Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

Summary

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

Abstract

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Introduction

Filamentous fungi are of great socioeconomic and ecological importance, being both crucial as industrial/agricultural tools for enzyme and antibiotic production^1,2 and as pathogens of crop plants³, pest insects⁴ and humans³. Moreover, filamentous fungi such as Aspergillus nidulans are widely used as model organisms for fundamental research, such as studies in genetics, cell and evolutionary biology as well as for the study of hyphal extension⁵. Filamentous fungi are highly polarized organisms that elongate through the continuous supply of membrane lipids/proteins and the de novo synthesis of cell wall at the extending tip⁶. A central role in the hyphal tip growth and polarity maintenance is a specialized structure named 'Spitzenkorper' (SPK), a highly ordered structure consisting mostly of cytoskeletal components and the polarized distribution of the Golgi^6,7,8.

Environmental stimuli/signals, such water-air interface, light, CO₂ concentration, and the nutritional status are responsible for the developmental decisions made by these molds⁹. In submerged (liquid) cultures the differentiation of A. nidulans is repressed and growth occurs by hyphal tip elongation⁶. During vegetative growth, asexual spores (conidia) germinate by apical extension, forming an undifferentiated network of interconnected hyphal cells, the mycelium, which may continue to grow indefinitely as long as nutrients and space are available. On the other hand, on solid media hyphal tips elongate and after a defined period of vegetative growth (developmental competence), asexual reproduction is initiated and aerial conidiophore stalks extend from specialized foot cells of the mycelium⁶. These give rise to specialized developmental multicellular structures called conidiophores, which produce long chains of haploid conidia¹⁰ that can restart growth under favorable environmental conditions.

A widely used method for measuring filamentous fungal growth is to inoculate spores on nutrient agar contained in a Petri dish and macroscopically measure the diameter of the colony a few days later¹¹. The diameter/area of the colony, most dependent on changes in mycelial growth rate and less on conidiophore density¹², is then used as a value of growth. Although, measuring fungal population (colony) size growing on solid surfaces is quite adequate, it is by no means the most accurate measure of growth. Compared to population level averages (averages of fungal colony size), single cell measurements can capture the heterogeneity of a cell population and allow identification of novel sub-populations of cells, states¹³, dynamics, pathways as well as the biological mechanisms by which cells respond to endogenous and environmental changes^14,15. Monitoring fungal cell growth and phenotype by time-lapse microscopy is arguably the most widely employed quantitative single cell observation approach.

Herein, we detail a label-free live imaging protocol using transmitted light microscopy techniques (such as phase-contrast, differential interference contrast (DIC), and polarized microscopy) to capture images, which independently of the combined use of fluorescence microscopy can be employed to analyze and quantify polar growth of A. nidulans strains in both submerged cultures and solid media.

Protocol

1. Inoculum preparation

NOTE: All steps should be performed under a laminar flow cabinet.

