Refinement of 3-photon microscopy excited at the 1700-nm window

Session

Poster Session

Authors

Gert-Jan Bakker (1), Maria Parlani (1), Peter Friedl (1)

Affiliations

1. Medical BioSciences department, Radboud University Medical Centre

Keywords

3-photon microscopy; bone; cancer biology; cell biology; human; mouse; nonlinear microscopy; third harmonic generation; tumor

Abstract text

Summary - We refined low-repetition-rate high-pulse-energy (highIR) excitation for 3-, 4-photon and higher harmonic microscopy in the 1700 nm window. By minimizing the pulse length under the microscope objective, the excitation efficiency was improved and the average power reduced, effectively reducing the thermal load on the sample. 

Introduction - Three-photon (3P) microscopy opens avenues for functional and structural imaging deep inside scattering tissues such as the brain, tumors, lymph nodes and bones[1-3]. The 1700 nm window offers most efficient 3P excitation of red fluorophores, based on reduced light scattering and attenuation as compared to 1300 nm and simultaneous excitation of a broad range of fluorophores and harmonic processes using a single beam[3]. However, the thermal load on the sample increases as compared to 3P excitation in the 1300 nm window, due to higher linear absorption of the excitation power. Therefore, further optimization of the excitation process and reducing phototoxicity during measurements is required. 

Methods/Materials - HighIR excitation was generated with a turn-key laser system with integrated  group delay dispersion (GDD) compensation (CRONUS-3P V2.0, Light Conversion, Lithuania). The excitation light travelled through an additional pair of prims to compensate for third order dispersion (TOD compressor, Light Conversion, Lithuania). Furthermore, the number of optical parts in the excitation path was minimized and the dichroic and multilayer mirrors in the scanhead (TrimScope II, Miltenyi Biotec, Germany) were replaced with silver mirrors and a custom scan and tubelens were installed. An autocorrelator with external sensor (Carpe IR, APE Angewandte Physik & Elektronik GmbH, Germany) was used to optimize the GDD setting and to verify dispersion properties of various parts in the optical path. 

Results and discussion – After setup optimization, pulse lengths of 57 fs (with TOD compressor) and 66 fs (without TOD compressor) were measured under the objective (1650 nm excitation, Sech² fit). Live cell experiments with tagRFP-expressing HT1080 fibrosarcoma cells showed that the increased excitation efficiency did not lead to increased 3-photon emission intensity, because fluorophore saturation effects and phototoxicity at 57 fs pulse width occurred at a lower energy thresholds. On the other hand, excitation efficiency was improved at pulse width of 57 fs, resulting in reduced pulse energy and average power, by a factor 1.5, to obtain a similar 3-photon emission strength as compared to our previous experiments performed with 89 fs pulse length[3]. 

Conclusion – By optimization of the highIR excitation pulse length of our microscope system, we were able to improve 3- and 4-photon excitation efficiency at 1650 nm, effectively reducing the average excitation power by a factor 1.5, in line with theory, thereby reducing heat buildup in the sample.

References

1.            Horton, N.G., et al., In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nature Photonics, 2013. 7(3): p. 205-209.

2.            Choe, K., et al., Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes. Nat Immunol, 2022. 23(2): p. 330-340.

3.            Bakker, G.J., et al., Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy. Elife, 2022. 11.