A single-objective light-sheet microscope
We are developing a novel microscopy modality, a single-objective light-sheet microscope with a high numerical aperture. It is an entirely novel design for a light-sheet microscope that combines the convenience of conventional sample mounting with sensitive subcellular and super-resolution imaging of cells and tissues. This single objective light-sheet fluorescence microscope started acquiring from biological samples only a few months ago and already several researchers across campus claimed interest in obtaining their own version, and there are plans to build a clone of the microscope for the imaging facility.
In an OPM, a single primary objective is used to both create the excitation light sheet and capture emitted light from the sample. Excitation light enters the objective sideways, resulting in an oblique light sheet on the sample, with an angle between 30°–45°. Emitted light from the tilted plane is collected by the same objective and optically refocused to a secondary objective, without introducing any relevant aberrations. It is subsequently re-imaged by a tilted tertiary objective onto a camera. The beauty of this approach lies in having a single high NA objective close to the sample, allowing for traditional sample mounting geometry (microscope slides, glass-bottom dish), and leaving accessible space around the sample for other perturbations and manipulations, such as microfluidic devices or optogenetic light stimulation.
As a proof of concept, we acquired images of Drosophila embryos, mESCs, and mouse gastruloids in their optimal growth conditions, showing that we achieve diffraction-limited resolution that, e.g., allows us to discriminate sister chromatids, follow their dynamics over time, and measure how they are transcriptionally, spatially, and temporally correlated. Moreover, we achieve a combination of high resolution, high contrast, and high speed that enables us to identify and track single mRNA molecules as they are released at the transcription site. We are currently developing analysis tools that will allow us to extract quantitative data from these images, e.g., to measure the diffusion and reach of individual mRNA molecules.