1. Streak out fungal strain of interest, from a glycerol stock (-80 °C) using a sterile inoculation loop, onto plates of minimal media (MM) supplemented with the appropriate nutritional requirements relevant to the strain examined [MM: 10.0 g/L glucose, 20 mL/L salt solution (salt solution: 26 g/L KCl, 26 g/L MgSO₄·7H₂O, 76 g/L KH₂PO₄, 2.0 mL/L chloroform) and 1 mL/L trace elements (trace elements: 40 mg/L Na₂B₄O₇·H₂O, 400 mg/L CuSO₄, 8 g/L ZnSO₄, 800 mg/L MnSO₄, 800 mg/L FePO₄), adjust pH to 6.8 with 1 M NaOH, add 1 % (w/v) agar and the required supplements as described in¹⁶ and autoclave] (Figure 1).
   NOTE: Salt solution, trace elements solution and supplements are autoclaved.
2. Incubate for 2-3 days at 37 °C.
3. Use a sterile toothpick (or an inoculation loop), transfer a small number of conidia, by gently touching a single colony, to plates of complete media (CM) [CM: 10.0 g/L glucose, 2.0 g/L peptone, 1.0 g/L yeast extract, 1.0 g/L casamino acids, 20 mL/L salt solution, 1 mL/L trace elements, 5 mL/L vitamin solution (vitamin solution: 0.1 g/L riboflavin, 0.1 g/L nicotinamide, 0.01 g/L p-amino benzoic acid, 0.05 g/L pyridoxine HCl, 1.0 mg/L biotin), adjust pH to 6.8 with 1 M NaOH, add 1 % (w/v) agar and the required supplements as described in¹⁶ and autoclave]. Autoclave and store the vitamin solution in a dark bottle at 4 °C.
   NOTE: In case fungal growth is too dense to identify and isolate individual colonies, re-streak onto a new agar plate to obtain single colonies.
4. Incubate for 3-4 days at 37 °C.
5. Obtain a conidial suspension of approximately 2 x 10⁶ cells/mL by scratching 1 cm from the surface of a conidiated fungal colony grown on CM agar plates, using a sterile toothpick (Figure S1).
   NOTE: When necessary, count conidia with a hemocytometer.
6. Harvest conidia of A. nidulans in a sterile 1.5 mL centrifuge tube with 1.0 mL of autoclaved distilled water containing 0.05% (v/v) Tween 80 for reducing the number of conidia clumps.
   NOTE: Conidia can be stored for up to 2-3 weeks at 4 °C without a relevant loss of viability (A. Athanasopoulos and V. Sophianopoulou, unpublished data). However, it is recommended to filter and/or to wash conidial suspension to remove mycelial parts and nutrients, in order to prevent conidial swelling.

2. Preparation for imaging filamentous fungi growing on agar (solid) mediums

NOTE: A modified version of the 'inverted agar method^17,18 is used.

1. Initially, spot 10 µL aliquots of vigorously vortexed conidial (approximately 2 x 10⁴ cells/mL) at several points onto Petri dishes (Ø9 cm) 15 mL of MM with 1% (w/v) agar (Figure 2).
   NOTE: Using the modified version of the 'inverted agar method', it is possible to image fungal samples for many hours without apparent deleterious effects on growing hyphae.
2. Incubate the experimental culture according to the developmental stage intended to be investigated.
3. Slice out a ≈0.8 mm² block of agar containing the colony using a sterile scalpel.
   NOTE: The dimensions of the agar block to be sliced out depends on the dimensions of the equipment to be placed afterwards. In the present work, 8 well µ-slides are used (see below).
4. Invert and place the agar block into a well of an µ-slide or similar 8 chambered coverglass with coverslip suitable for live imaging.
   ​NOTE: In case transmitted light microscopy will be used in combination with fluorescence (labeling) microscopy, the agar block can be inverted onto a droplet of liquid medium containing the live cell staining dye, just before imaging.

3. Preparation for imaging filamentous fungi growing on liquid medium

1. Transfer 10 µL aliquots of a vigorously vortexed conidial suspension (approximately 2 x 10⁴ cells/mL) in the wells of an 8 well µ-slide containing 200 µL of (liquid) MM with the appropriate supplements (see above).
2. Incubate for the desired time at the desired temperature (Figure 3).
   ​NOTE: If transmitted light microscopy will be used in combination with fluorescence (labeling) microscopy, liquid cultures have the great advantage that fluorescent dyes can be added at any desired time point during the experiment¹⁹.

4. Capture images

NOTE: The choice of microscope depends upon the available equipment. In any case the microscope setup should include an inverted stage, an environmental chamber or at least a room with precise air temperature control.

