Multiphoton microscopy is a form of nonlinear microscopy that relies on the simultaneous absorption of multiple low-energy photons (e.g., two or more) to excite a molecule. This technique has several advantages over conventional single-photon microscopy, including
Deeper excitation: due to the nature of, Infra-Red Light used in microscopy, is less scattered by samples allowing deeper penetration of the light into samples, particularly in thick tissues such as living organs,
Less photodamage and phototoxicity than visible LSM: as excitation is confined to the focal volume of the light beam. Moreover, the lower energy of the photons used results in reduced radiation exposure along the excitation path within the tissue compared to conventional visible-light microscopy. Another advantage of multiphoton microscopy is access to harmonic generation microscopy. Second harmonic generation (SHG) and third harmonic generation (THG) do not require external labeling, as they occur naturally in certain structures in biological tissues. These processes are exploited in multiphoton microscopy to visualize certain structural components (collagen, lipids).

The development and improvement of multicolor imaging technologies allow researchers to explore the complexity of the cellular composition of biological tissues, as accessible by cytometry techniques, while preserving information on the spatial organization of these tissues. Thus, these techniques offer information on the localization of cells of interest as well as spatial organization of cells within the tissue. Among these advanced techniques, modern microscopes equipped with multispectral imaging and linear unmixing are capable of simultaneously separating more than five fluorochromes, even when their emission spectra overlap, depending on the number of laser sources present in these systems [1]. Multispectral multiphoton microscopy is possible, but it traditionally requires multiple excitation wavelengths or complex pulse-synchronization schemes, which introduce significant optical alignment challenges and chromatic aberrations [2-3]. Moreover, spectral crosstalk and dispersion management complicate simultaneous excitation of several fluorophores, limiting robustness and accessibility of multicolor deep-tissue imaging. Single-wavelength multispectral strategies therefore offer an attractive alternative by simplifying the optical design while enabling stable, low-power excitation of multiple fluorophores [4]. To date, no study has presented a multiphotonic multispectral imaging approach that enables more than 3 fluorochromes separation with a single illumination source. Here we propose a 7-color multispectral imaging strategy allowing the localization of large cellular compartments in adipose tissue using only a biphoton approach and a single wavelength.

Biological protocol :
Paraformaldehyde (PFA) (4%) fixed adipose tissue from C57BL6 mice were embedded in agarose 2% overnight and sliced with Vibroslicer® 5100 mz (Campden instruments) (300 µm thick). Samples were simultaneously permeabilized and blocked using 0.2% Triton X-100 and horse serum in PBS. Tissue sections were incubated with fluorochrome coupled antibodies (table1) overnight at room temperature under constant agitation. Several washing with PBS with triton were performed, and then nuclei were stained with Draq5.

Images acquisition :
Images acquisition was performed on a ZEISS LSM880 NLO confocal microscope equipped with a gallium arsenide phosphide (GaAsP) spectral array detector tuning to 8.9 nm that allows 32 channel images ranging from 410 to 695 nm. Emission spectrum for each fluorochrome was extracted to be used in the “online fingerprinting” mode of ZEN Black imaging software. We used a High-Power Widely Tunable Femto second laser from Coherent (Chameleon Discovery NX) tuning range from 610 to 1320 nm to excite the samples.

Simultaneous imaging of multiple fluorescent labels within biological tissues is crucial for dissecting complex cellular and molecular interactions. We have overcome the main challenge: the ability to efficiently excite multiple fluorophores with distinct emission spectra using a single excitation wavelength. Since two-photon excitation is highly wavelength-dependent and nonlinear, identifying a wavelength that provides sufficient excitation efficiency for multiple fluorochromes simultaneously was non-trivial. To achieve this, we chose to use antibodies directly conjugated to fluorochromes. Several fluorescent conjugates were tested to identify those efficiently excited by two-photon microscopy at a single excitation wavelength.Here we propose a multiphotonic imaging approach of multilabel excitation (7 channels) with a unique excitation wavelength. This offers several advantages, including that of eliminating the impact of excitation wavelengths on chromatic aberrations.

In addition to enabling efficient fluorochrome separation, the use of an 800 nm laser also allowed the generation of a second harmonic generation (SHG) signal. This intrinsic signal, which does not require any exogenous labeling, provides complementary structural information, particularly on the organization and composition of fibrillar collagen. Given that collagen architecture is frequently altered in pathological conditions such as fibrosis, inflammation, or tumor progression, SHG imaging adds valuable context to the interpretation of the tissue pathophysiological state. This multimodal approach, combining fluorescence and label-free SHG, enhances the biological relevance of imaging data and supports more comprehensive tissue analysis.

Contrary to regular methods using several excitation wavelengths to acquire up to 7 colors [5], here we have demonstrated the capability to acquire up to seven colors or more using only a single excitation wavelength. This breakthrough opens new possibilities for simplifying laser setups in multiphoton imaging applications. Some infrared (IR) lasers provide a fixed emission line, which allows for the creation of more affordable and robust solutions for multicolor multiphoton imaging. By leveraging this innovation, researchers can utilize simpler laser systems without sacrificing imaging performance.
Furthermore, this work was performed on the LSM 880 system, which supports a maximum of eight simultaneously unmixable colors. In contrast, the new LSM 980 NLO and LSM 990 NLO systems are equipped with advanced spectral detection technology and are operated via ZEN Blue software. These platforms allow the use of all available detectors through the online fingerprinting mode, enabling the combination of a greater number of fluorophores within a single scan. As a result, highly multiplexed imaging can be achieved, leading to improved experimental efficiency and enhanced data quality.