Light field fluorescence microscopy (LFM) can provide three-dimensional (3D) images in one snapshot, but essentially lighting up the entirety of the sample, even though only a part of the sample is meaningfully captured in the reconstruction. Thus, entire illumination introduces extraneous background noise, degrading the contrast and accuracy of the final reconstructed images. In this paper, temporal focusing-based multiphoton illumination (TFMI) has the advantage of widefield multiphoton excitation with volume selective excitation. We implement the TFMI to LFM, illuminating only the volume of interest, thus significantly reducing the background. Furthermore, offering higher penetration depth in scattering tissue via multiphoton. In addition, the volume range can be varied by modulating the size of the Fourier-plane aperture of objective lens. 100 nm fluorescence beads are used to examine the lateral and axial resolution after phase space deconvolution from light field image, the experimental results show that the lateral resolution is around 1.2 μm and axial resolution is around 1.6 μm close to the focal plane. Furthermore, the mushroom body of drosophila brain which carried a genetic fluorescent marker GFP (OK-107) are used to demonstrate volumetric bioimaging capability.
In this study, we implement temporal-focusing multiphoton selective excitation (TFMPSE) to light field microscopy (LFM), illuminating only the volume of interest, thus significantly reducing the background noise and providing higher contrast and accuracy for the light field image reconstruction; furthermore, offering higher penetration depth in scattering tissue via multiphoton. 3D human-skin in situ immunofluorescence images are used to demonstrate volumetric bioimaging capability. The volume rate of the TFMPSE-LFM can achieve around 100 volumes per second
Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.
Light field technique at a single shot can get the whole volume image of observed sample. Therefore, the original frame rate of the optical system can be taken as the volumetric image rate. For dynamically imaging whole micron-scale biosample, a light field microscope with temporal focusing illumination has been developed. In the light field microscope, the f-number of the microlens array (MLA) is adopted to match that of the objective; hence, the subimages via adjacent lenslets do not overlay each other. A three-dimensional (3D) deconvolution algorithm is utilized to deblur the out-of-focusing part. Conventional light field microscopy (LFM) illuminates whole volume sample even noninteresting parts; nevertheless, whole volume excitation causes even more damage on bio-sample and also increase the background noise from the out of range. Therefore, temporal focusing is integrated into the light field microscope for selecting the illumination volume. Herein, a slit on the back focal plane of the objective is utilized to control the axial excitation confinement for selecting the illumination volume. As a result, the developed light field microscope with the temporal focusing multiphoton illumination (TFMPI) can reconstruct 3D images within the selected volume, and the lateral resolution approaches to the theoretical value. Furthermore, the 3D Brownian motion of two-micron fluorescent beads is observed as the criterion of dynamic sample. With superior signal-to-noise ratio and less damage to tissue, the microscope is potential to provide volumetric imaging for vivo sample.
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