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Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate

Femtosecond Spectroscopy Unit, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
Department of Physics, Indian Institute of Technology, Jodhpur, Rajasthan 342037, India
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(3), 65;
Received: 2 July 2019 / Revised: 24 July 2019 / Accepted: 25 July 2019 / Published: 28 July 2019
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
PDF [2969 KB, uploaded 28 July 2019]


Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community. View Full-Text
Keywords: two-photon microscopy; wide-field excitation; live imaging; temporal focusing two-photon microscopy; wide-field excitation; live imaging; temporal focusing

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Lisicovas, V.; Mariserla, B.M.K.; Sahoo, C.; Harding, R.T.; Man, M.K.L.; Wong, E.L.; Madéo, J.; Dani, K.M. Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate. Methods Protoc. 2019, 2, 65.

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