Real-time automated characterization of 3D morphology and mechanics of developing plant cells

Abstract

In this article, we introduce the real-time cellular force microscope (RT-CFM), a high-throughput microrobotic platform for mechanical stimulation and characterization of single cells. We developed computer vision algorithms that fully automate the positioning of target cells and localization of the sensor tip. The control and acquisition architecture dramatically increases the accuracy, speed, and reliability of force measurements. Pollen tubes provide an ideal model system for the study of plant mechanics at the single-cell level. To quantitatively obtain the physical properties of the plant cell wall, we generated topography and stiffness measurements from 3D scans of living, growing pollen tubes. We report techniques for real-time monitoring and analysis of intracellular calcium fluxes during mechanical intervention. Our platform is compatible with various imaging systems and enables a powerful screening technology to facilitate biomechanical and morphological characterization of developing cells.

Massively Parallelized Pollen Tube Guidance and Mechanical Measurements on a Lab-on-a-Chip Platform

“…The need for computer-vision assisted automation to ‘track multiple, overlapping pollen tube trajectories in fluorescent time-lapse images’ was raised at the Third Annual Pollen RCN Meeting in 2013 [4]. Real-time automation methods for micro-indentation and optical monitoring have recently been introduced [5,6], but they require costly hardware accessories to existing microscopes. Conventional in vitro assays lack the precise spatiotemporal control of electro-chemical stimuli in the microenvironment of the growing cells needed to study cell-cell signaling and chemo-electro tropism and guidance mechanisms, which are key to successful in vivo fertilization.…”

“…The throughput of most existing LoC-based assays is restricted, however, as only a limited number of pollen tubes could be incorporated, guided, and observed on the chip at a time. There have been attempts at LoC-based systems for mechanical characterization of pollen tubes, but they also suffer from low-throughput [14,17] and their closed-cell architecture does not allow interfacing to calibrated micro-indentation [5], micro-gripping [18,19], micro-injection [20], or nano-indentation [21] systems for quantitative biomechanical characterization of the cell wall and cytoplasm.…”

“…We have demonstrated the unidirectional growth of hundreds of lily and Arabidopsis pollen tubes with no significant changes in growth parameters such as morphology, germination, and growth rates as compared to conventional in vitro plate culture. We demonstrate the integration of the LoC device with the CFM [5,33,34] to characterize the cell wall stiffness of lily pollen tubes. The high-throughput mechanical measurements of the LoC-CFM combination in conjunction with FEM modeling allowed us to determine the uncertainty estimates of the linear elastic moduli of the lily pollen tube cell wall.…”

Effect of alignment angle on the alignment accuracy of a miniature rotation robot for microscopy imaging

“…For example, an automatic cellular force microscope is designed to measure topography and stiffness of tube-like pollen cells. 5,6 Optical microscope and scanning electron microscope (SEM) have been used to observe defects on different kinds of magnetic glass-coated microwires. 7,8 However, one problem about optical microscope and SEM is that optical microscope and SEM can only provide vision for samples from one fixed direction.…”

Applications of Micro/Nano Automation Technology in Detecting Cancer Cells for Personalized Medicine

“…A famous example is the automotive industry, where full robot automation is usually found for car body assembly, press tending, painting, coating and to some extent for engine and power assembly [11], considerably improving the efficiency of automotive production since 1970s [12]. In recent years, many autonomous robotic systems at the microscale have been developed for diverse biomedical applications, such as assembly of vascular-like microtube [13], 3D morphological and mechanical characterization of single plant cells [14], cell motility analysis [15], cellular sample preparation [16], tissue-and organ-level physiology [17], and drug discovery [18]. The applications of R&A technology conversely promote the development of automation science and engineering.…”