- Light-sheet fluorescence microscopy (LSFM), also known as selective plane illumination microscopy (SPIM), is an advanced imaging technique that allows rapid, high-resolution, and minimally invasive three-dimensional visualization of biological specimens. It achieves optical sectioning by illuminating a thin sheet of light perpendicular to the detection objective, thereby exciting fluorescence only within a narrow focal plane. This selective illumination minimizes photodamage, reduces background fluorescence, and significantly enhances imaging speed and contrast compared to traditional widefield or confocal microscopy.
- The fundamental principle of LSFM lies in decoupling the illumination and detection pathways. A thin plane of laser light—typically generated using cylindrical lenses or scanned Gaussian beams—is projected laterally through the specimen, illuminating only a single optical slice at a time. The emitted fluorescence from the illuminated plane is then collected perpendicularly by a detection objective and recorded by a sensitive camera (often a sCMOS or CCD detector). By moving the specimen through the light sheet (or vice versa), successive planes can be imaged and computationally reconstructed into a 3D volume.
- One of the major advantages of LSFM is its ability to image large and thick specimens—such as embryos, organoids, and whole tissues—over long periods with minimal phototoxicity. This makes it ideal for live imaging of dynamic developmental processes, neuronal activity, and cell migration in intact biological systems. Furthermore, LSFM supports multi-view imaging, where the sample is rotated to acquire datasets from different angles, improving resolution and compensating for light scattering and absorption in dense samples.
- Several LSFM variants have been developed to accommodate specific imaging needs. These include dual-view LSFM, lattice light-sheet microscopy for enhanced axial resolution, and adaptive optics-enhanced LSFM for imaging in heterogeneous or optically challenging environments. LSFM is also increasingly integrated with clearing methods (e.g., CLARITY, iDISCO) to image whole organs or organisms post-fixation.
- In conclusion, light-sheet fluorescence microscopy represents a powerful and versatile tool in modern biological imaging. Its combination of optical sectioning, low phototoxicity, and high temporal and spatial resolution makes it particularly valuable for 3D and 4D (time-resolved) imaging of complex biological systems in their native context.
