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

Abstract

Dual-color two-photon excitation (TPE)-fluorescence imaging is used in conventional temporal-focusing multiphoton excitation microscopy (TFMPEM) to observe specimens with different fluorophore labels. However, concerns have been raised about the excitation efficiency and selectivity of the fluorophores under fixed-wavelength excitation. This study presents a wavelength-switching approach using a scanning mirror, beam expander, and diffraction grating in the TFMPEM to switch the excitation wavelengths and match the optimal absorption of the fluorophores to acquire dynamic dual-color TPE-fluorescence images. The presented TFMPEM system was demonstrated to have an axial excitation confinement of 2.3–5.0 μm for excitation wavelengths of 730–1000 nm, and was used to visualize three-dimensional images of the vasculature of a mouse brain. The TPE efficiencies of different fluorophores were evaluated through TFMPEM imaging with excitation wavelength scanning to obtain their TPE spectra. Consequently, time-lapsed dual-color TFMPEM imaging was performed on rhodamine 6G (R6G)–poly(lactic-co-glycolic acid) (PLGA) nanoparticles and enhanced-yellow-fluorescent protein (EYFP)-tagged clathrin using excitation wavelengths at the maximum TPEs of R6G and EYFP, respectively. Our results revealed the PLGA-nanoparticle uptake of live cells via long-lived clathrin-coated plaques in clathrin-mediated endocytosis.

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.…”

Temporal‐Focusing Multiphoton Excitation Single‐Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores

“…This technique results in a laser pulse with high peak power and a short pulse width, which enables wide-field and opticalsection fluorescence excitation on the objective focal plane. [26][27][28][29][30][31][32] Overall, capturing MPE fluorescence images with a high-speed camera considerably increases the temporal resolution of images, thus meeting the requirements for recording the blinking fluorescence of fluorophores. Therefore, TFMPE is applied for localization imaging as an alternative optical-section activation source in photoactivated localization microscopy (PALM) to obtain localization images with a lateral resolution higher than 50 nm for cell samples.…”

“…As an alternative, temporal‐focusing MPE (TFMPE) microscopy recombines all the frequencies of the laser pulse in phase on the objective focal plane through a diffraction grating and the 4 f configuration of collimating and objective lenses. This technique results in a laser pulse with high peak power and a short pulse width, which enables wide‐field and optical‐section fluorescence excitation on the objective focal plane [26–32] . Overall, capturing MPE fluorescence images with a high‐speed camera considerably increases the temporal resolution of images, thus meeting the requirements for recording the blinking fluorescence of fluorophores.…”