent in the paper, textile and detergent industry, its benefits for the clinical diagnosis of fungal infections was realized early on 21,22 . Because CFW intercalates irreversibly into the nascent chitin chain it disturbs normal chitin microfibril assembly during cell wall biogenesis thereby generating cell wall stress 23 . This in turn triggers a cell wall damage repair mechanism leading to locally heightened cell wall deposition as the result of glucan and chitin synthase activation 24,25 . This phenomenon can occur with any dye that operates by stably binding to cell wall polymers, is concentration-dependent and is most noticeable at hyphal tips which represent the most prolific growing and thus most sensitive parts of the mycelium (Figure 3). A comprehensive summary of the molecular machinery that responds to cell wall damage has recently been provided 26 .
Overdosed dye in combination with phototoxicity can lead to rapid cell lysis of hyphal compartments (Movie 1). Nonetheless, increased sensitivity to dye concentrations that are "vital" in the wild type can be exploited to identify defects in cell wall biosynthesis of gene loss-offunction mutants 9 . For CFW and Congo Red (CR), another textile colorant also known as Direct Red 28 and employed as α-and β-chitin-specific cell wall stain for fungi and insects 27,28 , threshold concentrations that strongly induce chitin synthases have been determined with > 60 µM CFW and > 70 µM CR, respectively, whereas concentrations <15 µM of either dye did not alter or inhibit fungal growth 29,30,31 . Hickey et al. placed this threshold concentration for CFW at 25 µM 2 . Therefore, it is advisable to use dye concentrations ≤ 5 µM to exclude stress-related artefacts and ensure using these molecules as truly "vital fluorescent dyes" 2,32 . This equally applies to Solophenyl Flavine 7GFE 500 (SPF) and Pontamine Fast Scarlet 4B (PFS), synonymous to Direct Yellow 86 and Direct Red 23, respectively, two other useful cell wall dyes whose application for fungi has been reported for the first time more than a decade ago 33 . But despite their remarkable spectral properties 34,35 , the use of both dyes has since then been very limited 36,37 . As previously shown for 1.5 µM CFW 2 , 2 µM SPF are sufficient to resolve cell wall dynamics under native conditions with very high temporal resolution (Movie 2). The same results can be obtained with 2 µM CR or PFS.
Together, these four dyes, CFW, SPF, PFS and CR, comprise a set of cell wall-selective fluorescent markers that cover almost the complete visible emission light spectrum (400-700 nm) utilized on modern fluorescent microscopes (Figure 4). The significant increase in fluorescence intensity upon binding to cell wall polymers is inherent to all four and generates excellent S/N-ratios. This in turn permits to keep dye concentrations and excitation light intensity very low and allows to perform cell wall staining as "low dose" live-cell imaging technique 2 . Because these cell wall dyes are plasma membrane impermeable, they simultaneously function as live/dead stains. Notably, due to their extremely wide emission light spectra, some limitations regarding the co-imaging properties of CFW and SPF with other fluorophores need to be carefully considered.

Cultivation of fungal colonies
1. Using a sterile scalpel, cut a small 3 mm x 3 mm agar block carrying non-sporulating mycelium from the colony edge of the pre-culture. 2. Place the agar block at the center of a fresh solid medium plate to inoculate the experimental culture. 3. Incubate the experimental culture according to the developmental stage intended to be investigated. For instance, the wild-type T. atroviride requires 20-22 hours at 25 °C in the dark to develop colonies of about 2 cm diameter on PDA, whereas the wild-type N. crassa reaches colony diameters of about 4 cm after 14-16 h of incubation at 30 °C in the dark on VMM. NOTE: Incubation in the dark prevents the formation of pigments which might introduce autofluorescence. In order to eliminate medium background fluorescence from the experimental culture, replace agar with 1.5% w/v of a transparent solidifying agent (see the Table of  Materials), and any complex medium with a defined minimal medium.
3. Cultivation of solid germling cultures 1. Use 5 mL of sterile physiological salt solution (0.9% w/v NaCl) to harvest conidial spores from the pre-culture plate and collect the resulting spore suspension in a 15 mL screw cap tube. 2. Mix the spore suspension well by vigorous vortexing and subsequently filter it over a 1 cm x 5 cm strip of sterile filter fabric (see the

Preparation of dye working solutions
1. To guarantee full solubility of each dye, prepare 2 mM stock solutions in dimethyl sulfoxide (DMSO) by adding the appropriate amount (see exact weights in Table 1) to 1 mL of 100% DMSO and mix well by vortexing. CAUTION: Make sure to take the DMSO from a septum-sealed bottle; it should be a clear transparent liquid. Upon contact with air, DMSO turns brown-probably due to the oxidation of trace impurities-and might negatively affect cell growth or dye staining. 2. Filter sterilize the stock solution through a 0.2 µm syringe membrane filter into a fresh sterile 1.5 mL reaction tube. To minimize dye bleaching, wrap the tube in aluminum foil. NOTE: The dye stock solution can be aliquoted into smaller volumes to avoid thawing/freezing cycles, and kept at 4 °C for several months. 3. With a clean scalpel, cut out a 15 mm x 15 mm sample from the periphery of the colony or solid germling culture and place it vertically beside the medium drop onto the cover slip. 4. Using the scalpel to support the upper edge of the block and a finger to hold the rear side of the block in place, slowly lower the side carrying the mycelium or germlings onto the liquid. The sample is now ready for transfer onto the microscope stage. CAUTION: It is essential to do this slowly and very carefully in order to minimize mechanical stress on the cells and to avoid air bubbles being trapped between sample and cover slip.

