Abstract
.Significance: Two-photon (2P) fluorescence imaging can provide background-free high-contrast images from the scattering tissues. However, obtaining a multiplane image is not straightforward. We present a two-photon volume holographic imaging (2P-VHI) system for multiplane imaging.Aim: Our goal was to design and implement a 2P-VHI system that can provide the high-contrast optically sectioned images at multiple planes.Approach: A 2P-VHI system is presented that incorporates angularly multiplexed volume holographic gratings and a femtosecond laser source for fluorescence excitation for multiplane imaging. A volume hologram with multiplexed gratings provides multifocal observation, whereas nonlinear excitation using a femtosecond laser helps in significantly enhancing both depth resolution and contrast of images.Results: Standard fluorescent beads are used to demonstrate the imaging performance of the 2P-VHI system. Two-depth resolved optical-sectioning images of fluorescently labeled thick mice intestine samples were obtained. In addition, the optical sectioning capability of our system is measured and compared with that of a conventional VHI system.Conclusions: Results demonstrated that 2P excitation in VHI systems provided the optical sectioning ability that helps in reducing background noise in the images. Integration of nonlinear fluorescence excitation in the VHI provides some unique advantages to the system and has potential to design multidepth optical sectioned spatial–spectral imaging systems.
Highlights
Light-induced fluorescence imaging has widely been used for biomedical and clinical applications.[1,2] Two-photon (2P) microscopy is a well-established technique and preferred for biomedical imaging.[3]
Results demonstrated that 2P excitation in volume holographic imaging (VHI) systems provided the optical sectioning ability that helps in reducing background noise in the images
Integration of nonlinear fluorescence excitation in the VHI provides some unique advantages to the system and has potential to design multidepth optical sectioned spatial–spectral imaging systems
Summary
Light-induced fluorescence imaging has widely been used for biomedical and clinical applications.[1,2] Two-photon (2P) microscopy is a well-established technique and preferred for biomedical imaging.[3] Two-photon excitation is a nonlinear fluorescence process in which the simultaneous absorption of two photons occurs.[4,5,6] Typically, a 2P excitation in microscopy is achieved using an intense ultrashort infrared light source It provides deep penetration into the tissue in comparison to confocal or conventional fluorescence microscopy. One of the main advantages of a 2P microscope is its ability to maintain high contrast within the thick samples, which helps in studying biological structures deep within the tissues. The longer excitation wavelength (twice the wavelength with single photon) scatters less and allows deep penetration
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