1. Preheat the thermostated microscope chamber at 37 °C (unless otherwise indicated or as suitable for the used fungal species) to stabilize the temperature before starting. This chamber allows temperature modulation of the microscope optics and sample stage during time-lapse experiments. Be aware that optical aberrations²⁰ are introduced when normal immersion oils (designed for use at 23 °C) oils are used at 37 °C or above.
   NOTE: On a tight budget, incubation chambers can be made out of cardboard and insulating packing material²¹ or by using a 3D printer²².
2. Turn on the microscope, the scanner power, the laser power and computer, and load the imaging software. Place the µ-slide (prepared previously) in the microscope stage and focus.
3. Find fields of view that contain isolated/not overlapping cells (or at least not overcrowded), in order to facilitate growth measurements during image analysis. Capture at least 50 growing cells per sample to allow robust statistical analysis.
4. Select the desired transmitted light microscopy approach. Reduce the exposure time or laser power and pixel dwell time and/or increase pinhole diameters, in order to minimize photobleaching of fungal cells, as described elsewhere ^23,24.
5. Set microscope to acquire images at desired time intervals and start time series acquisition.
   ​NOTE: To correct for focal drift over time (especially for long experiments), due to thermal drift, diverse cell sizes, and cell motion use an autofocus strategy if available in your microscope software.

5. Image Analysis

NOTE: This section describes the key steps of processing time-lapse microscopy images for measuring growth rate of A. nidulans. Opening, visualization and processing of images is accomplished with the open source ImageJ/Fiji software²⁵.

1. Import the images to Fiji using Plugins | Bio-Formats | Bio-Formats Importer from the Fiji menu with default settings (Figure 4A).
   NOTE: Check whether the Bio-Formats Importer properly recognize the image calibration. The picture dimension shown in the upper information field image window, must be equal to the original picture dimensions (Figure 4B). Press Shift + P to display and change image properties in ImageJ/Fiji software.
2. Where needed, use histogram matching²⁶ for illumination correction between different frames (Image | Adjust | Bleach Correction | Histogram Matching) (Figure 4C).
3. Where needed, use SIFT-algorithm (Plugins | Registration | Linear Stack Alignment with SIFT) for aligning or matching image stacks (Figure 4D). Selecting "Translation" from the expected transformation menu should be enough to correct any x-y drift.
   NOTE: Other plugins can be also used to align a stack of image slices, such as Image Stabilizer (https://imagej.net/Image_Stabilizer) or StackReg (http://bigwww.epfl.ch/thevenaz/stackreg/).
4. Select hyphae that grow parallel to coverslip, avoiding those that are tilted. Be sure to select hyphae that propagate by polar extension and avoid hyphae presenting lateral and/or apical branching.
5. Use MTrackJ (Plugins | MTrackJ) plugin to track growing hyphal tips (Figure 4E)²⁷. To add a track, select the Add button in the toolbar and place the first point at a hyphal tip using the left click of the mouse. The time series will automatically move to the next frame. To complete the tracking process, double click the mouse on the final point (or press the Esc key) (Figure 4F). Move to another point of interest (i.e., growing hyphal tip) in the initial time frame and restart the procedure by measuring growth rate of another hypha.
   NOTE: To install MTrackJ follow the instructions presented at https://imagescience.org/meijering/software/mtrackj/
6. Click the Measure button in the MTrackJ dialog (Figure 4G) to open the output table. Save track measurements (File | Save As) to the desired file format (e.g., csv), analyze and plot them (Figure 4H).
   NOTE: By selecting the Movie button, a movie is produced showing the image and track progression.

Representative Results

Following this protocol, we captured and analyzed various images corresponding to different growth/developmental stages of the filamentous fungus A. nidulans. The data presented in this study were processed and analyzed using the Fiji software. Measurements were saved as csv files, statistically analyzed and prepared as graphs using commercial statistical software and/or Python programming language using software libraries like pandas, numpy, statsmodels, matplotlib and seaborn. More details can be found in the cited original publications.

