Adaptive multi-photon imaging

Multi-photon excitation scanning microscopy such as two-photon excitation fluorescence (TPEF) is one of the most powerful tools for imaging structures deep in biological tissues.  It uses infrared excitation wavelengths, which are less prone to scattering compared to visible light and can penetrate significantly farther through seemingly opaque tissues like brain and skin. Also, in TPEF there is no need to sharply image the signal – which is  distorted as it propagates through the specimen – onto a camera. Instead, all of the light is collected by a bucket detector and an image is generated by scanning the excitation beam Below is a TPEF image of a microglia cell in mouse brain tissue taken in our lab.

Figure 1 TPEF image of GFP-labelled microglia cell inside fixed mouse brain tissue


However, even TPEF imaging can only be used up to a finite depth. Beyond this, too many excitation photons undergo multiple scattering events, which destroys the imaging focus as shown in Fig. 1.

Figure 2 Effect of scattering tissue on a focused laser beam.


In this project, we investigate methods to counteract these scattering events using spatial light modulation and therefore to significantly push the limits of deep tissue imaging. Our focus lies on bringing the technology closer to biological real-world applications. This requires improving on certain key aspects such as correction speed, photon-efficient algorithms and enlarging the corrected field of view. 

Figure 3 TPEF imaging is already improved by using adaptive optics to correct low order aberrations in the imaging system. The next step is to tackle the high order aberrations caused by scattering.

Results of this project have been recently highlighted in a contribution to the "Sculptured Light in the Brain Online Series 2021":

Fast converging compensation for deep tissue imaging

We are currently hiring a master’s student on this project. If you would like to learn more or apply for the position, please email Assoc. Prof. Alexander Jesacher at to arrange a meeting.


Researchers on this project at our institute

-) Molly May, PhD (postdoc)
-) Prof. Monika Ritsch-Marte
-) Alexander Jesacher, PhD (PI)


“Deep brain vision: 3D adaptive 2-photon microscopy” FWF P32146,  2019 – 2023.


  • Michaela Kress, Dept. of Physiology, Medical University of Innsbruck


[1] Martin Bawart, Molly A. May, Thomas Öttl, Clemens Roider, Stefan Bernet, Michael Schmidt, Monika Ritsch-Marte, and Alexander Jesacher, "Diffractive tunable lens for remote focusing in high-NA optical systems," Opt. Express 28, 26336-26347 (2020)

[2] Molly A. May, Martin Bawart, Michiel Langeslag, Stefan Bernet, Michaela Kress, Monika Ritsch-Marte, and Alexander Jesacher, "High-NA two-photon single cell imaging with remote focusing using a diffractive tunable lens," Biomed. Opt. Express 11, 7183-7191 (2020)

[3] M.A. May, K.K. Kummer, M-L. Edenhofer, J. L. Choconta, M. Kress, M. Ritsch-Marte, and A. Jesacher: Simultaneous scattering compensation at multiple points in multi-photon microscopy. Biomedical Optics Express 12, 12, 7377-7387 (2021),

[4] M.A. May, N. Barré, K.K. Kummer, M. Kress, M. Ritsch-Marte and A. Jesacher: Fast holographic scattering compensation for deep tissue biological imaging. Nature Communications 12, 4340 (2021)