(A) A 0.3 mm floral bud from the HTA10:RFP reporter line was embedded in media with low melting point agarose within the closed FEP/capillary system and imaged at time point 0 (MIP, upper panel; lower panel: detail of one anther). The bud was imaged again after 96 hr of cultivation in the dark at 21°C. Scale bars 100 µm. (B) A 0.3 mm bud from the HTA10:RFP reporter line was placed into the closed FEP/capillary system and imaged continuously every 1 hr. Time points 0 hr, 60 hr (onset of first meiotic division) and 96 hr were selected to compare developmental progression under regular laser illumination (MIP upper panel; lower panel: detail of one anther). Scale bars 100 µm. (C) Gantt chart depicting the developmental progression of anthers cultivated in closed capillaries. C 01–04 are four independent flowers grown outside of the microscope and imaged only at the beginning and the end of the experiment. Colors of rectangles indicate the most prominent stage of pollen development in individual anther lobes. The experiment was started with eight individual lobes (indicated by numbers 1–8), but not all of them could be scored due to technical reasons at the end of the experiment. Anther lobes that could not be scored at 96 hr are in white. S01, 03, and 04 depict the development of continuously imaged floral buds. S01 is a bud from the HTA10:RFP line, S03 and S04 from the H2B:mRuby2 ASY1:YFP line. Abbreviations: premeio. – a premeiotic stage; lepto. – leptotene, dia. – diakinesis, micro. cent. – microspore with a centrally localized nucleus. Data interpretation: In this experiment we cultivated floral buds in the closed capillary for 96 hr. Within this time, PMCs developed from the premeiotic stage to tetrads and microscpores with centrally localized nuclei. Staging of floral buds showed that tetrads and microspores with the centrally localized nuclei are prevalent in 0.6 mm floral buds grown on plants (Figure 1D). The growth dynamics experiment in Figure 1—figure supplement 1B shows that on plant, floral bud grows from the size of 0.3 mm to 0.6 mm approximately 84 hr. Thus, floral buds grown on plants and in the capillary reach the same developmental stage after 84 hr and 96 hr, respectively, indicating that cultivation in capillary leads to only a slight delay in anther development. Furthermore, there is no major difference in the development of floral buds that are continuously imaged (S01, S03, and S04) and the controls grown in capillaries outside the microscope (C01-04). Thus, phototoxicity is negligible during continuous imaging.