Three dimensional fluorescence imaging using multiple light-sheet microscopy

Abstract

We developed a multiple light-sheet microscopy (MLSM) system capable of 3D fluorescence imaging. Employing spatial filter in the excitation arm of a SPIM system, we successfully generated multiple light-sheets. This improves upon the existing SPIM system and is capable of 3D volume imaging by simultaneously illuminating multiple planes in the sample. Theta detection geometry is employed for data acquisition from multiple specimen layers. This detection scheme inherits many advantages including, background reduction, cross-talk free fluorescence detection and high-resolution at long working distance. Using this technique, we generated 5 equi-intense light-sheets of thickness approximately 7.5 μm with an inter-sheet separation of 15 μm. Moreover, the light-sheets generated by MLSM is found to be 2 times thinner than the state-of-art SPIM system. Imaging of fluorescently coated yeast cells of size 4±1 μm (encaged in Agarose gel-matrix) is achieved. Proposed imaging technique may accelerate the field of fluorescence microscopy, cell biology and biophotonics. © 2014 Mohan et al.