Simultaneous imaging of multiple focal planes using a two-photon scanning microscope

Abstract

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.

High-Resolution, 2- and 3-Dimensional Imaging of Uncut, Unembedded Tissue Biopsy Samples

“…For instance, multiphoton imaging approaches exist that incorporate the use of inexpensive diode lasers, significantly reducing the overall cost of an MPM system to that of a modern motorized microscope. 15,16 Alternatives to galvanometer-based single-point laser raster scanning-including multibeam scanning systems, 17 spatiotemporal multiplexing, 18 and temporal focusing 19,20 -promise to improve scanning times by an order of magnitude or more, making real-time video imaging a possibility. Finally, there are several potential approaches to speed up the otherwise diffusion-limited process of clearing, including the use of established microwave technology for histologic processing.…”

Remote focusing for programmable multi-layer differential multiphoton microscopy

“…When compared to other multi-focal techniques, this system is also advantageous in that the beams are spatially separated by several millimeters, allowing for manipulation of the individual beams with little difficulty. These characteristics produce an extremely flexible imaging system that is capable of simultaneously imaging specimens at multiple different layers [17], employing different excitation polarizations [18,19], or different pulse shapes [20,21]. As a result of the myriad of possible applications for this imaging technology, we refer to the general approach as "differential multiphoton microscopy".…”

Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy

“…Apart from the advantages of simplicity and efficiency, this simple shaping scheme turns out to be particularly well suited for use in combination with an efficient multiplexing scheme previously demonstrated in the context of multifocal multiphoton microscopy [8][9][10] and of coherent antiStokes Raman scattering (CARS) microscopy [11,12]. We thus report in vivo dual-excitation 2PEF imaging in developing embryos with a 5 μs pixel acquisition time and a switching rate of 150 MHz between the two pulse shapes.…”

“…The final step of rapid switching between τ 1 ðωÞ and τ 2 ðωÞ is achieved by time multiplexing [8][9][10][11][12], as discussed in the following. The experimental setup is shown in Fig.…”

“…This arrangement results in a 150 MHz pulse train with a different pulse shape every other pulse, which corresponds to a 150 MHz switching rate. The 2PEF is then acquired using a photon-counting scheme synchronized with the 150 MHz pulse train, with two different counters for even and odd pulses [8][9][10].…”