Year: 2025
Type: article
Abstract: Light-sheet fluorescence microscopy (LSFM) has generated significant interest in biological research as a result of its ability to volumetrically image biological specimens with high optical sectioning and low photobleaching and phototoxicity. While its imaging speed can be high, the spatial resolution is often modest, as the numerical aperture of the used objectives is limited. This is due to the orthogonal arrangement of illumination and detection objectives, which have to split the available solid angle. Typically, a numerical aperture of 1.1 or less is used for detection, which limits the sensitivity and lateral resolution in LSFM. Furthermore, the orthogonal imaging geometry sterically occludes many commonly used sample mounting methods (e.g., microfluidics and multi-well plates), and typically one cannot freely change the objectives to adapt the system to different imaging tasks. Oblique plane microscopy (OPM), a subset of LSFM, uses a single primary objective lens to illuminate the sample with a tilted light-sheet and to detect the fluorescence emission. As such, OPM is compatible with conventional sample mounting and is not limited in the choice of objectives, opening up the use of very high NA objectives. In this presentation we show recent progress in the field of OPM, which pushes the spatial and temporal resolution, and widens its application space. The use of a TIRF objective as a primary objective allowed us to build an OPM that has sub 300nm lateral and 600nm axial resolution (raw values before deconvolution) for sensitive imaging of single cells. The axial resolution in an LSFM is the product of the detection PSF and the light-sheet intensity distribution. While considerable efforst have been invested into creating thinner light-sheets, our “TIRF-OPM” system achieves high axial resolution by shrinking the detection Point spread function. To improve the temporal resolution in OPM, we found that a deliberate amount of undersampling in the acquisiton of a 3D stack can lead to a form of aliasing that can be fully recovered; i.e. the full spatial resolution is preserved and no aritifacts are introduced. Combined with the low inertia (no sample or objective movement) volumetric acquisiton in OPM, this enables 3D whole cell imaging at up to 10 volumes per second with subcellular resolution. For even faster imaging, we developed a method to form projections with an OPM under varying viewing angles from a three-dimensional sample. Since each projection can be integrated in a single camera frame, this enables observation of dynamics that would otherwise be too quick for conventional three-dimensional stack acquisitions. Lastly, we show that we can adapt an OPM system to use different objectives, such as high NA oil immersion, long-working distance water immersion and even air objectives to perform aberration free 3D imaging. As such, the OPM system can be adapted from sensitive high resoluiton imaging of single cells, to whole organism imaging and to rapid multi-well imaging. This drastically improves the versatility of the system compared to traditional LSFM architecture that are optimized to a specific sample and mounting technique.
Source: Microscopy and Microanalysis
Authors: Reto Fiolka, Bingying Chen
Institution: The University of Texas Southwestern Medical Center
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4412908430