Abstract
Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination.
Highlights
Two-photon excited fluorescence (TPEF) microscopy is a powerful and widely utilized tool for three-dimensional (3D) biological imaging, microsurgery, and microstructure fabrication [1,2,3]
Through the advantages of natural optical sectioning capability, minimum invasiveness, lower photobleaching and deeper penetration depth, this type of microscopy is suitable when imaging thick tissues for in vivo studies [6]. Both TPEF and second harmonic generation (SHG) are nonlinear optical phenomena that only occur with a high excitation photon density achieved with a high numerical aperture (NA) objective lens to spatially and temporally focus the ultrafast laser beam to generate a strong electromagnetic field
Since the size of the fluorescent bead is known and the location is fixed, we can show that the axial average fluorescence intensity profile indicates the measured bead size and analyze the scanned images to ensure all acquired image positions are stable during overall volumetric imaging
Summary
Two-photon excited fluorescence (TPEF) microscopy is a powerful and widely utilized tool for three-dimensional (3D) biological imaging, microsurgery, and microstructure fabrication [1,2,3]. Through the advantages of natural optical sectioning capability, minimum invasiveness, lower photobleaching and deeper penetration depth, this type of microscopy is suitable when imaging thick tissues for in vivo studies [6] Both TPEF and SHG are nonlinear optical phenomena that only occur with a high excitation photon density achieved with a high numerical aperture (NA) objective lens to spatially and temporally focus the ultrafast laser beam to generate a strong electromagnetic field. It should be noted that the excitation area depends on the initial laser beam size, the diffraction element, and the overall system magnification This system allows for high throughput illumination and detection capability, which could be used for widefield fluorescence lifetime imaging [21], large area multiphoton-induced ablation [22], and capturing dynamic events at high-frame rate [19]. The 3D Brownian motion of one-micron fluorescent beads is shown at 30 volume/sec video rate
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