High-resolution Portable Microscope for in situ Real-time Monitoring of Stomata and other Biological & Non-biological Applications

Abstract

Commercial high-resolution optical microscopes are essential for microscopy imaging; however, they are expensive and bulky, which limits their use in point-of-care devices, resource-limited areas, and real-time imaging of a sample in a large apparatus. In this study, we report a novel compact lightweight submicron-resolution reflected and inverted optical microscope at low cost. Our technique utilizes the proximity of the image sensor to a commercial microscope objective lens for compactness of the microscope. The use of an image sensor with a small pixel size helps to reduce the information loss, which provides high-resolution images. Moreover, our technique offers a freedom to tailor the design of microscope according to the required resolution, cost, and portability for specific applications, which makes it a suitable candidate for affordable point-of-care devices. Unlike conventional reflected microscope, which consists of downward facing objective lens, our portable reflected microscope is designed with upward facing objective lens. Such configuration with high-resolution imaging capability and portability makes it suitable for imaging of abaxial (lower side) surface of a plant leaf. Stoma, functionally specialized micrometer-sized pores on the epidermis of leaves (mainly on the lower epidermis) were observed and analyzed. Since stomata 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. 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 μm) 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. Therefore, this portable and inexpensive microscopeic technique provides the essential functionalities of a bulky expensive high-performance microscope along with the specific applications at a lower cost. Images of several micron-to-submicron scale patterns and spherical beads are acquired to observe the resolution and quality of the images obtained using our microscope. In addition, we demonstrate the applications of our microscope in various fields such as recording of high-speed water microdroplet formation inside a microfluidic device, high-resolution live cell imaging inside an incubator, and real-time imaging of crack propagation in a sample under stretching by a material testing system (MTS).