Thin laser light sheet microscope for microbial oceanography

Abstract

Despite a growing need, oceanographers are limited by existing technological constrains and are unable to observe aquatic microbes in their natural setting. In order to provide a simple and easy to implement solution for such studies, a new Thin Light Sheet Microscope (TLSM) has been developed. The TLSM utilizes a well-defined sheet of laser light, which has a narrow (23 micron) axial dimension over a 1 mm x 1 mm field of view. This light sheet is positioned precisely within the depth of field of the microscope's objective lens. The technique thus utilizes conventional microscope optics but replaces the illumination system. The advantages of the TLSM are two-fold: First, it concentrates light only where excitation is needed, thus maximizing the efficiency of the illumination source. Secondly, the TLSM maximizes image sharpness while at the same time minimizing the level of background noise. Particles that are not located within the objective's depth of field are not illuminated and therefore do not contribute to an out-of-focus image. Images from a prototype system that used SYBR Green I fluorescence stain in order to localize single bacteria are reported. The bacteria were in a relatively large and undisturbed volume of 4ml, which contained natural seawater. The TLSM can be used for fresh water studies of bacteria with no modification. The microscope permits the observation of interactions at the microscale and has potential to yield insights into how microbes structure pelagic ecosystems.