Live-cell microscopy
1. Adjust the basic image acquisition settings. The following image acquisition settings allow to capture staining dynamics in individual hyphae and are applicable to both of the following assays 1. Apply 5-10% laser power of 20% of the full output power of the device. 2. Use a Plan Apo 60x-63x glycerol or water immersion objective with a high numerical aperture ≥ 1.2.
3. Restrict the image acquisition area to the outline of the hyphae by setting an image size of 1024 x 256 pixels and by using an optical zoom factor of 2-3. 4. Use bidirectional scanning with 400 Hz. Adjust the pinhole size to 1 Airy unit. 5. Set the gain of the most sensitive detector to 100%. 6. For time laps recording, start image acquisition with one frame every 15 s to allow reasonable temporal resolution without producing dye bleaching or photo stress. 7. For 3D recording, set the upper and lower spatial limit to the boundary of the hyphae and space optical sections 1 µm apart to allow reasonable spatial resolution. NOTE: Due to the fast growth of hyphae, high spatial resolution in the Z axis has often been sacrificed for high temporal resolution in the X/Y axis or the other way around. Only very modern confocal laser scanning microscopes are fast enough to satisfy both demands.
2. Endocytosis uptake assays 1. Consult Figure 1 and Table 1 to identify the best excitation/emission settings for FM 1-43 and/or FM 4-64 available on the microscopy system and adjust accordingly. NOTE: With the recommended 2 µM concentration, incorporation of FM dye into the plasma membrane is instant in normal healthy cells. The whole process from initial plasma membrane staining to dye appearance in tubular vacuoles is usually completed within 30-45 min at room temperature. Increasing the FM dye concentration increases the S/N-ratio and thus produces higher contrast images quicker. However, it also speeds up the labeling process making it more difficult to differentiate the chronological succession of organelle staining. 2. Start image recording using the above recommended basic image acquisition settings and evaluate the results. 3. Optimize 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. 4. For instance, in order to capture very fast dynamics in X/Y, decrease the overall image size, image only one focal plane and increase the scanning rate to 1 fps. For higher resolution in the Z axis, decrease resolution in X/Y, decrease image size and decrease the distance between optical sections to 0.5 µm.
3. Cell wall dynamics 1. Consult Figure 4 and Table 1 to identify the best excitation/emission settings for the applied cell wall dye available on the microscopy system and adjust accordingly. NOTE: Due to their broad emission spectra, CFW and SPF are not well suited for simultaneous co-imaging with other fluorophores, predominantly GFP. Some restrictions do even apply for sequential imaging approaches with these dyes, and thus have to be optimized individually. 2. Start image recording using the above recommended basic image acquisition settings and evaluate the results. NOTE: With the recommended 2 µM concentration, incorporation of dye into the cell wall is not necessarily instant but reasonably fast. The whole process of septum formation, for instance, takes on average about 5-7 min at room temperature 20 . Increasing the cell wall dye concentration increases the S/N-ratio and thus produces higher contrast images quicker. However, it also rapidly introduces artefacts due to induced cell wall damage repair. 3. Optimize image acquisition settings to the spatial and temporal resolution required to capture the aspect of cell wall morphogenesis the experiment is focused on, as outlined in section 4.2.3.

