What Do You Use For Microscopic Photography Of Plants

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Time-lapse microscopic-photography allows in-depth phenotyping of microorganisms. Here we written report evolution of such a organization using a microfluidic device, generated from polydimethylsiloxane and glass slide, placed on a motorized stage of a microscope for conducting fourth dimension-lapse microphotography of multiple observations in 20 channels simultaneously. Nosotros take demonstrated the utility of the device in studying growth, germination and sporulation in
Fusarium virguliforme
that causes sudden decease syndrome in soybean. To measure the growth differences, we developed a polyamine oxidase
mutant in this fungus that fails to grow in minimal medium containing polyamines as the sole nitrogen source. Using this system, we demonstrated that the conidiospores of the pathogen have an average of v hours to germinate. During sporulation, information technology takes an average of 10.5 h for a conidiospore to mature and become detached from its conidiophore for the first time. Conidiospores are developed in a single conidiophore one afterward some other. The microfluidic device enabled quantitative fourth dimension-lapse microphotography reported here should be suitable for screening compounds, peptides, micro-organisms to identify fungitoxic or antimicrobial agents for decision-making serious plant pathogens. The device could also be applied in identifying suitable target genes for host-induced gene silencing in pathogens for generating novel illness resistance in crop plants.


Time-lapse microscopic-photography (micro-photography) allows taking images over a period of time for in-depth phenotyping of micro-organisms1,two,iii,four,5,6,7,eight,9. For example, this applied science has been proven useful in studying bacterium-host interactions more accurately than through electron microscopy9. Microfluidic technology allows conducting experiments in controlled environments with limited sample volumes ideal for studying responses of micro-organisms to peptides, chemical compounds or
in vitro
transcribed double-stranded RNA (dsRNA) mediated RNAi for identifying target pathogen genes for host-induced gene silencing10. Microfluidic chips often contain small channels made of biocompatible silicone (polydimethylsiloxane or PDMS), an optically clear material allowing for piece of cake visualization of biological samples in the channels. Microfluidic devices can also exist formed by attaching PDMS channels to the surfaces of other substrates such as drinking glass slide. Based on the blueprint of the channels, desired micro-environmental conditions tin can be generated on the chip. Microfluidic chips take been used in cell analysis11, drug screening12,thirteen,14, found phenotyping15,16, and microbial technologyix. Recently, microfluidic technology has besides been used to report fungi17,18,19,xx,21,22,23,24,25,26, such as
Pycnoporus cinnabarinus
Neuospora crassa

Hither we have adult a microfluidic device and showed its application in quantitative phenotyping of growth, germination, and sporulation processes of a fungal plant pathogen with the aid of a stereoscopic microscope. The microfluidic device, containing twenty channels from PDMS and drinking glass slide, was placed on a motorized stage of a stereoscopic microscope for conducting time-lapse photography of multiple observations simultaneously. We have shown that phenotypic data can be caused through fourth dimension-lapse microscopic-photography (microphotography) for growth, formation and sporulation in
Fusarium virguliforme, a fungal pathogen that causes sudden decease syndrome in soybean27,28,29,30. The quantitative mycelia growth differences were recorded for a polyamine oxidase
mutant and the complemented
mutant that grow differentially in minimal media containing polyamines as the sole nitrogen source. This organisation immune us to accurately determine the time taken by conidiospores to germinate. Furthermore, we showed that it takes x.5 h for a conidiospore to mature and get discrete from its conidiophore for the showtime time. The time-lapse video created in this study also documented that conidiospores are developed in a single conidiophore one later another. The possible applications of this microfluidic device enabled quantitative time-lapse microphotography reported here are also discussed.


Microfluidic device enabled time-lapse microphotography

The microfluidic device was developed from silicone PDMS and glass slide. It contains 20 channels, each with a dimension of 0.04 × 3 × 13 mmiii
(Fig. 1a and b). The device was manufactured using toll-effective microfabrication techniques described in Methods. The device was placed on a programmable motorized IsoPro XY stage (Leica, Wetzlar, Deutschland) interfaced with a stereoscopic MZ205 Leica microscope (Leica, Wetzlar, Germany). A digital camera DFC310 FX (Leica, Wetzlar, Germany) was fastened to the microscope (Fig. 1c). The motorized IsoPro XY stage was programmed to capture images of selected microscopic fields at a regular time-interval for collecting phenotypic observations. Thus, the microscope allowed taking fourth dimension-lapse photos of many microscopic fields for quantitative conclusion of vegetative and reproductive phenotypes of microorganisms, peculiarly fungi or oomycetes.

