Abstract
Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.
Highlights
Selective plane illumination microscopy (SPIM) is one of the most suitable techniques for fast, three-dimensional imaging
Since the observation plane is at an angle with respect to the sample container, the field of view for flat samples, such as a monolayer of cells, is limited, i.e., the full field of view of the detector cannot be utilized. Another approach to using high numerical aperture (NA) lenses is reflected light sheet microscopy, in which the light sheet is generated by reflecting a beam incident from the top by 90° with a small mirror mounted on an atomic force microscope cantilever [5]
We converted one of our sample chambers to a fluidic device to look at biofilm dynamics under flow
Summary
Selective plane illumination microscopy (SPIM) is one of the most suitable techniques for fast, three-dimensional imaging. Since the observation plane is at an angle with respect to the sample container, the field of view for flat samples, such as a monolayer of cells, is limited, i.e., the full field of view of the detector cannot be utilized Another approach to using high NA lenses is reflected light sheet microscopy, in which the light sheet is generated by reflecting a beam incident from the top by 90° with a small mirror mounted on an atomic force microscope cantilever [5]. SPIM implementations using a single lens do not suffer from opto-mechanical constraints of two lens designs but are limited in spatial resolution and/or imaging depth [12,13,14,15,16] This manuscript describes a new selective plane illumination method in the conventional sample geometry. Since the observation volume can be very small, high throughput 3D imaging of multiple wells is possible by fast and precise stage movement
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call