Noble Elizabeth, Kumar Sunil, Görlitz Frederik G, Stain Chris, Dunsby Chris, French Paul M W
Photonics Group, Department of Physics, Imperial College London, London, SW7 2AZ UK.
Department of Chemistry, Imperial College London, London, SW7 2AZ UK.
Plant Methods. 2017 Jun 15;13:48. doi: 10.1186/s13007-017-0201-7. eCollection 2017.
In order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe.
Our studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (μm), single point (1 mm) and macroscopic (~8 × 6 mm of a leaf). Multiphoton excited fluorescence lifetime imaging of leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC-a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620-710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For all these in vivo plant autofluorescence lifetime measurements, the plants were not dark-adapted.
We demonstrate that the local impact of a photosystem II herbicide on living plant leaves can be conveniently mapped in space and time via changes in autofluorescence lifetime, which we attribute to changes in chlorophyll fluorescence. Using portable fibre-optic probe instrumentation originally designed for label-free biomedical applications, this capability could be deployed outside the laboratory for monitoring the distribution of herbicides in growing plants.
为了更好地理解和改进农用化学品的作用模式，能够在体内以非侵入性方式（理想情况下是在田间）可视化它们的吸收和分布是很有用的。在这里，我们利用多光子激发荧光寿命成像、宽场单光子激发荧光寿命成像以及通过光纤探头进行的单点荧光寿命测量，探索植物自发荧光（特别是叶绿素荧光）在不同尺度上提供除草剂作用读数的潜力。
我们的研究表明，叶绿素荧光寿命的变化可以作为一种间接读数，用于在三种不同尺度上检测活体植物叶片中抑制光系统II的除草剂活性：细胞尺度（约微米）、单点尺度（约1毫米）和宏观尺度（约8×6毫米的叶片）。叶片的多光子激发荧光寿命成像表明，用抑制光系统II的除草剂氟草酮乳油处理的叶片，其空间平均叶绿素荧光寿命有所增加。未处理的叶片平均寿命为560±30皮秒，而处理后2小时成像的叶片在不同视野中寿命增加到2000±440皮秒。在440纳米激发下的活体宽场单光子激发荧光寿命成像结果表明，用氟草酮乳油处理同一叶片后，叶绿素荧光寿命从未处理叶片的521皮秒增加到3分钟后的1000皮秒，27分钟后增加到2150皮秒。活体单点荧光寿命测量也显示叶绿素荧光寿命有类似增加。在440纳米激发的叶绿素通道CH4（620 - 710纳米）中，未处理的叶片荧光寿命为435皮秒。处理后的前5分钟内，平均荧光寿命增加到1纳秒，60分钟后增加到1.3纳秒。对于所有这些活体植物自发荧光寿命测量，植物未进行暗适应处理。
我们证明，通过自发荧光寿命的变化（我们将其归因于叶绿素荧光的变化），可以方便地在空间和时间上绘制光系统II除草剂对活体植物叶片的局部影响。利用最初为无标记生物医学应用设计的便携式光纤探头仪器，这种能力可以在实验室外用于监测生长植物中除草剂的分布。