Figure 1: Microfluidic device-enabled fourth dimension-lapse microphotography system for studying mycelial growth, spore formation and sporulation processes in
F. virguliforme.

(a) Microfluidic device (top view) with xx channels. Each channel is three mm wide and 13 mm long. (b) Side view of the microfluidic device. Arrow shows a 40 μm deep aqueduct (cerise brown color). The spore suspensions are loaded into each channel through its inlet. The entire device is sealed with a piece of glass (1.2 mm thick) to prevent evaporation. (c) Gear up of the microfluidic assay. The sealed microfluidic device is placed on the motorized stage (shown past a white pointer) of a Leica stereoscopic microscope.

Full size paradigm

Investigation of the germination process in
F. virguliforme

To investigate the time of germination of conidiospores, spore suspensions in polyamine (PA) media, h2o, and i/3 PDB were prepared just before loading into private channels. Photos were taken in each selected microscopic field every 15 min for up to 24 h (Fig. S1; Fig. ii). Nosotros observed that the spores take three and a one-half to nine and a half hours to germinate (Supplementary Table 1). The average germination time for spores grown in PA media with spermine germinated v.29 h after being exposed to the media. In the PA media containing spermidine, the spores germinated in v.eleven h following add-on to the media. In water, the spores started to germinate in 5.17 h and in one/3 PDB spores germinate in 4.99 h following interruption to the media (Table 1). We observed no meaning difference in the average formation time of spores among the 4 media (p = 0.49). This is consistent with the germination time recorded on a microscope slide. On an average, 35% of spores were germinated 5 hours after following their suspension in the media (Fig. S2; Supplementary Table 2).

Figure 2: Germination and mycelial growth of
F. virguliforme.

figure 2

(a) Fourth dimension-lapse microscopic images showing germ tube development, elongation, and mycelial growth. (b) Growth of
F. virguliforme
Mont-1 post-obit germination of conidiospores shown in digital pixels. Arrow shows when majority of the spores started to germinate (Table 1). Data are mean ± standard errors of observations collected every 15 min. (c) Fourth dimension taken (h) past the spores to evidence first sign of germination (Supplementary Table 1).

Full size image

Table i Fourth dimension taken to for the first visible sign of formation among conidiospores.

Full size table

Nosotros likewise used the germination time to determine the variation in experimental weather condition across twenty channels. Mean and standard errors were calculated for each of the 20 channels from an experiment with PA media containing either spermine or spermidine and are presented in Fig. 3. Results indicate that in general, formation time of the coniodiospores was uniform with no pregnant differences in germination time within (p = 0.83) and between channels (p = 0.36).

Effigy iii: Variation in time taken to prove first sign of germination amidst and within channels.
figure 3

Channel numbers are shown in the left console. Data are mean and standard errors of three observations in each channel of a typical experiment. Details of the data are presented in Supplementary Tabular array one. Data are not significantly different (p = 0.83 for observations within channel and
p = 0.37 for observations amid 20 channels).

Full size paradigm

Investigation of the mycelial growth in
F. virguliforme

To investigate the utility of the device in studying the vegetative growth of the pathogen, we generated a knockout
mutant (Δfvpo1) of the
cistron encoding a polyamine oxidase that metabolizes polyamines to H2O2
and nitrogen31. A complemented
mutant (Δfvpo1::FvPO1) was also generated (Fig. S3). The mutant grows very slowly in minimal PA media containing either spermidine or spermine every bit the sole nitrogen source. The conidiospores of
were grown in randomly selected channels for up to 48 h. Digital photos of each observation were taken every 30 min and for up to 48 h; the relative mycelial growth of the ii isolates in pixels are presented in Fig. 4. As expected, retarded growth was observed in the
mutant since it cannot metabolize either spermidine or spermine as efficiently equally the
Δfvpo1::FvPO1. There is a second gene,
FvPO2, encoding polyamine oxidase in the
F. virguliforme
genome. We speculate that this gene might have been responsible for some growth that we observed in the

Effigy 4
figure 4

Differential mycelial growth of
and complemented
isolate in minimal media containing spermine.