In order to interpret the response of populations, it is important to determine heterogeneity of key parameters within the populations and to efficiently identify physiologically distinct subpopulations²⁸. Figure 5 shows growth of the azhAΔ ngnAΔ mutant strain, bearing deletions in two genes, the products of which are implicated in the detoxification and assimilation of the toxic phytoproduct L-azetidine-2- carboxylic acid²⁹, in comparison with the WT strain. Measuring their growth rate in submerged liquid cultures, we detect a statistically significant lower growth rate of the double mutant compared to the WT strain (t(715) = 20.61, p = <0,0001) (Figure 5A-B). This difference in growth was not detectable measuring only the colony area of these strains (Figure 5C-D), emphasizing that microscopic single cell analysis is more effective in determining small differences in growth rates that are difficult to detect with macroscopic observation of the colony.

Single cell long-term live imaging is of significant value in the effort to obtain spatial and temporal information of cellular protein dynamics. Long-term live cell imaging (Figure 6, Video 1) of conidial germination co-expressing the GFP-labeled core eisosomal protein PilA and the mRFP histone H1 shows that PilA³⁰ forms static structures with low mobility at fungal plasma membrane^31,32. Moreover, Figure 7 shows that fluorescent dyes can be used for live imaging of strains grown on agar medium, in order to study dynamics of organelles and cell structures. Here, FM4-64³³ was used to visualize mitochondrial network and the vacuolar system in A. nidulans.

Figure 1: Schematic representation of the inoculum preparation procedure. Please click here to view a larger version of this figure.

Figure 2: Schematic representation of the procedure followed for imaging filamentous fungi growing on agar medium. Please click here to view a larger version of this figure.

Figure 3: Schematic representation of the procedure followed for imaging filamentous fungi growing in liquid medium. Please click here to view a larger version of this figure.

Figure 4: Schematic representation of image analysis procedure. Steps for processing time-lapse microscopy images and measuring growth rate of A. nidulans. Please click here to view a larger version of this figure.

Figure 5: Comparing traditional population measurements with single cell measurements. (A) Representative confocal microscopy images of azhAΔ ngnAΔ double deletion and WT strains, grown in submerged liquid cultures containing urea as sole nitrogen source at 25 °C. All strains were incubated for a total of 18 h, at 25 °C and frames were captured at 15 min intervals. Images of 2048 × 2048 pixels were collected with a pixel size of 115.02 x 115.02 nm using a TCS SP8 MP (Leica, Germany) microscope equipped with HC Plan APO 63x, N.A. 1.40 oil immersion objective. (B) Measurements of (A) are plotted in box-and-whiskers plots. Statistical significance was analyzed via t-Test and is depicted with asterisks (***), indicating p < 0.001. (C) Growth at 25 °C of WT and double deletion strain on solid MM. Colonies were photographed at different time intervals (18 h, 36 h and 72 h), spatially calibrated and measured manually with a ruler in ImageJ program. (D) Measurements of the area of colonies presented in (C) are plotted as box-and-whiskers plots. Differences of the area of colonies were not statistically significant between the different time points of the double mutant compared to the WT strain (18h: t(8) = 0.69, p = 0.50, 36h: t(6) = 0.27, p = 0.5068, 72h: t(6) = 0.39, p = 0.70 ). Please click here to view a larger version of this figure.

Figure 6: Long-term live cell imaging of conidial germination co-expressing proteins of interest fused with genetically encoded fluorescent tags. (A) Maximum intensity projections (MIP) of 4 slices generated in the z plane, of a strain co-expressing PilA-GFP and H1-mRFP. Conidia were spotted on agar medium and incubated for approximately 1 h at 30 °C in order to help adhesion of the cells to the agar. The agar block containing conidia was sliced out, placed into wells of a µ-slide and incubated for a total of 18 h, at 30 °C. Frames were captured at 25 min intervals, using a TCS SP8 MP (Leica, Germany) microscope equipped with HC Plan APO 63x, N.A. 1.40 oil immersion objective (with a pixel size of 92.26 x 92.26 nm). Images were obtained by exciting GFP at 488-nm wavelength and detecting fluorescence in the spectral band ranging from 495 to 558 nm, and by exciting mRFP at 561-nm wavelength and detecting fluorescence in the spectral band ranging from 580 to 660 nm. (B) Measurements (velocity of germination and hyphal tip extension) of (A) are plotted as line graph. Please click here to view a larger version of this figure.

