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Marked Glass Surfaces for Identifying and Studying Individual Particles

Paul Zhu, Jose M. Moran-Mirabal*, Harold G. Craighead, and Larry Walker*

*Department of Biological and Environmental Engineering, Cornell

Abstract

We use patterned glass surfaces to immobilize pre-treated biomass particles that are rich in cellulose. By dividing a glass cover slip into more than a hundred identifiable areas, we can not only count the total number of immobilized particles, but also address individual particles of interest, and observe them using optical microscopy, as well as scanning electron microscopy.

Research Summary

Figure 1.

In studying cellulase enzyme and cellulose interactions, one method involves fluorescence microscopy, in which fluorescently-labeled cellulases bind to immobilized bacterial cellulose fibers, and fluorescence is recorded over time. Binding kinetics is then derived from time-trace of fluorescence intensity [1].

We study similar cellulase-cellulose interactions with pretreated biomass particles originating from switch grass and wood. After mechanical and thermal treatment, particles present different morphologies and cellulosic structures which may influence enzyme binding and cellulose depolymerization. In addition, thickness and pore sizes associated with each particle may be different as well. As a result, we want to be able to address individual particles, and observe them using different microscopy methods, and at different times.

To accomplish this goal, a 170um fused silica glass is patterned with 16-by-16 squares, each row and and column are addressed with hexadecimal numbering. A conventional lift-off technique was used to pattern the substrate. Briefly, a layer of positive photo-resist is spun on fused silica substrate, and exposed to develop into 16-by-16 numbered squares. A layer of gold is evaporated onto the surface and resist is cleaned off with solvent, leaving gold-patterns (Fig 1).

A water-suspension of pre-treated cellulosic biomass is dropped on the aforementioned substrate. The water is allowed to dry, leaving biomass material to adhere to the surface. When there are a few particles remaining on the surface, each individual particle can be easily numbered by its location among the 256 squares (Fig 1). We can subsequently zoom in, address and study each particle by its location designation, observing it using optical microscopy (Fig 2), as well as scanning electron microscopy (Fig 3).

Figure 2.

Figure 3.

References

  1. "Immobilization of Cellulose Fibrils on Solid Substrates for Cellulase-Binding Studies Through Quantitative Fluorescence Microscopy," J.M. Moran-Mirabal, N. Santhanam, S. C. Corgie, H.G. Craighead, L.P. Walker, Biotechnology and Bioengineering 101 (6) (2008) 1129-1141. doi:10.1002/bit.21990. Abstract