Total size image

Investigation of the sporulation process in
F. virguliforme

To study the sporulation in
F. virguliforme, photos were taken every 1.5 h, from 24 to 120 h, following intermission of the spores in PA media. Collected images were stringed together to generate time-lapse videos (Fig. S4). The videos were analyzed individually to decide the sporulation procedure. The overall growth patterns of mycelia in two PA media from 44 to 80 h are presented in Fig. 5. Nosotros observed that the second conidiospore started to develop immediately post-obit detachment of the outset conidiospore from the conidiophore; i.due east., the conidiospores were developed on the same conidiophore one subsequently another continuously during the 120 h studied period. On boilerplate, the first conidiospore was discrete from the conidiophore in 64 h when grown in PA medium containing spermine, and in 69 h when grown in PA medium containing spermidine. The difference in time however was non statistically meaning (Tabular array 2). It took
F. virguliforme
x.7 h to develop a mature conidiospore in PA medium with spermine and 10.5 h in PA medium containing spermidine (Table 2).

Figure v: Sporulation in
F. virguliforme

figure 5

(a) Microscopic time-lapse images showing the development of a conidiospore from the outset sign of its development on a conidiophore (shown by arrows in the magnified sections of the selected area) grown in PA medium containing spermine. (b) Mycelial growth plot showing the time of the beginning disengagement of conidiospores from their conidiophores with arrows (mean ± standard error [horizontal line]). (c) Fourth dimension taken from evolution to disengagement of individual conidiospore.

Full size image

Table 2 Conidiospore formation in
F. virguliforme.

Full size table


In this study, nosotros have investigated a knockout
and complemented
mutant isolates developed through homologous recombination for studying quantitative differences in mycelial growth (Fig. S3). The mutant defective the
gene failed to produce polyamine oxidase that metabolizes polyamines such every bit spermidine or spermine and grew very slowly (Fig. 4). Our information demonstrated that the system is suitable for growing the pathogen and gathering data from 60 locations of xx channels of the microfluidic device for quantification of the vegetative growth. The pathogen was able to grow in PA media and germinate in around 5 h, which is comparable to 8 h observed for
F. graminearuim, a closely related fungus, when its spores were suspended in liquid germination medium32.

The microfluidic device adult for this written report contains 20 channels for replicated experiments. Considering the miniature nature of the device, experimental variations among and inside channels were expected to be minimal or nonexistent (Fig. 3). The variations in germination time among and within channels presumably resulted from the biological variation in the spores. Although the pathogen propagates asexually, there are physiological differences amid the spores that can impact the time of germination. We observed a range in the time taken by the conidiospores to mature (Fig. 5c). The spores could vary in size and their physiological condition, which presumably may have caused some variation in germination fourth dimension.

PDMS microfluidic devices have been widely used for the development of microfluidic platforms for cell culture and various assays33,34,35,36,37. Previous studies have shown that jail cell zipper, proliferation, differentiation, and growth rates could be affected and regulated by many factors, including the wettability (hydrophilicity and hydrophobicity), the mixing ratio of curing agent to base, and the topography and stiffness of PDMS, due to microenvironmental effects38,39,40,41. Withal, as displayed in Fig. 1b, the channels of our device were formed at the bottom of PDMS and attached to the drinking glass slide. Thus, fungi were gown on the drinking glass surface. It is noteworthy that glass is a commonly used material for cell and fungi culture plates and the chemical and mechanical properties of glass surface are stable, and then the glass substrate of our microfluidic channels is not expected to influence the growth of fungi even though the elevation and side walls of the channels were made of PDMS. In our experiment, the surface of PDMS was modified from its original hydrophobic (~110° contact angle for static water drops) to hydrophilic state via oxygen plasma handling. The surface hydrophobicity of PDMS was partially recovered as indicated by the increase in contact angle from 0 to 77° (Fig. S5). This change did not lead to whatever noticeable effects on the growth of fungi, because, as mentioned to a higher place, the fungi were actually grown on the glass surface of glass, not on the PDMS surface. In fact, when we compared the mycelial growth on glass and PSMS surfaces, no meaning deviation was observed (Fig. S6).

