Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence

Abstract

We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity.

Transcutical imaging with cellular and subcellular resolution

“…Recently, another research group used a micro-electro-mechanical (MEMS) SLM to maximize local 3P fluorescence signal of neurons and blood vessels in a feedback-based optimization scheme [10]. They demonstrated up to 4x increase in relative intensity within a few seconds.…”

“…The nonlinearity orders are 3.10 and 3.07 with standard deviation of 0.10 and 0.34 in these two cases. In addition, the same excitation power generated much stronger fluorescence emission when D 2 Refractive aberrations affect 3P imaging more severely than 1P or 2P imaging due to the higher order non-linearity [10]. In order to characterize the aberration-induced signal loss, we used the DM to introduce primary spherical aberrations with different amplitudes, and measured the intensity of fluorescence emitted by microspheres.…”

“…Refractive aberration increases the focal volume and hence decreases twophoton fluorescence emission. One can use adaptive optics (AO) to correct refractive aberration to increase the imaging depth [6][7][8][9][10][11]. Three-photon (3P) excitation enables imaging of fluorescent proteins (e.g., mCherry) and neuronal activity sensors (e.g., red calcium indicators) at approximately 3 times the wavelength of their one-photon excitation peaks.…”

Characterization of wavefront errors in mouse cranial bone using second-harmonic generation

“…AO correction using these correction elements will allow us to image fine structures in the bone marrow (e.g., subcellular features, such as mitochondria) that would not be resolvable using a lower NA, lower magnification objective lens to reduce distortions; 57 or by moving to longer wavelength two-photon or three-photon excitation that requires an exotic optical assembly and fluorophores. 6 Our bone characterization approach can also be used to directly estimate wavefront aberrations and be applied to a correction algorithm; however, it should be noted that the illumination wavelength used to estimate aberrations should match the wavelength to be corrected.…”

“…Multiphoton excitation microscopy has the potential to acquire deep tissue optical sectioned images with minimal damage in highly scattering media at depths greater than 150 μm. [1][2][3][4][5][6] This potential has led to widespread use in the bone biology and neuroscience fields. Two-photon microscopy commonly exploits light with longer wavelengths in optimal windows for in vivo tissue imaging (near-IR and IR) with reduced Rayleigh scattering and absorbance to penetrate deeper inside biological tissue; but nonhomogeneous wave propagation through highly scattering turbid media, such as bone, dramatically reduces image resolution even at moderate depths of ∼100 μm.…”