Figure 7: FM4-64 labeling of hyphae growing on solid medium. Conidia of a WT strain were cultivated on 1% agar MM Petri dishes for 16 h at 30 °C. The growing germlings were incubated for 20 min with 10 µL of MM containing 10 µM FM4-64. Following incubation, an agar block containing stained germlings was sliced out, placed into 8 µ-slide followed by a 47 min chase at 30 °C. Frames were captured every 7 min at 30 °C using a TCS SP8 MP microscope equipped with HC Plan APO 63x, N.A. 1.40 oil immersion objective (with a pixel size of 184.52 x 184.52 nm). Images were obtained by exciting FM4-64 at 514-nm wavelength and detecting fluorescence in the spectral band ranging from 596 to 682 nm. The FM4-64 fluorescence signal is shown as inverted image. Please click here to view a larger version of this figure.

Figure 8: Early stages of conidiophore formation. (A) Conidia of WT strain were cultured on agar medium for 136 h at 30 °C, sliced out and placed into wells of a µ-slide. Frames were captured every 20 min. Images of 2048 × 2048 pixels were collected with a pixel size of 245.2 x 245.2 nm using a TCS SP8 MP (Leica, Germany) microscope, equipped with HC Plan APO 63x, N.A. 1.40 oil immersion objective. Images show the formation of conidiophore vesicles (black arrow) as well as the formation (brown arrow) and maturation of metulae (blue arrow). (B) Measurements (velocity of germination and unipolar hyphal tip extension) of (A) are plotted as line graph. Please click here to view a larger version of this figure.

Video 1: Long-term live cell imaging of conidial germination co-expressing PilA- GFP and H1-mRFP. Please click here to download this Video.

Video 2: Early stages of conidiophore formationin A. nidulans. Please click here to download this Video.

Figure S1: Preparation of conidial suspension. By scraping with a sterile toothpick an approximately 1 cm² surface of a conidiated plate, a suspension of approximately 2 x 10⁶ conidia/ml is obtained. Please click here to download this File.

Discussion

Monitoring fungal cell growth and phenotype by time-lapse microscopy is a powerful approach to assess cellular behavior in real-time and quantitatively and accurately determine whether a particular drug treatment and/or genetic intervention results in detectable cell growth or phenotypic differences over time.

In this study, a reliable live-cell imaging methodology was described to measure and quantitatively analyze fungal development, including the dynamics of germ tube and hyphal tip growth in A. nidulans. Monitoring morphological changes over time, using transmitted light microscopy techniques, can be highly helpful to identify cells that are stressed, dying or dead. A detailed knowledge of such dynamic processes at the single-cell level allows to assess heterogeneity within a mixed population of cells and in a long-time perspective, permits the identification of pathways and detailed mechanisms by which cells respond to endogenous and environmental signals.

One advantage of this method is that it is based on a label free imaging approach (which nevertheless can be easily combined with fluorescence microscopy) that uses inherent light refraction properties of cells to create image contrast without introducing dyes/labels, which may confound the results. While fluorescent markers can be used to tag cellular compartments and proteins and significantly facilitate the segmentation and tracking of cells, transmitted light microscopy circumvents the need for genetically engineering cells, enabling researchers to avoid the cost and the time-consuming dye/label optimization and also to avoid probable phototoxicity effects coming from fluorescence imaging^13,34.