Time-lapse microscopy was applied in studying growth and sporulation in the bacterium,
Streptomyces coelicolor
42. In that study, however, the fourth dimension-lapse images were collected for 1 microscopic field at a time. Microfluidic device-enabled quantitative time-lapse microscopic-photography organisation reported here can capture images from xx samples simultaneously at a set interval through commands from a estimator that are integrated to a digital camera and motorized stage. Thus, this invention volition facilitate rapid objective phenotyping of non-motile microorganisms including fungi, oomycetes, bacteria, and nematodes. The device should be suitable as well for screening compounds, peptides, micro-organisms to identify fungitoxic or antimicrobial agents for controlling serious plant pathogens. The device could besides be applied in identifying suitable target genes for host-induced factor silencing in pathogens for generating novel disease resistance in crop plants.


Microfluidic Device Fabrication

The microfluidic devices used in this study were made using conventional soft lithography43. The first step was to make a master mold with photoresist SU-eight (Model SU-viii-5; Microchem, Westborough, MA, U.s.a.). The mold was fabricated past spin-coating the photoresist on a 3-inch silicon wafer (University Wafer, Boston, MA, USA) at 500 rpm for 5 sec and so at 1,000 rpm for 30 sec. The resulting thickness of the photoresist layer was 35 μm. Afterward, the wafer was prebaked on a hotplate at 65 °C for 3 min and and then at 95 °C for 7 min. The patterns of microfluidic channels were designed by AutoCAD software (Autodesk, San Rafael, CA, U.s.a.) and printed on a transparency film photomask (3,600 dpi) using a loftier-resolution plotter (Fineline Imaging, Colorado Springs, CO, USA). The photomask was put on elevation of the photoresist and exposed under ultraviolet lights (150 mJ/cmtwo) to course the patterns of microfluidic channels. Next, the patterned photoresist was further broiled on a hotplate at 65 °C for 1 min and and then at 95 °C for another 3 min. The wafer was so immersed into the developer solution of SU-8 (Microchem, Westborough, MA USA) for 6 min, followed by washing off the wafer with deionized water for vi min and then drying under nitrogen gas for 3 min. Later that, two-parts cure polydimethylsiloxane (PDMS, Sylgard 184; Dow Corning, Midland, MI, USA) forerunner solution (weighing ratio of function A:B = 10:i) was mixed and and then degassed in a bootleg chamber for 30 min under vacuum (x−iv Torr). The degassed PDMS solution was poured over the made photoresist mold in a disposable polystyrene Petri dish (100 mm diameter; Sigma-Aldrich, St. Louis, MO, USA) and baked on a hotplate at eighty °C for one h. Later on thermally cured, the PDMS device was peeled off from the master mold. For loading spore suspensions into the microfluidic channels, an inlet and an outlet were manually punched at the ii ends of each channel. Next, the PDMS slab was bonded to a glass slide (expanse: 50 mm × 75 mm; thickness: 0.9 mm; Dow Corning, Midland, MI, USA) by treating with oxygen plasma for 1 min. The device was then baked on a hotplate at 70 °C for 30 min. Finally; some other oxygen plasma treatment was given to the whole device for i min to brand the channels hydrophilic in order to facilitate loading of conidiospores into individual channels.

Microfluidic Assay

PA medium (volume: 600 μl) containing either spermidine or spermine was used to suspend the conidiospores to a final concentration of around 16 spores per microliter. Approximately 5 μl of spores were added to each of the 20 channels. The spore samples prepared from different genotypes were loaded into the channels at random. Once all channels were loaded, a second drinking glass slide (area: l mm × 75 mm; thickness: 0.9 mm; Dow Corning, United states) was sealed on superlative of the device to minimize any possible evaporation from the channels. And so the device was placed on the motorized stage of the stereomicroscope, which was connected to a computer (Dell Precision 3000, Circular Rock, TX, United states of america). LAS 10 software installed in the computer was used to program the motility trajectory of the stage and locations of microscopic fields for time-lapse imaging. 3 microscopic fields per channel containing 1 or two spores were marked using the LAS Ten software (Fig. 1). The same software was used to take images from the selected 3 microscopic fields from each of the 20 channels simultaneously at a set up interval using a digital DFC310 FX, Leica camera (Leica, Wetzlar, Germany).

