Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters

Abstract

Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinearity in the fluorescence response induced by the SAX. Calculation of the optical transfer functions and the edge responses for SAX microscopy revealed the contrast improvement of high-spatial frequency components in the sample structure and the effective reduction of background signals from the out-of-focus planes. Experimental observations of the edge response and x-z cross-sectional images of stained HeLa cells agreed well with theoretical investigations. We applied SAX microscopy to the imaging of three-dimensional cultured cell clusters and confirmed the resolution improvement at a depth of 40 μm. This study shows the potential of SAX microscopy for super-resolution imaging of deep parts of biological specimens.

3D super-resolved in vitro multiphoton microscopy by saturation of excitation

“…Until recently, saturation has been considered harmful for imaging and has been avoided [9,10]. It is possible, however, to show that in a saturation regime, the relation between the intensity of excitation and the emitted fluorescence intensity is no longer linear and contains nonlinearities that can be used to improve the lateral as well as the axial resolution [5,11,12].…”

Introduction to super-resolution microscopy

“…Therefore, the use of the non-linear signals enables the effective suppression of the detection of fluorescence signals from out-of-focus planes. The high background elimination property of SAX allows us to observe finer structures in a thick sample with high spatial resolution and imaging contrast [91,92]. Figure 11 shows the result of volumetric imaging of α-tubulin in a cell cluster obtained with SAX microscopy.…”

Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application

“…Nowadays, the fluorescent confocal laser scanning microscopy (FCLSM) that combines the fluorescent labels and CLSM has been one of the most powerful and versatile methods for studying cells, tissues and organisms. Furthermore, various super resolution microscopies have been developed based on FCLSM, such as stimulated emission depletion (STED) [2][3][4], reversible saturable optical fluorescence transitions (RESOLFT) [5][6][7], and saturated excitation (SAX) [8,9].…”