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FRINATEK-Fri prosj.st. mat.,naturv.,tek

Development of Real Volumetric Microscopy through Single Objective Light-Sheet Imaging System (SOLIS)

Alternative title: Utvikling av ekte volumetrisk mikroskopi gjennom én-objektivs lys-ark system (SOLIS)

Awarded: NOK 8.0 mill.

In this project, a completely new type of optical microscope will be developed that combines the best aspects of traditional confocal with light-sheet microscopy. Confocal microscopy is one of the most successful imaging methods in biomedicine and suitable to visualise the structure of cells and tissues in 3D space. However, as in confocal microscopy a single point is raster-sampled through the volume of interest, its acquisition speed is very slow. Furthermore, only a fraction of the fluorescent light generated inside the sample is actually captured by the microscope?s photodetector, which limits this technique?s application to living samples, which tolerate only a limited amount of light exposure. Light-sheet microscopy, on the other hand, excels in imaging speed and low light-dose, both of which are critical for studies of living samples. In light-sheet microscopy, a thin slice of the sample is illuminated at a time and the produced fluorescence is imaged by a second lens, which is positioned at a right angle to the light-sheet. Although this arrangement is very efficient in collecting light from the sample, the requirement for a second lens limits access to the imaging volume and thus excludes the use of standard sample preparation protocols. The new microscope, dubbed SOLIS (single-objective light-sheet imaging system), keeps the resolution and ease of use of confocal microscopy, i.e. only a single objective lens near the sample is required, while increasing its gentleness akin to light-sheet microscopy. Additionally, thanks to its unique optical design, SOLIS manages to surpass the imaging speed of classical light-sheet microscopy as, in essence, it records fully sectioned volumes in an instant. One application that will be enabled by the new system is the exploration of the repair mechanism of heart cells.

Biology happens in 3D, in living organisms. Fluorescence microscopy of cellular processes should thus be performed in cells residing in their native environment. Yet, many studies rely on 2D imaging or 3D confocal microscopy, which has limited live-cell compatibility due to low imaging speed and high phototoxicity. Light-sheet microscopy (LSM) may be a solution. It uses two objective lenses oriented at right angles to each other, instead of the single objective used in conventional microscopy. The first objective produces a thin sheet of light that only excites fluorophores within the focal plane of the second objective, thus avoiding the generation of out-of-focus light. Scanning the sample through the imaged plane enables optically sectioned volumetric microscopy. Crucially though, the requirement of two objective lenses near the sample has the tremendous drawback of obstructing conventional sample mounting techniques and has hence severely hindered a more widespread use of LSM. SOLIS solves this. In SOLIS, a high-performance objective with large numerical aperture (NA) is used for generation of a scannable light-sheet and, in stark contrast to conventional LSM, this same objective is used for collection of the sample’s fluorescence signal. SOLIS is thus fully compatible with conventional sample mounting. To align the focal plane of the objective with the illumination plane, SOLIS uses a dedicated optical system downstream from the objective, and hidden from the user, to effectively “tilt” the sample volume so it can be recorded by a camera without optical aberrations. As only a single objective is near the sample, the NA of the collection objective in SOLIS can be much larger than in conventional LSM, thus increasing achievable resolution by 15% and photon collection efficiency by over 30%. Furthermore, by using novel multiplane optical elements, SOLIS can record at rates in the kilohertz regime, while still providing optically-sectioned volumetric imaging.

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FRINATEK-Fri prosj.st. mat.,naturv.,tek