Discussion
This article continues the ground breaking work that established the use of various fluorescent dyes as vital organelle markers for filamentous fungi in the early 2000s 2,4,43 , and attempts to discuss the photophysical and cell biological properties of FM dyes and selected cell wall dyes in greater detail than before. Especially with respect to unwanted cellular effects, such as membrane saturation or cell wall damage, that occur above certain dye concentrations. What previously has been considered non-toxic on the cellular level is now regarded toxic on the molecular level. Even though these effects might be very subtle and not directly evident by obvious changes in organelle or cell behavior, any possible interference of dye application other than visualization has to be minimized for the investigation of native molecular function. Fortunately, improved sensitivity and quantum efficiency of modern detectors, such as silicon avalanche photodiode detectors (Si-APDs) 44 or the Airyscan area detector 45 , facilitate the use of even lower dye amounts than before. Another key objective of the article is to exemplify the co-imaging properties of these dyes with other fluorophores, most importantly, those of GFP as the most frequently used fluorescent protein in biology. This should aid the design of imaging experiments that aim to correlate the subcellular localization dynamics of fluorescent fusion proteins to those of the fungal cell wall, the plasma membrane or the endo-and exocytosis pathway etc.
Imaging under natural and stress-free conditions is key for the acquisition of reliable data. Some practical considerations regarding culture medium and sample preparation aim at providing a starting point for finding the optimal conditions that allow artefact-free, long time observation of healthy, unstressed cells with the highest S/N-ratio possible for any given sample. There is no universal way of achieving reliable and meaningful imaging results. It is inherent to the approach that biological variation of the sample, subjectivity and expectations of the microscopist, as well as image post processing have significant influence on data acquisition and interpretation, respectively. Hence, the practical experience of the microscopist, her/his intimate knowledge about the cell biology of the fungus under investigation, as well as skillful sample preparation to create conditions as 'natural' and undisturbed as possible in a lab setting, are paramount for acquiring and evaluating imaging data that truthfully reflects the studied cellular phenomena. As a rule of thumb, the occurrence of unwanted side effects of fluorescent dyes, ranging from the subtle and thus not obviously visible activation of plasma membrane or cell wall re-modelling stress response pathways to the straightforward cytotoxic induction of cell autolysis, can only be securely prevented by applying low dye concentrations ≤2 µM.
The application of fluorescent dyes is simple, yet their specificities are poorly characterized. A key strength of using fluorescent dyes is the preparative simplicity of the experimental protocols. Cultivation and sampling of the fungus, addition of the dye(s), and mounting onto the microscope stage are (with practice) straightforward. Adjustment of the basic imaging settings, including excitation and emission wavelengths, exposure times, time course settings etc., follow simple biophysical rules of the microscope and biological rules of the used fluorescent dyes inside the cells. Table 1 intends to support the identification of the most suitable dye or dye combination for experimentation. Furthermore, fluorescent dyes are reasonably priced, readily available with dependable high quality and thus ensure highly reproducible application.
Two major restrictions of using membrane or cell wall selective fluorescent dyes are the (often) limited knowledge of their precise staining properties, which in most cases are non-specific on the organelle and molecular level, and their concentration-dependent unwanted side effects. FM dyes are specific for lipid bilayers participating in endo-and exocytosis. However, precisely which subcellular organelles become successively labeled under the tested conditions is not immediately evident and requires comparison of different FM dye variants, and colabeling with additional organelle-specific markers. The preference of FM 1-43 for mitochondrial membranes, in comparison to FM 4-64, is one example. Cell wall selective dyes display varying specificity for the three major polymers of the fungal cell wall. CFW is thought to be a nonspecific stain for β-glucans and chitin, SPF is thought to be most selective for β-1,4-glucans, and CR is thought to be highly selective for α-and β-chitins. Information on the binding specificity of PFS to fungal cell wall polysaccharides is currently not available. To what ratio which fungal cell wall polymer is most effectively labeled at a given dye concentration in the fungal species under investigation is not easily answered, and the application of detailed measurements acquired in vitro or in vivo in other organisms or other fungal species must be considered very carefully. Unfortunately, this information is sparse and highly scattered in the literature 35,42,46 . More recent records that would follow on previous studies 33 to provide new insights into the precise staining properties of the featured dyes specifically in fungi are currently not available.
Imaging controls are essential to evaluate staining patterns and cellular responses accurately. Probably the most challenging part, however, is to know the cell biology of the fungus so well that the recorded changes in the subcellular localization of membrane and cell wall selective fluorescent dyes, alterations in cellular architecture or hyphal growth pattern can exclusively and confidently be related to the intended effects of the experimental treatment. For this, it is crucial to have good controls alongside any new live-cell imaging experiment. These include the untreated wild type as negative imaging control to exclude background autofluorescence and detector noise from the acquired image, and to have a morphological comparator when working with mutants. Furthermore, a positive imaging control, for example a strain that expresses GFP or RFP in the cytoplasm or another well-known fluorescent marker protein, is essential to set the excitation light intensity to the required minimum and have a cell vitality control. Once these controls are set, the use of fluorescent dyes is not restricted to visualization tasks only, but their concentration-dependent staining dynamics, as well as concentration-dependent side effects can be analytically exploited; for instance, for quantitatively monitoring cell wall biosynthesis in real time or for the identification of mutant-specific phenotypes in susceptibility assays 47 . Improved future applications depend on a detailed functional analyses of dye staining properties. A major ongoing challenge is to further improve and automate quantitative image analyses in order to advance functional evaluation of the subcellular dynamics of membrane and cell wall selective fluorescent dyes in filamentous fungi. For this, extensive, quantitative co-localization studies of these dyes with known organelle and cell wall polymer markers in combination with mutant strains deficient in particular transport pathways or that lack specific structural components are first required. Several endocytosis markers for comparative analyses with FM dyes are available 48,49 , and with respect to the still poorly characterized binding specificities of cell wall dyes in fungi, the application of fluorescently-labeled glucan-specific antibodies 50 might provide one possibility to address this issue.

Disclosures
The authors declare that they have no competing financial interests and nothing to disclose.