Engineering a perfusion-enabled mechanical compressor for long-duration immobilization and microscopy of cells and small organisms

Abstract

The study of living specimens is essential to the understanding of organismal behavior. Unfortunately, a major difficulty in the study of live organisms is that many move in and out of the field of view or focal plane during microscopy. The present work seeks to combat this considerable problem by developing a mechanical microcompressor that immobilizes living cells and small organisms for long-duration optical microscopy. The device, dubbed the "Commodore Compressor," features two key innovations: (1) the integration of a perfusion system to keep the trapped specimen alive over several hours, as well as permitting the addition of chemoattractants, drugs, and other chemicals; (2) the incorporation of an optional patterned PDMS platform to improve the efficacy of immobilization in a targeted, organism-specific manner. One application of the Commodore Compressor is in monitoring the change in protein bioluminescence intensity in many trapped Saccharomyces cerevisiae cells during synchronized cell cycles. The experiment's feasibility and key techniques have been well demonstrated, although bioluminescence cannot currently be visualized. A second application involves fluorescence imaging of the neural network development of immobilized Caenorhabditis elegans over many hours. The development of new patterned PDMS platform designs, aided by the innovative use of established techniques, has driven the present work toward accomplishing the goal, but true long-term viability remains elusive. The Commodore Compressor may be directly used or easily adapted for many other specimen types and experimental scenarios.