This protocol is suitable to study growth kinetics of fungi that form hyphae or pseudohyphae. Furthermore, this approach is highly suitable for imaging different cell structures of different strains and under different developmental stages. In Figure 8 and Video 2, we present the initial stages of A. nidulans conidiophore (i.e., structures bearing asexual spores) development. It must be noted, however, that, this method is not the most suitable for imaging conidiophore formation, since carbon/nitrogen starvation and air exposure, both necessary for the development of conidiophores, cannot be standardized by our approach³⁵.

In addition, this protocol is poorly suited to track fungal biofilms, which are subject to vertical extension forming a dense heterogeneous, surface-associated colony comprised of filamentous hyphae, pseudohyphal cells, yeast-form cells, and various forms of extracellular matrix³⁶. Another limitation of the technique is that although label free microscopic analysis represents a precise method for the qualitative and quantitative analysis of germination/differentiation dynamics, manual evaluation of such data is time-consuming especially when toο many strains or/and conditions are examined. Thus, data from high-throughput time-lapse imaging microscopy should be analyzed by suitable computational software enabling automated or semi-automated image analysis of hyphal development and conidial germination^37,38,39.

In summary, herein we present a protocol for analyzing fungal growth kinetics in a reproducible and reliable manner without the need of any prior image analysis experience from the user. This protocol allows objective and accurate quantification of fungal growth and differentiation, and provides a complementary imaging approach to study fungal life cycles and fungal pathogenicity³⁷.

Materials

Name	Company	Catalog Number	Comments
µ-Slide 8 Well	Ibidi	80826	Imaging slides
4-Aminobenzoic acid	Merck	A9878
azhAΔ ngnAΔ			Genotype: zhAΔ::pyrGAf; ngnAΔ::pyrGAf; pyroA4 pantoB100 / References:Laboratory collection, Athanasopoulos et al., 2013
Bacto Casamino Acids	Gibco	223030
Biotin	Merck	B4639
Chloroform	Merck	67-66-3
Copper(II) sulfate pentahydrate	Merck	C8027
Glucose	Merck	G8270
GraphPad Prism 8.0	GraphPad Software		Statistical Software
ImageJ	NIH		Image processing and analysis software
Inoculating Loop	Merck	I8263-500EA
Iron(III) phosphate	Merck	1.03935
Leica Application Suite X	Leica Microsystems		Microscope software
Magnesium sulfate heptahydrate	Merck	63138
Manganese(II) sulfate monohydrate	Merck	M7899
Microscope Leica TCS SP8	Leica Microsystems
Nicotinamide (Niacinamide)	Supelco	47865-U
Peptone	Millipore	68971
Petri Dishes for Microbiology Culture	KISKER	G090
Potassium chloride	Merck	P4504
Potassium phosphate monobasic	Merck	P5655
Pyridoxine hydrochloride	Merck	P6280
Quali - Microcentrifuge Tubes, 1,7 mL, DNase-, RNase and pyrogen free, sterile	KISKER	G052-S
Quali - Microcentrifuge Tubes, 2.0 mL, sterile	KISKER	G053-S
Quali - Standard Tips, Bevelled, 100-1000 µL	KISKER	VL004G
Quali - Standard Tips, Bevelled, 1-200 µL	KISKER	VL700G
Quali Microvolume Tips, DNase-, RNase free, 0,1-10 µL/clear	KISKER	GC.TIPS.B
Riboflavin (B2)	Supelco	47861
Scalpel blades NO. 11	OdontoMed2011	S2771
Sodium chloride	Merck	S7653
Sodium hydroxide	Merck	S8045
Sodium tetraborate decahydrate	Merck	S9640
VS151 (PilA-GFP and H1-mRFP)			Genotype: pyrG89; pilA::sgfp::AfpyrG+ argB2 nkuAΔ::argB+  pyroA4 hhoA::mrfp::Afribo+ riboB2 / References:Laboratory collection, Biratsi et al., 2021
WT			Genotype: nkuAΔ::argB; pyrG89; pyroA4;pyrG89 / References: TN02A3 -FGSC A1149
Yeast Extract	Millipore	70161
ZnSO4