Rakhymzhan et al. – Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo

Sci Rep. 2017 Aug 2;7(1):7101

by Rakhymzhan A, Leben R, Zimmermann H, Günther R, Mex P, Reismann D, Ulbricht C, Acs A, Brandt AU,  Lindquist RL, Winkler TH, Hauser AE, Niesner RA

(DIAL in cooperation with DRFZ AG Niesner)

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.

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