Abstract
High spatial resolution imaging of the mouse brain through the intact skull is challenging because of the skull-induced aberration and scattering. The research group of Dan Zhu from Huazhong ...University of Science and Technology has developed a skull-clearing technique that provides a long-term (~ weeks), stable, transparent window for high resolution optical imaging over a large field of view.
Three-photon microscopy has been increasingly adopted for probing neural activities beyond the typical two-photon imaging depth. In this review, we outline the unique properties that differentiate ...three-photon microscopy from two-photon microscopy for in vivo imaging in biological samples, especially in the mouse brain. We present a systematic summary of the optimization of three-photon imaging parameters for neural imaging, based on their effects on calcium imaging quality and perturbation to brain tissues. Furthermore, we review the existing techniques for volumetric imaging and discuss their prospects in mesoscale three-photon imaging in deep tissue.
Optical imaging is important for understanding brain function. However, established methods with high spatiotemporal resolution are limited by the potential for laser damage to living tissues. We ...describe an adaptive femtosecond excitation source that only illuminates the region of interest, which leads to a 30-fold reduction in the power requirement for two- or three-photon imaging of brain activity in awake mice for improved high-speed longitudinal neuroimaging.
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.
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.
Clinician moral distress has been documented over the past several decades as occurring within numerous healthcare disciplines, often in relation to clinicians’ involvement in patients’ end-of-life ...decision-making. The resulting harms impact clinician well-being, patient well-being, and healthcare system functioning. Given Covid-19’s catastrophic death toll and associated demands on end-of-life decision-making processes, the pandemic represents a particularly important context within which to understand clinician moral distress. Thus, we conducted a convergent mixed methods study to examine its prevalence, associations with clinicians’ demographic and professional characteristics, and contributing circumstances among Veterans Health Administration (VA) clinicians. The study, conducted in April 2021, consisted of a cross-sectional on-line survey of VA clinicians at 20 VA Medical Centers with professional jurisdiction to place life-sustaining treatment orders working who were from a number of select specialties. The survey collected quantitative data on respondents’ demographics, clinical practice characteristics, attitudes and behaviors related to goals of care conversations, intensity of moral distress during “peak-Covid,” and qualitative data via an open-ended item asking for respondents to describe contributing circumstances if they had indicated any moral distress. To understand factors associated with heightened moral distress, we analyzed quantitative data using bivariate and multivariable regression analyses and qualitative data using a hybrid deductive/inductive thematic approach. Mixed methods analysis followed, whereby we compared the quantitative and qualitative datasets and integrated findings at the analytic level. Out of 3,396 eligible VA clinicians, 323 responded to the survey (9.5% adjusted response rate). Most respondents (81%) reported at least some moral distress during peak-Covid. In a multivariable logistic regression, female gender (OR 3.35; 95% CI 1.53–7.37) was associated with greater odds of moral distress, and practicing in geriatrics/palliative care (OR 0.40; 95% CI 0.18–0.87) and internal medicine/family medicine/primary care (OR 0.46; 95% CI 0.22–0.98) were associated with reduced odds of moral distress compared to medical subspecialties. From the 191 respondents who completed the open-ended item, five qualitative themes emerged as moral distress contributors: 1) patient visitation restrictions, 2) anticipatory actions, 3) clinical uncertainty related to Covid, 4) resource shortages, and 5) personal risk of contracting Covid. Mixed methods analysis found that quantitative results were consistent with these last two qualitative themes. In sum, clinician moral distress was prevalent early in the pandemic. This moral distress was associated with individual-, system-, and situation-level contributors. These identified contributors represent leverage points for future intervention to mitigate clinician moral distress and its negative outcomes during future healthcare crises and even during everyday clinical care.
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.
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.
We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of ...fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 mum are performed.
Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult ...zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model.
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