Space-multiplexed multifocal nonlinear microscopy

Standard forms of nonlinear microscopy rely on single beam scanning, but th e usually weaker signal and the need to image in real-time call for paralle lization of the image formation. Since the nonlinear susceptibilities neces sitate a comparatively large illumination power, with current laser systems the brightness or field of view of any parallelized nonlinear microscope i s limited by the brightness of the laser. For example, by producing an arra y of high aperture foci, multifocal multiphoton microscopy (MMM) provides r eal-time, light-efficient three-dimensional fluorescence imaging at high-re solution. The available power limits the degree of parallelization and henc e codetermines the field of view. As the utilization of all the laser power is imperative, the focal intensity can be adjusted only through altering t he number of foci, This compromises to some extent the flexibility to adjus t the focal intensity to benign and effective levels, Here we introduce spa ce-multiplexing (SMX) as a novel option in parallelized nonlinear microscop y, which enables an improved exploitation of the total laser power and faci litates changing the intensity levels in selected regions, without attenuat ing the total laser power. The basic idea of SMX is to overlap arrays of sl ightly offset coherent focal fields whose interference modulates the intens ity across the sample. For a given degree of parallelization and power, SMX increases the two- and three-photon excited signal of parallelized nonline ar microscopy by a factor of up to 1.5 and 2.5, respectively. To some exten t, sensitive regions may be spared out, whereas in regions with weaker nonl inear susceptibilities the intensity is increased. SMX is relevant to all m odes of nonlinear microscopy, including parallelized second- and third-harm onic imaging, coherent anti-Stokes Raman scattering, and widefield multipho ton excitation.