Optically Accessible Microfluidic Flow Channels for Noninvasive High-Resolution Biofilm Imaging Using Lattice Light Sheet Microscopy

Abstract

Imaging platforms that enable long-term, high-resolution imaging of biofilms are required to study cellular level dynamics within bacterial biofilms. By combining high spatial and temporal resolution and low phototoxicity, lattice light sheet microscopy (LLSM) has made critical contributions to the study of cellular dynamics. However, the power of LLSM has not yet been leveraged for biofilm research because the open-on-top imaging geometry using water-immersion objective lenses is not compatible with living bacterial specimens; bacterial growth on the microscope's objective lenses makes long-term time-lapse imaging impossible and raises considerable safety concerns for microscope users. To make LLSM compatible with pathogenic bacterial specimens, we developed hermetically sealed, but optically accessible, microfluidic flow channels that can sustain bacterial biofilm growth for multiple days under precisely controllable physical and chemical conditions. To generate a liquid-and gas-tight seal, we glued a thin polymer film across a 3D-printed channel, where the top wall had been omitted. We achieved negligible optical aberrations by using polymer films that precisely match the refractive index of water. Bacteria do not adhere to the polymer film itself, so that the polymer window provides unobstructed optical access to the channel interior. Inside the flow channels, biofilms can be grown on arbitrary, even nontransparent, surfaces. By integrating this flow channel with LLSM, we were able to record the growth of S. oneidensis MR-1 biofilms over several days at cellular resolution without any observable phototoxicity or photodamage.

BCM3D 2.0: Accurate segmentation of single bacterial cells in dense biofilms using computationally generated intermediate image representations

“…High cell density and low SBR datasets are encountered often in biofilm research, especially when cells touch each other and biofilms extend farther into the vertical ( z -) dimension, so that light scattering becomes a pronounced background contribution 52 . We therefore sought to specifically improve bacterial cell segmentation accuracy for high cell density and low SBR biofilm images.…”

“…However, every fluorescence imaging modality is subject to trade-offs between the achievable spatial and temporal resolution, image contrast (SBR), and phototoxicity and photodamage. If reducing the total radiation dose delivered to the cells is an experimental necessity, light sheet-based microscopy approaches offer substantial advantages over confocal microscopy 52 .…”

“…Whether this behavior is part of a negative phototaxis response of S. oneidensis remains to be investigated more quantitatively in future work. If reducing the total radiation dose delivered to the cells is an experimental necessity, light sheet-based microscopy approaches offer substantial advantages over confocal microscopy 52 . In our lattice light sheet microscope, a high (30 s) time resolution could possibly be maintained at a lower, more tolerated radiation dose by further decreasing the excitation laser intensities at the sample and acquiring images at even lower SBRs.…”

BCM3D 2.0: accurate segmentation of single bacterial cells in dense biofilms using computationally generated intermediate image representations

“…However, every fluorescence imaging modality is subject to trade-offs between the achievable spatial and temporal resolution, image contrast (SBR), and phototoxicity and photodamage. If reducing the total radiation dose delivered to the cells is an experimental necessity, light sheet-based microscopy approaches offer substantial advantages over confocal microscopy 32 .…”

“…High cell density and low SBR datasets are encountered often in biofilm research, especially when cells touch each other and biofilms extend farther into the vertical (z-) dimension, so that light scattering becomes a pronounced background contribution 32 . We, therefore, sought to specifically improve bacterial cell segmentation accuracy for high cell density and low SBR biofilm images.…”