Optical Microscopy and Wavefront Shaping

Group leader: Alexander Jesacher

Our group develops new methods for deep-tissue optical microscopy and complex light shaping, combining adaptive optics and computer-generated holography to extend the limits of what can be visualized inside biological specimens.

Deep-Tissue Imaging via Nonlinear Microscopy and Adaptive Optics

Optical microscopy allows us to visualize cells and tissues with sub-micrometer resolution and rich functional contrast. In our group, we develop advanced imaging methods that extend the current limits of optical microscopy, with the goal of increasing imaging depth, improving image quality inside scattering tissue, and extracting more information from biological samples.

A central component of our work is dynamic wavefront shaping, which enables precise control of light fields at megapixel resolution and kilohertz update rates. These technologies allow us to correct distortions introduced by biological tissue and to restore diffraction-limited imaging performance deep inside complex specimens.

Multi-Photon Microscopy

Understanding how cells function inside intact tissue requires imaging techniques that remain effective even in strongly scattering environments. Multi-photon microscopy meets this challenge by using ultrashort laser pulses in the near-infrared spectral range (typically 800–1300 nm), where light penetrates deeper into biological tissue.

Because nonlinear optical signals are generated only at the focal point, multi-photon microscopy naturally provides optical sectioning and enables high-contrast three-dimensional imaging hundreds of micrometers deep—and in favorable cases approaching millimeter depths—in challenging specimens such as brain tissue or organoids.

**Figure 1.1** shows an axial image stack of GFP-expressing microglia in a fixed mouse brain slice (hippocampus) down to a depth of 500 µm. Third-harmonic generation (red) highlights axons, blood vessels, and cellular interfaces. (Sample provided by Kai Kummer, Dept. of Physiology at the Medical University of Innsbruck)

**Figure 1.2** shows neurons expressing GFP-progerin inside a human brain organoid. Third-harmonic signals reveal cellular boundaries and internal structural organization without additional labeling. (Sample provided by Elisa Gabassi and Frank Edenhofer, Institute of Molecular Biology at the University of Innsbruck)

**Figure 1.3** shows a cross-section of a human meningioma. Second-harmonic generation from myosin highlights muscle tissue surrounding a blood vessel. The image was acquired entirely without fluorescent labels, demonstrating the power of label-free nonlinear microscopy. (Sample provided by Prof. Adelheid Wöhrer, Institute of Neuropathology, Medical University of Innsbruck)

Adaptive Optics

Imaging at even greater depths requires compensating optical distortions introduced by tissue inhomogeneities. In our research, we address this challenge using adaptive optics and dynamic wavefront shaping.

By shaping the excitation wavefront before it enters the specimen, aberrations and scattering effects can be partially compensated, allowing a tight focus to be restored deep inside the tissue. This improves signal strength, resolution, and contrast in otherwise inaccessible regions.

**Figure 1.4** illustrates the principle of adaptive optics. Without correction, tissue distorts the excitation beam and prevents the formation of a sharp focus. After wavefront correction, a diffraction-limited focus can be recovered. The inset images show two-photon fluorescence from a 4 µm microbead beneath 0.5 mm of chicken breast tissue before and after correction.

**Figure 1.5** shows GFP expressing endothelial cells inside mouse lung tissue imaged before and after aberration correction. The scale bar indicates 10 µm.

**Figure 1.6** shows a GFP-expressing microglia cell in fixed mouse brain tissue. After correction, fine cellular processes become clearly visible.

Complex Light Shaping and Computer-Generated Holography

Computer-generated holography (CGH) allows us to design complex light fields numerically and generate them experimentally using programmable diffractive optical elements. This enables precise control over the amplitude and phase of light and opens new possibilities for adaptive optics, structured illumination, and point-spread-function engineering in advanced microscopy.

**Figure 2.1** shows an example of multi-plane light conversion (MPLC). By applying multiple phase-modulation steps in consecutive planes, nearly arbitrary amplitude and phase distributions can be generated. In this example, a Gaussian beam is transformed into a user-defined arrangement of geometric intensity patterns.

**Figure 2.2** demonstrates a two-plane light-conversion configuration that reshapes incoherent illumination from a green LED into user-defined intensity distributions. This illustrates that wavefront-shaping approaches can also be applied to non-laser light sources, enabling flexible illumination control in compact optical systems.

**Figure 2.3** shows a computer-designed volume hologram fabricated inside fused silica using direct femtosecond laser writing. The structure consists of hundreds of thousands of precisely arranged phase voxels and converts incident Gaussian beams into Hermite–Gaussian modes depending on the angle of incidence.

Selected publications

Nam K., Borozdova M., Park J., Jesacher A., Fast converging iterative wavefront sensing for scatter compensation in multi-photon fluorescence microscopy, J. Phys. Photonics, DOI:10.1088/2515-7647/adeb1f

Maloberti J. G. et al., Joint estimation of point spread function and molecule positions in SMLM informed from multiple planes, Biomed. Opt. Express, 1310, DOI:10.1364/BOE.551278

Muñoz-Bolaños J. et al., Confocal Raman Microscopy with Adaptive Optics, ACS Photonics, DOI:10.1021/acsphotonics.4c01432

Sohmen M., Borozdova M., Ritsch-Marte M., Jesacher A., Complex-valued scatter compensation in nonlinear microscopy, Phys. Rev. Applied, DOI:10.1103/physrevapplied.22.044036

Barré N. et al., Direct laser-written aperiodic photonic volume elements for complex light shaping with high efficiency: inverse design and fabrication, Advanced Photonics Nexus, 036006-036006, DOI:10.1117/1.APN.2.3.036006

May M. et al., Fast holographic scattering compensation for deep tissue biological imaging, Nature Communications, 4340, DOI:10.1038/s41467-021-24666-9