The images were arranged in the correct sequence and compressed into the Windows Media Video (.wmv) format. A MATLAB lawmaking (http://www.memslab.internet/uploads/1/1/five/v/11554938/matlab_codes.docx) was used to analyze the images past comparing changes in pixels from paradigm to image to determine how much growth occurred in each marked field. A graph was produced to show the growth changes over time and videos were analyzed to study germination and sporulation processes in
F. virguliforme.

F. virguliforme

The virulent
F. virguliforme
Mont-1 strain was obtained a unmarried-spore isolate of
F. virguliforme
nerveless from Illinois in 1991. The isolate was initially obtained on modified Nash and Snyder medium (MNSM; four) and then maintained on Bilay medium. Before inoculation, cultures of the isolate were grown on potato dextrose agar at room temperature (24 ± 2 °C) in darkness for 14 days. Knockout
mutants were created using the homologous recombination protocol described by Pudake and co-workers44. Two regions, approximately 1 kb five′- and 3′-end of the
gene were PCR amplified (Supplemental Tabular array 4) using Cx PFU Turbo Taq polymerase (Agilent Technologies, Santa Clara, CA). The ii PCR fragments were cloned into the pRF-HU2 binary vector44
using the USER enzyme mix (New England Biolabs, Inc, Ipswich, MA). The resultant plasmid was transformed into
Agrobacterium tumefaciens
EHA-105 strain. Positive EHA-105 colonies were grown overnight at 28 °C in Aye medium (Common cold Spring Harbor Protocol, 2006) amended with kanamyacin sulfate (50 μg/ml) and rifamipicin (25 μg/ml). The cultures were used to inoculate IMAS medium44
containing kanamycin sulfate (fifty μg/μl) and grown till OD600
between 0.five and 0.7. The cultures were then mixed with
F. virguliforme
spores (2 × tenhalf-dozen
spores/ml) in a i:1 ratio. The mixture was then plated on blackness filter paper (Whatman, GE Healthcare Bio-Sciences, Pittsburgh, PA) layered over IMAS plates. The Filters were transferred to DFM plates44
containing hygromycin (150 μg/ml) and cefotaxime (300 μg/ml) later 3 days co-civilization. After three–5 days on the DFM plates, the filters were moved to new DFM plates containing hygromycin (150 μg/ml) and cefotaxime (300 μg/ml). Colonies were selected and screened using PCR and Southern absorb assaytwo
for presence of the hygromycin resistance
gene and absence of the
gene. For
complementation, a fragment containing 3,594 bp including the
five′-cease region, gene and 3′-end region and the 1 kb fragment containing the
3′-end sequence was amplified using the Cx PFU Turbo Taq polymerase. The fragments were cloned into the pRF-HU2 vector with the hygromycin resistance factor replaced with the geneticin resistance gene using the USER enzyme mix. The resulting plasmid was transformed into
A. tumefaciens
strain EHA-105 and positive colonies were selected and confirmed for stability of the plasmid construct in the
A. tumefaciens
by conducting brake digestion and gel assay of the restriction enzyme-digested plasmid.
mutant spores were transformed with the construct following the protocol described above for generating the
mutant. The transformants were selected on geneticin (150 μg/ml) containing plates. The resulting colonies were verified using PCR to demonstrate the presence of the
and geneticin resistance genes. Knockout mutant and complemented
F. virguliforme
isolates were checked for polyamine oxidase activity on minimal media containing spermine and spermidine as the sole nitrogen source to confirm the loss of polyamine oxidase function in the knockout
mutant and regain of the activity in a complemented a
mutant (Fig. S4).

