Non-degenerate 2-photon excitation in scattering medium for fluorescence microscopy

Abstract

Non-degenerate 2-photon excitation (ND-2PE) of a fluorophore with two laser beams of different photon energies offers an independent degree of freedom in tuning of the photon flux for each beam. This feature takes advantage of the infrared wavelengths used in degenerate 3-photon excitation (D-3PE) microscopy to achieve increased penetration depths, while preserving a relatively high 2-photon excitation cross section in comparison to that of D-3PE. Here, using spatially and temporally aligned Ti:Sapphire laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, we employ ND-2PE and provide a practical demonstration that a constant fluorophore emission intensity is achievable deeper into a scattering medium using ND-2PE as compared to the commonly used degenerate 2-photon excitation (D-2PE). References and links1. K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50(6), 823-839 (2006). 2. J. N. Kerr and W. Denk, "Imaging in vivo: watching the brain in action," Nat. Rev. Neurosci. 9(3), 195-205 (2008). 3. W. Denk and K. Svoboda, "Photon upmanship: why multiphoton imaging is more than a gimmick," Neuron 18(3), 351-357 (1997). 4. F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2(12), 932-940 (2005 11. D. Kobat, N. G. Horton, and C. Xu, "In vivo two-photon microscopy to 1.6-mm depth in mouse cortex," J. Biomed. Opt. 16(10), 106014 (2011). 12. R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, "Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser," Sci. Rep. 3, 1014 (2013). 13. P. Theer and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am.A adaptive optical recovery of optimal resolution over large volumes," Nat. Methods 11(6), 625-628 (2014).

Short-Wave Infrared Confocal Fluorescence Imaging of Deep Mouse Brain with a Superconducting Nanowire Single-Photon Detector

“…41 For confocal fluorescence imaging, and taking into account the Stokes shift, Figure 5b,c shows that the l con reached the maximum values with three pairs of excitation and emission wavelengths: (1) excitation in the 1300 nm window and emission in the 1700 nm window (this work), (2) excitation in the 1700 nm window and emission in the 2200 nm window, Therefore, in addition to the results shown here, there are additional opportunities for long-wavelength confocal fluorescence imaging if appropriate fluorescent labels exist. We note that the considerations for the wavelength selections discussed here are also applicable to nonlinear excitations where two or more wavelengths are employed for excitation such as nondegenerate two-photon excitation, 48 stimulated Raman scattering, 49 and pump−probe imaging. 50,51 With the estimated confocal signal attenuation length, we can determine the absolute imaging depth limit of fluorescence confocal microscopy in the following section.…”

Efficient non-degenerate two-photon excitation for fluorescence microscopy

“…We also showed that the increase in tissue heating, due to excitation with longer wavelengths, is not as large as water absorption increase for longer wavelengths. This increase in heating may be an acceptable trade-off for leveraging enhanced ND-TPACS and other advantages of ND-2PE [22] in deep tissue imaging applications.…”

“…4. The linear dependence of the fluorescence signal generated by ND-TPE on the excitation power of each beam is the characteristic signature of the ND-TPE [22,29].…”

“…Alternatively, the same energy needed for the transition to the excited state can be delivered via absorption of two photons of different energy (i.e., different color) [12]. This non-degenerate two-photon excitation (ND-TPE) regime has been explored in two-photon excitation microscopy aiming to extend the excitation wavelength range [12][13][14], reduce out-of-focus fluorescence [15][16][17][18], increase spatial resolution [19,20], and increase penetration depth in scattering media [21,22].…”

Development of a beam propagation method to simulate the point spread function degradation in scattering media

“…Higher-order moments and other details of the phase function are also ignored, which will be studied in the future by incorporating other forms of power spectral densities using different local phase smoothing functions [21]. Nonetheless, we expect that this model will be useful to guide further developments of optical microscopy techniques to overcome scattering such as non-degenerate two-photon microscopy [22], and three-photon microscopy [23].…”