Multifocal nonlinear microscope with single element detector for multidepth imaging

We present a laser scanning microscope capable of producing multiple focal volumes. These volumes can be displaced vertically, to acquire simultaneous images from multiple planes, or superimposed at the same depth but with different polarization states. We call this last implementation, differential multiphoton laser scanning microscopy (dMPLSM). To our knowledge, this constitutes the first report of a multifocal microscope with this capacity. The microscope is able to take images in different modalities, two-photon excited fluorescence, second, and third harmonic generation. In this work, we demonstrate several capabilities of our microscope: simultaneous acquisition of two and six images from two focal planes separated by several microns, and a pair of simultaneous images taken at the same focal plane but with two different polarizations. Some potential applications include following microorganism motion, studies of phase matching in microscopic environments, studies of blood flow, etc. The microscope is based on a pulsed ultrafast laser. The pulses are split, manipulated and recombined in an interlaced pattern in order to generate a sequence of pulses with different divergences, and possibly different polarizations. This pulse train is sent to the objective and focused at different depths. The signal is recorded using a photoncounting photomultiplier tube. Images from different foci are separated using time demultiplexing based on a low cost field programmable gate array. The use of a single element detector, instead of a multi-element (CCD camera), allows for imaging of scattering media. The use of photon counting leads to lower signal to noise ratio in the images.