Advancing Cotton Fiber Research with Variable-Pressure Scanning Electron Microscopy (VP-SEM)

Fang Bai ^1* Michael Jansen ^2,3* Fang Bai ^4*

• ¹ Crop Genetics Research Unit, Agricultural Research Service (USDA), Stoneville, Mississippi, United States
• ² Beltsville Agricultural Research Center, Agricultural Research Service (USDA), Beltsville, Maryland, United States
• ³ USDA, Beltsville Agricultural Research Center, Systematic Entomology Laboratory, Electron and Confocal Microscopy Unit, Beltsville, United States
• ⁴ University of Florida, Gainesville, Florida, United States

Cotton fibers, as highly extended, thickened epidermal seed structures, are a crucial renewable resource in textile production. Cotton plants produce two main types of fiber cells: wide, hemisphere-shaped fibers and narrow, tapered fibers. Both types stabilize through secondary cell wall development, with the mature narrow fibers being particularly valued for spinning into fine, strong yarns, suitable for premium cotton fabrics. Traditional methods for studying fiber development and cell types, such as scanning electron microscopy (SEM), are often time-intensive and costly. SEM preparation steps, including fixation, dehydration, and sputter coating, can cause shrinkage and other image distortions, limiting the accuracy of observations. Variable-pressure scanning electron microscopy (VP-SEM) offers an alternative approach, operating under low pressure rather than a high-vacuum environment, which can be advantageous for imaging live samples with minimal sample preparation. In this study, we applied VP-SEM to observe fiber cell initiation and early elongation in the conventional upland cotton cultivar UGA 230 at 0 and 1-day post-anthesis. Two SEM detectors, the ultra-variable-pressure detector and backscattered electrons, were used to capture detailed images. Optimal imaging conditions were identified with a 15 keV accelerating voltage and a 50 Pa pressure setting, enabling clear visualization of early fiber development without the need for extensive preparation. This VP-SEM protocol not only facilitates high-resolution imaging of cotton fibers at early developmental stages but also reduces time and expense, minimizing sample damage. Additionally, this optimized approach can be adapted for other fresh biological samples, making it a versatile tool for real-time imaging across various studies in plant biology and beyond.