Simultaneous detection of multiple cellular and molecular players in their
native environment, one of the keys to a full understanding of immune
processes, remains challenging for in vivo microscopy. Here, we present a
synergistic strategy for spectrally multiplexed in vivo imaging composed of
(i) triple two-photon excitation using spatiotemporal synchronization of two
femtosecond lasers, (ii) a broad set of fluorophores with emission ranging
from blue to near infrared, (iii) an effective spectral unmixing algorithm.
Using our approach, we simultaneously excite and detect seven fluorophores
expressed in distinct cellular and tissue compartments, plus second harmonics
generation from collagen fibers in lymph nodes. This enables us to visualize
the dynamic interplay of all the central cellular players during germinal
center reactions. While current in vivo imaging typically enables recording
the dynamics of 4 tissue components at a time, our strategy allows a more
comprehensive analysis of cellular dynamics involving 8 single-labeled
compartments. It enables to investigate the orchestration of multiple cellular
subsets determining tissue function, thus, opening the way for a mechanistic
understanding of complex pathophysiologic processes in vivo. In the future,
the design of transgenic mice combining a larger spectrum of fluorescent
proteins will reveal the full potential of our method
