Three-photon imaging of mouse brain structure and function through the intact skull.

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-µm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain.

High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

Direct visualization of functional heterogeneity in hepatobiliary metabolism using 6-CFDA as model compound.

Hepatobiliary metabolism is one of the major functions of the liver. However, little is known of the relationship between the physiological location of the hepatocytes and their metabolic potential. By the combination of time-lapse multiphoton microscopy and first order kinetic constant image analysis, the hepatocellular metabolic rate of the model compound 6-carboxyfluorescein diacetate (6-CFDA) is quantified at the single cell level. We found that the mouse liver can be divided into three zones, each with distinct metabolic rate constants. The sinusoidal uptake coefficients k1 of Zones 1, 2, and 3 are respectively 0.239 ± 0.077, 0.295 ± 0.087, and 0.338 ± 0.133 min-1, the apical excreting coefficients k2 of Zones 1, 2, and 3 are 0.0117 ± 0.0052, 0.0175 ± 0.0052, and 0.0332 ± 0.0195 min-1, respectively. Our results show not only the existence of heterogeneities in hepatobiliary metabolism, but they also show that Zone 3 is the main area of metabolism.

Dispersion compensation in three-photon fluorescence microscopy at 1,700 nm.

Signal generation in three-photon microscopy is proportional to the inverse-squared of the pulse width. Group velocity dispersion is anomalous for water as well as many glasses near the 1,700 nm excitation window, which makes dispersion compensation using glass prism pairs impractical. We show that the high normal dispersion of a silicon wafer can be conveniently used to compensate the dispersion of a 1,700 nm excitation three-photon microscope. We achieved over a factor of two reduction in pulse width at the sample, which corresponded to over a 4x increase in the generated three-photon signal. This signal increase was demonstrated during in vivo experiments near the surface of the mouse brain as well as 900 µm below the surface.

Measurements of multiphoton action cross sections for multiphoton microscopy.

We report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. These values indicate that the optimum repetition rate for deep tissue 3-photon microscopy is approximately 1 to 2 MHz. We further demonstrate that it is feasible to perform 4-photon fluorescence microscopy of GFP labeled microglia in mouse brain in vivo at 1700 nm. 4-photon excitation increases the accessibility of fluorophores at the long wavelength spectral window of 1700 nm.

In vivo three-photon microscopy of subcortical structures within an intact mouse brain.

Two-photon fluorescence microscopy (2PM)1 enables scientists in various fields including neuroscience2,3, embryology4, and oncology5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue3 or the insertion of optical probes6,7. Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.

Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy.

We demonstrate a fiber-based, three-color femtosecond source for simultaneous imaging of three fluorescent proteins (FPs) using two-photon fluorescence microscopy (2PM). The three excitation wavelengths at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation (SHG) of the 1550-nm pump laser and the 1728-nm and 1900-nm solitons generated through soliton self-frequency shift (SSFS) in a large-mode-area (LMA) fiber. These energetic pulses are well matched to the two-photon excitation peaks of red, cyan and yellow fluorescent proteins (TagRFPs, TagCFPs, and TagYFPs) for efficient excitation. We demonstrate simultaneous 2PM of human melanoma cells expressing a "rainbow" combination of these three fluorescent proteins.

In vivo two-photon microscopy to 1.6-mm depth in mouse cortex.

Deep tissue in vivo two-photon fluorescence imaging of cortical vasculature in a mouse brain using 1280-nm excitation is presented. A record imaging depth of 1.6 mm in mouse cortex is achieved in vivo, approximately reaching the fundamental depth limit in scattering tissue.