Customizable live-cell imaging chambers for multimodal and multiplex fluorescence microscopy

Abstract

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires provision of appropriate experimental conditions in a format that meets the microscopy’s optical requirements. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized live-cell imaging chambers that are compatible with a range of imaging modalities including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and which supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting PDMS microfluidic devices. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with modern microscopy techniques.

SARS-COV-2 NSP5 Antagonizes MHC II Expresion by Subverting Histone Deacetylase 2

“…Cells of interest were seeded at a density of 1000 cells/mm 2 into either 18 mm circular coverslips placed into the wells of a 12-well plate, or into the wells of a custom-printed 15-well imaging chamber [105]. Cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature.…”

“…The resulting plasmids were labeled with ATTO647N and fragmented using a ATTO647N NT Labeling Kit, producing fragments averaging 200 nucleotides. Primary human macrophages or A549 cells were plated into the 7.5 mm wells of a customized imaging chamber [112], transduced with lentiviral vectors (macrophages) or transfected (A549 cells) with NSP5 or an empty vector, and treated with siRNA as described above. These cells were stained for immune-FISH as per the protocol of Ye et al [117], including wells which were left unstained, or stained only with the donor (acetyllysine-Cy3) or acceptor (FISH probes).…”

Lightsheet Microscopy

“…Russell and Rees (2020) present 3D CAD files to print a versatile chamber that can hold coverslips as well as small embryos and tissues. A relevant concern is the potential for cell-toxic materials to be present in 3D printing substrates; this issue has been investigated by Tepperman et al (2020).…”

Microfluidic Live Imaging Technology to Perform Research Activities in 3D Models

“…Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes [80]. Technological advances have enabled a broad array of live-cell imaging approaches to investigate cellular processes, many of which require optically demanding imaging modalities to achieve the necessary resolution and sensitivity [83]. A new technology based on newly developed microfluidics and imaging techniques can enable the management and identification of the phenotype, the biological activities of the present populations of the present populations without destroying the 3D of an organoid or derived in culture or co-culture of progenitor organs and/ or donors who self-organize in space/time like the in vivo [74,84,85].…”