In-situ Real-time Field Imaging and Monitoring of Leaf Stomata by High-resolution Portable Microscope

Stomata, functionally specialized micrometer-sized pores on the epidermis of leaves (mainly on the lower epidermis), control the flow of gases and water between the interior of the plant and atmosphere. Real-time monitoring of stomatal dynamics can be used for predicting the plant hydraulics, photosensitivity, and gas exchanges effectively. To date, several techniques offer the direct or indirect measurement of stomatal dynamics, yet none offer real-time, long-term persistent measurement of multiple stomal apertures simultaneously of an intact leaf in a field under natural conditions. Here, we report a high-resolution portable microscope-based technique for in situ real-time field imaging and monitoring of stomata. Our technique is capable of analyzing and quantifying the multiple lower epidermis stomal pore dynamics simultaneously and does not require any physical or chemical manipulation of a leaf. An upward facing objective lens in our portable microscope allows the imaging of lower epidermis stomatal opening of a leaf while upper epidermis being exposed to the natural environment. Small depth of field (~ 1.3 m) of a high-magnifying objection lens assists in focusing the stomatal plane in highly non-planar tomato leaf having a high density of trichome (hair-like structures). For long-term monitoring, the leaf is fixed mechanically by a novel designed leaf holder providing freedom to expose the upper epidermis to the sunlight and lower epidermis to the wind simultaneously. In our study, a direct relation between the stomatal opening and the intensity of sunlight illuminating on the upper epidermis has been observed in real-time. In addition, real-time porosity of leaf (ratio between the areas of stomatal opening to the area of a leaf) and stomatal aspect ratio (ratio between the major axis and minor axis of stomatal opening) along with stomatal density have been quantified.