Abstract
Demands of higher spatial and temporal resolutions in linear and nonlinear imaging keep pushing the limits of optical microscopy. We showed recently that a multiphoton microscope with 200 kHz repetition rate and wide-field illumination has a 2-3 orders of magnitude improved throughput compared to a high repetition rate confocal scanning microscope. Here, we examine the photodamage mechanisms and thresholds in live cell imaging for both systems. We first analyze theoretically the temperature increase in an aqueous solution resulting from illuminating with different repetition rates (keeping the deposited energy and irradiated volume constant). The analysis is complemented with photobleaching experiments of a phenolsulfonphthalein (phenol red) solution. Combining medium repetition rates and wide-field illumination promotes thermal diffusivity, which leads to lower photodamage and allows for higher peak intensities. A three day proliferation assay is also performed on living cells to confirm these results: dwell times can be increased by a factor of 3×10(6) while still preserving cell proliferation. By comparing the proliferation data with the endogenous two-photon fluorescence decay, we propose to use the percentage of the remaining endogenous two-photon fluorescence after exposure as a simple in-situ viability test. These findings enable the possibility of long-term imaging and reduced photodamage.
Highlights
To image living cells, it is required that the optical interactions that give rise to an image does not induce damage [1,2,3,4,5]
Kalies et al [19] showed that the photobleaching half-life of exogenous 2PF fluorophores matches the half-saturation value of the reactive oxygen species (ROS) production, and Tirlapur et al [31] showed that the ROS production is increased when illuminating with a fs laser pulse leading to cell apoptosis
We determined the optical parameters that allow for damage- and label-free imaging of live cells using wide-field medium-repetition-rate multiphoton microscopy
Summary
It is required that the optical interactions that give rise to an image does not induce damage [1,2,3,4,5]. We consider the effects of these three pathways to optical damage and model photodamage using the heat transport equation, paying particular attention to the effects induced by changing from scanning to wide-field imaging and lowering the repetition rate [13]. We combine this analysis with two-photon fluorescence (2PF) experiments of solutions and endogenous in living cells. We propose an in-situ viability test based on the decay of endogenous 2PF signals
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
