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.
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.
Two-photon fluorescence microscopy (2PM)
enables scientists in various fields including neuroscience
, embryology
, and oncology
to visualize
and
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 tissue
or the insertion of optical probes
. Here we demonstrate non-invasive, high resolution,
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.
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.
Optical imaging plays a major role in both basic biological research and clinical diagnostics, providing noninvasive or minimally invasive microscopic imaging capability to investigate biological ...tissues. Optical image acquisition through significant depths of biological tissues, however, presents a major challenge since tissue is extremely heterogeneous and the strong scattering of the various tissue components has restricted high-resolution optical imaging to superficial layers. Multiphoton microscopy (MPM) has significantly extended the penetration depth of high-resolution optical imaging, particularly for in vivo applications. Multiphoton imaging critically depends on ultrafast technologies, particularly pulsed excitation sources. In this paper, the basics of deep tissue MPM and its improvements utilizing soliton self-frequency shift (SSFS) are reviewed. Wavelength tunable, high-energy soliton generation through SSFS in large-mode-area (LMA) fibers and photonic crystal rods is presented. The application of these solitons to MPM enables noninvasive imaging in biological tissues with unprecedented depth. The main characteristics of the excitation source for deep tissue MPM, such as wavelength, pulse energy, and repetition rate, are discussed.
Multiphoton microscopy (MPM) is widely used for optical sectioning deep in scattering tissue,
1-2. Phosphorescence lifetime imaging microscopy (PLIM) 3 is a powerful technique for obtaining ...biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching 4-5. Point-measurement PLIM 6 of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM 7, 8. However, the maximum fluorescence generation rate, which is inversely proportional to the phosphorescence lifetime, fundamentally limits PLIM pixel rates. Here we experimentally demonstrate a parallel-excitation/parallel collection MPM-PLIM system that increases pixel rate by a factor of 100 compared with conventional configurations while simultaneously acquiring lifetime and intensity images at depth
. Full-frame three-dimensional in vivo PLIM imaging of phosphorescent quenching dye is presented for the first time and defines a new platform for biological and medical imaging.
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.
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.
Confidence intervals provide a way to determine plausible values for a population parameter. They are omnipresent in research articles involving statistical analyses. Appropriately, a key statistical ...literacy learning objective is the ability to interpret and understand confidence intervals in a wide range of settings. As instructors, we devote a considerable amount of time and effort to ensure that students master this topic in introductory courses and beyond. Yet, studies continue to find that confidence intervals are commonly misinterpreted and that even experts have trouble calibrating their individual confidence levels. In this article, we present a 10-min trivia game-based activity that addresses these misconceptions by exposing students to confidence intervals from a personal perspective. We describe how the activity can be integrated into a statistics course as a one-time activity or with repetition at intervals throughout a course, discuss results of using the activity in class, and present possible extensions. Supplementary materials for this article are available online.
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.
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