Journal: Scientific Reports

Article Title: RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

doi: 10.1038/s41598-024-63226-1

Figure Lengend Snippet: RoPod, a toolkit comprising plant growth protocol and microscopy chamber optimized for long-term time-lapse imaging of Arabidopsis roots. ( a ) the RoPod toolkit allows to grow, treat and image seedlings within the same chamber, thus keeping plants under continuous conditions that closely resemble their standard growth in the laboratory. The use of RoPods minimizes perturbations associated with remounting seedlings from growth medium onto microscopy slides. Arabidopsis seeds are plated directly into the chamber so that emerging roots grow between the agar medium and a microscope cover slide. Roots are thus immobilized in a position suitable for microscopy. The chamber itself can be either commercial ( b ), or 3D printed custom-designed ( c–f ). Custom designed chambers are optimized specifically for Arabidopsis root growth and various types of experimental layouts. For example, a RoPod chamber v24 has lanes to guide the direction of the root growth and for easy tracking of biological replicates ( d ), a larger RoPod v26 can accommodate more seedlings and enable imaging of seedlings older than 7 days ( e ), RoPod v5 is liquid-tight and allows to perform treatment of seedlings in a vertical position ( f ). ( g–i ) Growth conditions in RoPod chambers are equivalent to standard growing conditions on Petri plates, as demonstrated by comparing root growth on the top of the medium, under the medium inside the standard Petri plate and in RoPod chambers. Seeds of wild-type (WT) and autophagy-deficient atg5-1 Arabidopsis plants expressing GFP-ATG8 were sown on the agar medium in Petri plates or RoPods. Plates and chambers were placed on SPIRO under long day conditions and imaged for a week. ( h ) Zoomed-in insets shown in ( g ). ( i ) The chart represents data from two independent experiments. The root lengths were measured on the 7th day after seed plating. One-Way ANOVA test revealed no difference in root length of seedlings for the WT seedlings grown in RoPods and on Petri plates, significance level of 0.05, n = 103.

Article Snippet: Both RoPod v24 with seedlings and glass bottom chamber with transferred seedlings were imaged at different end points using CLSM 800 (Carl Zeiss), Objective 40x/1.2W, excitation light 488 nm, and emission 505–560 nm.

Techniques: Microscopy, Imaging, Expressing

Journal: Scientific Reports

Article Title: RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

doi: 10.1038/s41598-024-63226-1

Figure Lengend Snippet: Prolonged incubation of Arabidopsis seedlings on microscopy glass upregulates autophagy in the epidermal root cells. ( a ) Arabidopsis seedlings expressing fluorescent marker for autophagy (pHusion-ATG8) in the wild-type (WT) or autophagy-deficient ( atg5/7 KO) backgrounds were grown on standard Petri plates or in the RoPod v23. 5 days-old seedlings grown on a Petri plate were mounted between a standard microscopy-grade sample slide and a cover slip in a liquid 0.5xMS medium and incubated on the bench for the designated amount of time prior to imaging using confocal microscope. Seedlings of the same genotypes grown in the RoPod were imaged using the same settings (left panel). ( b ) Quantification of pHusion-positive puncta in the root cells illustrated in ( a ) reveals gradual upregulation of autophagic activity in the roots mounted on microscopy glass. The chart comprises representative data from one out of three individual experiments. Two-tailed t-test with unequal variants, n = 35; *, p -value < 0.05.

Article Snippet: Both RoPod v24 with seedlings and glass bottom chamber with transferred seedlings were imaged at different end points using CLSM 800 (Carl Zeiss), Objective 40x/1.2W, excitation light 488 nm, and emission 505–560 nm.

Techniques: Incubation, Microscopy, Expressing, Marker, Imaging, Activity Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

doi: 10.1038/s41598-024-63226-1

Figure Lengend Snippet: Diffusion of chemical compounds in the RoPod. ( a ) Representative time lapse images of roots treated with fluorescein in the RoPod. Arabidopsis seedlings were grown in RoPods using the described protocol. Liquid MS medium containing 2 μg.ml -1 of fluorescein dye was added to the chambers immediately prior to the start of imaging. Maximum intensity projections of the fluorescent signal are shown on the top row, corresponding transmitted light images are shown in the bottom row. The circles indicate three types of regions of interest (ROI) analyzed for each root hair to produce the data presented in ( b ). Magenta circle, root hair tip; green circle, root hair base; white circle, background. ( b ) Quantification of the data illustrated in ( a ). Diffusion rate of fluorescein in the RoPod chamber demonstrates that chemical compounds reach root hair cells within 15 min of application. Data for two independent experiments is plotted for each type of analyzed ROI. ( c ) Dynamics of fluorescence accumulation at the bottom coverslip of RoPod. In this experiment liquid MS medium containing 140 μg.ml −1 fluorescein was added to RoPods containing only growth medium. The fluorescence was recorded in four separate chambers, with 2 to 3 fields of view chosen at random positions in each RoPod.

Article Snippet: Both RoPod v24 with seedlings and glass bottom chamber with transferred seedlings were imaged at different end points using CLSM 800 (Carl Zeiss), Objective 40x/1.2W, excitation light 488 nm, and emission 505–560 nm.

Techniques: Diffusion-based Assay, Imaging, Fluorescence

Journal: Scientific Reports

Article Title: RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

doi: 10.1038/s41598-024-63226-1

Figure Lengend Snippet: Monitoring sucrose effect on root hair elongation illustrates applicability of RoPod for long-term time-lapse imaging combined with treatments. Arabidopsis Col-0 WT seedlings were grown for one week in a RoPod5 using the described protocol. The growth of root hairs was firstly recorded for 4 h under control conditions. After that, the chambers were flooded with liquid 0.5 × MS (control treatment) or liquid 0.5 × MS supplemented with 1% of sucrose (sucrose treatment). ( a ) Representative images showing roots at 0 h and 8 h of treatment. Each panel is a z-projection covering 240 µm. ( b,c ) Result of root hair tip tracking for one representative root for control ( b ) and for sucrose ( c ) treatments. The final root hair length ( d ), the root hair growth rate (e) and the growth duration ( f ) were calculated as described in the Supplementary Methods. ( d–f ) The sample size used for each measurement is shown in the tables below the charts. A Dunn’s multiple comparison test, different letters designate significantly different groups, p < 0.01. The dot in the violin plots is the median of the distribution.

Article Snippet: Both RoPod v24 with seedlings and glass bottom chamber with transferred seedlings were imaged at different end points using CLSM 800 (Carl Zeiss), Objective 40x/1.2W, excitation light 488 nm, and emission 505–560 nm.

Techniques: Imaging, Control, Comparison