Preparation of
F. virguliforme

F. virguliforme
isolate Mont-1 was grown on solid Bilay medium (0.1% KH2PO4
[wt/vol], 0.one% KNO3
[wt/vol], 0.05% MgSOfour
[wt/vol], 0.05% KCl [wt/vol], 0.02% starch [wt/vol], 0.02% glucose [wt/vol], and 0.02% sucrose [wt/vol]) and transferred to solid ane/three PDA medium (0.9% PDA [wt/vol])45
a beta-1,3-glucan synthase, is involved in prison cell wall integrity, hyperosmotic pressure tolerance and conidiation in Metarhizium acridum. Plates were incubated at room temperature in the dark. After 2–3 weeks the plates were washed with ii mL autoclaved, double distilled water and placed in a clean one.seven mL Eppendorf tube. The tubes were and so centrifuged for ten sec at maximum speed to pellet the spores. The pellets were and then re-suspended in autoclaved, double distilled water (dd H2O), repeated two more times to eliminate whatsoever leftover nutrients from the 1/3 PDA plates and finally resuspended in 1 ml dd H2O. A 1:100 dilution of the spore pause was prepared in the respective PA medium to count in a hemocytometer. Two readings were taken and averaged to become a more accurate concentration. The spore suspension was diluted in PA medium (one% glucose [wt/vol], 0.02% MgSO4
7H2O [wt/vol], 0.3% KH2PO4 [wt/vol], 0.1 mL Trace Elements) containing spermine (500 μM) or spermidine (690 μM), 1/3 PDB (8.8 g spud dextrose broth in 1 L dd H2O), or dd HiiO. The diluted spore suspensions were immediately loaded into individual channels of the microfluidic device.

Fungal growth comparison

Glass Petri dishes (xc mm diameter) with PDMS were created past pouring ii ml of PDMS precursor solution (weighing ratio of part A:B = 10:i) into each drinking glass Petri dish and cured at 80 °C for 1 h. Spores were harvested from two-week onetime
F. virguliforme
civilization grown on 1/three PDA plates. The spores were diluted to a final concentration of i × 10iv
spores per ml in 1/iii PDB. The spore break of 12 ml was added to each of six drinking glass Petri dishes or six glass Petri dishes carrying a layer of PDMS. The plates were incubated at room temperature in night atmospheric condition. Fungal mycelia were harvested 48 and 72 hours following incubation. For fungal DNA quantification, 5 ml of the mycelial intermission was centrifuged at full speed and the supernatant was removed. To determine fungal dry out weights, 5 ml of the homogenized mycelial break from each plate was centrifuged to obtain mycelial pellets. Samples were transferred to pre-weighed 1.7 ml Eppendorf tubes. The samples were washed three times with 1 ml double distilled HiiO. The samples were placed in an 80 °C oven and dried until constant weight was obtained. Weights were taken after 24 and 48 hours of drying. Pupil’southward t-test was conducted to determine the statistically significant difference in mycelial growth betwixt treatments. 3 replications were conducted for each treatment.

Boosted Information

How to cite this commodity:
Marshall, J.
et al. Microfluidic device enabled quantitative time-lapse microscopic-photography for phenotyping vegetative and reproductive phases in
Fusarium virguliforme, which is pathogenic to soybean.
Sci. Rep.
7, 44365; doi: ten.1038/srep44365 (2017).

Publisher’s annotation:
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


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This work was supported by the National Institute of Food and Agriculture (NIFA), U.s. Department of Agriculture (Grant no. 2022-68004-20374), U.S. National Science Foundation (Grant no. NSF-DBI-1353819), Department of Agronomy and the Plant Sciences Establish at the Iowa State University.

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1000.1000.B. and L.D. conceived the idea. J.1000. and J.B. performed the biology experiments. X.Q. and 50.D. built the hardware platform for the methodology. 10.Q. and J.X. developed the software for the methodology. J.M., M.K.B., and J.B. analyzed data. J.G., Thousand.K.B., J.B., X.Q. and L.D. wrote the manuscript.

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Correspondence to Liang Dong or Madan Yard. Bhattacharyya.

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Marshall, J., Qiao, X., Baumbach, J.
et al.
Microfluidic device enabled quantitative time-lapse microscopic-photography for phenotyping vegetative and reproductive phases in
Fusarium virguliforme, which is pathogenic to soybean.
Sci Rep
44365 (2017). https://doi.org/ten.1038/srep44365

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