Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
Abstract
:1. Introduction
2. Materials and Methods
2.1. Temporal Focusing Microscopy Setup
2.2. Imaging Samples
3. Results
3.1. Signal Intensity Increases Linearly with the Repetition Rate
3.2. Photobleaching Rate Increases with Peak Intensity, but Not with the Repetition Rate
3.3. Temperature Constraints Indicate an Optimum Repetition Rate Between 0.5 and 5MHz
3.4. Live Sample Imaging at 100 kHz Yields High Signal Intensities without a Photomultiplier
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Model Param. | Description | Values and Units | Ref. |
---|---|---|---|
Fa | Fraction of absorbed photons that do not result in two-photon emission | — | — |
Na | Number of absorbed photons | — | — |
Nτ | Total number of photons | — | — |
N1λ | Number of photons absorbed due to one-photon absorption of the medium | — | — |
N2λ-Nem | Number of absorbed photons that do not result in two-photon emission | — | — |
µa | Absorption coefficient | 1 cm−1 | [25,26,27] |
l | Propagation length (two Rayleigh lengths) | 5 µm | — |
I | Peak power density | W·cm−2 | — |
S | Coherence parameter | — | [28] |
δ2λ | Two-photon cross-section at 960 nm | 31 × 10−50 cm4·s−1·molec. −1·photon−1 | [11] |
C | Typical concentration of fluorophores | 6 × 1018 molec.·cm−3 | [29] |
λ | Wavelength used for two-photon imaging | 960 nm | — |
h | Planck constant | 6.62607015 × 10−34 J·s−1·photon−1 | — |
c | Speed of light in vacuum | 299,792,458 m·s−1 | — |
Φ | Quantum efficiency | 0.67 | [11] |
ε | Heat source | J·m−3 | — |
PA | Average laser power | W | — |
V | Volume of the excitation cylinder | µm3 | — |
qr | Heat | J | — |
T | Temperature | K | — |
k | Specific thermal conductivity of brain tissue | 0.5918 W·K−1·m−1 | [30,31] |
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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. https://doi.org/10.3390/mps2030065
Lisicovas V, Mariserla BMK, Sahoo C, Harding RT, Man MKL, Wong EL, Madéo J, Dani KM. Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate. Methods and Protocols. 2019; 2(3):65. https://doi.org/10.3390/mps2030065
Chicago/Turabian StyleLisicovas, Viktoras, Bala Murali Krishna Mariserla, Chakradhar Sahoo, Reuben T. Harding, Michael K. L. Man, E Laine Wong, Julien Madéo, and Keshav M. Dani. 2019. "Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate" Methods and Protocols 2, no. 3: 65. https://doi.org/10.3390/mps2030065