Efficient generation of diffraction-limited multi-sheet pattern for biological imaging

Abstract

We demonstrate a new technique to generate multiple light-sheets for fluorescence microscopy. This is possible by illuminating the cylindrical lens using multiple copies of Gaussian beams. A diffraction grating placed just before the cylindrical lens splits the incident Gaussian beam into multiple beams traveling at different angles. Subsequently, this gives rise to diffraction-limited light-sheets after the Gaussian beams pass through the combined cylindrical lens-objective sub-system. Direct measurement of field at and around the focus of objective lens shows multi-sheet pattern with an average thickness of 7.5 μm and inter-sheet separation of 380 μm. Employing an independent orthogonal detection sub-system, we successfully imaged fluorescently-coated yeast cells (≈4 μm) encaged in agarose gel-matrix. Such a diffraction-limited sheet-pattern equipped with dedicated detection system may find immediate applications in the field of optical microscopy and fluorescence imaging.

Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array

“…This is a critical requirement that ensures high-degree of viability of a biological sample. However, existing techniques of parallelization are either restricted to two dimensions (e.g., LSFM 3,7,8 ) or sparse sampling in three dimensions (c.f., multi-focal [9][10][11] or multi-light-sheet imaging [12][13][14][15][16][17][18] ). In particular, the current multi-light-sheet fluorescence imaging systems, including the recent advances in lattice light-sheet microscopy, commonly resort to coherent beam interference, beam split, or wavefront shaping (in the spatial or Fourier domain) [12][13][14][15]19 .…”

Optimization method to suppress background for imaging multiple planes

“…In addition, the method introduces spherical aberrations in two outside light sheets. Another method has also been suggested to illuminate specimen with multiple light sheets which are arranged at focusing position in the optical axis of detection lens, such as di®raction grating 24 and Daperture. 10 This method enables simultaneous imaging through a 3D volume using di®raction 25 or refraction optics.…”