Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

Abstract

Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore–labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 ?? lines / mm . TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0 ± 0.7 ?? ? m and the shifting distance of the temporal focal plane was less than 0.95 ?? ? m within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

An excitation wavelength switching to enhance dual-color wide-field temporal-focusing multiphoton excitation fluorescence imaging

“…Compared with the conventional TFMPEM system, which uses only a single fixed excitation wavelength for multicolor imaging, the present strategy enables each fluorophore to substantially enhance the SNR of the TPEfluorescence signals. High-contrast merged TPE images of the multifluorophore-labeled specimen using the excitation wavelengths at the maximum TPEs of different fluorophores are also demonstrated [40]. The stronger TPE-fluorescence signals play a key role in deep imaging of the biotissue specimens [54][55][56].…”

“…Hence, for multicolor imaging of multiple fluorophores, the presented TFMPEM can acquire TPE-fluorescence images of each fluorophore by rapidly switching the excitation wavelength to obtain maximum TPE-fluorescence signals according to their TPE spectral measurement. Higher TPE efficiency of fluorophores is critical for higher SNR of TPE images [40]. Compared with the conventional TFMPEM system, which uses only a single fixed excitation wavelength for multicolor imaging, the present strategy enables each fluorophore to substantially enhance the SNR of the TPEfluorescence signals.…”

“…The first-order diffraction beams of all excitation wavelengths can propagate through the following 4f configuration to achieve temporal-focusing excitation. The excitation wavelength can be rapidly switched using the rotation of the scanning mirror, as compared to using the shift of a motorized translation stage [40]. The axial excitation confinement and optical-section imaging were measured using excitation wavelengths of 730-1000 nm.…”

Multicolor two-photon light-patterning microscope exploiting the spatio-temporal properties of a fiber bundle

“…Apart from the advantage of making the system more compact and straightforward the major benefit of this configuration is that it allows for tuning the excitation wavelength or coupling multiple colors simultaneously without modifications to the optical system [ 41 , 42 ]. The use of a grating, in fact, requires adjusting the angle of incidence or exit for each wavelength, and for multicolor excitation, it would necessitate the use of two gratings and therefore two distinct optical paths and multiplexing SLMs.…”

“…This requires to readjust the angle between the incident or the reflected beam and the grating to maintain a constant deflection for the different wavelengths. Although dual-color 2PE with TF was used to image specimens labeled with different fluorophores [ 41 , 42 ], the experimental system needed a switching element (such as galvanometric mirrors) to compensate for the different grating angles, increasing the system’s complexity. Moreover, this compensation is limited to a narrow range of excitation wavelengths.…”