Measurement of third order coherence by in situ autocorrelation for determining three-photon cross-sections.

We show theoretically that the third order coherence at zero delay can be obtained by measuring the second and third order autocorrelation traces of a pulsed laser. Our theory enables the measurement of a fluorophore's three-photon cross-section without prior knowledge of the temporal profile of the excitation pulse by using the same fluorescent medium for both the measurement of the third order coherence at zero delay as well as the cross-section. Such an in situ measurement needs no assumptions about the pulse shape nor group delay dispersion of the optical system. To verify the theory experimentally, we measure the three-photon action cross-section of Alexa Fluor 350 and show that the measured value of the three-photon cross-section remains approximately constant despite varied amounts of chirp on the excitation pulses.

Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions.

In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.

Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo.

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 µm into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 µm of the murine spinal cord, in vivo.

A Large Field-of-view, Single-cell-resolution Two- and Three-Photon Microscope for Deep Imaging.

In vivo imaging of large-scale neuron activity plays a pivotal role in unraveling the function of the brain's network. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to overcome constraints on the field of view in three-photon microscopy and to perform deep imaging that is inaccessible to two-photon microscopy. These innovations enable us to image neuronal activities in a ~3.5-mm diameter field-of-view at 4 Hz with single-cell resolution and in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into any multiphoton microscope for large-field-of-view imaging for system-level neural circuit research.

Measurement of three-photon excitation cross-sections of fluorescein from 1154†nm to 1500†nm.

Measurements of three-photon action cross-sections for fluorescein (dissolved in water, pH ∼11.5) are presented in the excitation wavelength range from 1154 to 1500 nm in ∼50 nm steps. The excitation source is a femtosecond wavelength tunable non-collinear optical parametric amplifier, which has been spectrally filtered with 50 nm full width at half maximum band pass filters. Cube-law power dependance is confirmed at the measurement wavelengths. The three-photon excitation spectrum is found to differ from both the one- and two-photon excitation spectra. The three-photon action cross-section at 1154 nm is more than an order of magnitude larger than those at 1450 and 1500 nm (approximately three times the wavelength of the one-photon excitation peak), which possibly indicates the presence of resonance enhancement.

Addressing clinician moral distress: Implications from a mixed methods evaluation during Covid-19.

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.

Simultaneous multimodal three-photon and optical coherence microscopy of the mouse brain in the 1700 nm optical window in vivo.

Multimodal microscopy combining various imaging approaches can provide complementary information about tissue in a single imaging session. Here, we demonstrate a multimodal approach combining three-photon microscopy (3PM) and spectral-domain optical coherence microscopy (SD-OCM). We show that an optical parametric chirped-pulse amplification (OPCPA) laser source, which is the standard source for three-photon fluorescence excitation and third harmonic generation (THG), can be used for simultaneous OCM, 3-photon (3P) fluorescence and THG imaging. We validated the system performance in deep mouse brains in vivo with an OPCPA source operating at 1620 nm center wavelength. We visualized small structures such as myelinated axons, neurons, and large fiber tracts in white matter with high spatial resolution non-invasively using linear and nonlinear contrast at >1 mm depth in intact adult mouse brain. Our results showed that simultaneous OCM and 3PM at the long wavelength window can be conveniently combined for deep tissue imaging in vivo.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

Impact of the COVID-19 Pandemic on Providing Recommendations During Goals-of-Care Conversations: A Multisite Survey.

Background: Goals-of-care conversations (GoCCs) are essential for individualized end-of-life care. Shared decision-making (SDM) that elicits patients' goals and values to collaboratively make life sustaining treatment (LST) decisions is best practice. However, it is unknown how the COVID-19 pandemic onset and associated changes to care delivery, stress on providers, and clinical uncertainty affected SDM and recommendation-making during GoCCs. Aim: To assess providers' attitudes and behaviors related to GoCCs during the COVID-19 pandemic and identify factors associated with provision of LST recommendations. Design: Survey of United States Veterans Health Administration (VA) health care providers. Setting/Participants: Health care providers from 20 VA facilities with high COVID-19 caseloads early in the pandemic who had authority to place LST orders and practiced in select specialties (n = 3398). Results: We had 323 respondents (9.5% adjusted response rate). Most were age ≥50 years (51%), female (63%), non-Hispanic white (64%), and had ≥1 GoCC per week during peak-COVID-19 (78%). Compared with pre-COVID-19, providers believed it was less appropriate and felt less comfortable giving an LST recommendation during peak-COVID-19 (p < 0.001). One-third (32%) reported either "never" or "rarely" giving an LST recommendation during GoCCs at peak-COVID-19. In adjusted regression models, being a physician and discussing patients' goals and values were positively associated with giving an LST recommendation (B = 0.380, p = 0.031 and B = 0.400, p < 0.001, respectively) at peak-COVID-19. Conclusion: Providers who discuss patients' preferences and values are more likely to report giving a recommendation; both behaviors are markers of SDM during GoCCs. Our findings suggest potential areas for training in conducting patient-centered GoCCs.

Whole-brain optical access in a small adult vertebrate with two- and three-photon microscopy.

Although optical microscopy has allowed scientists to study the entire brain in early developmental stages, access to the brains of live, adult vertebrates has been limited. Danionella, a genus of miniature, transparent fish closely related to zebrafish has been introduced as a neuroscience model to study the adult vertebrate brain. However, the extent of optically accessible depth in these animals has not been quantitatively characterized. Here, we show that both two- and three-photon microscopy can access the entire depth and rostral-caudal extent of the adult wildtype Danionella dracula brain without any modifications to the animal other than mechanical stabilization. Three-photon microscopy provides higher signal-to-background ratio and optical sectioning of fluorescently labeled vasculature through the deepest part of the brain, the hypothalamus. Hence, we use multiphoton microscopy to penetrate the entire adult brain within the geometry of this genus' head structures and without the need for pigment removal.

Optical clearing of the mouse skull.

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 Adaptive Optics for Mouse Brain Imaging.

Three-photon microscopy (3PM) was shown to allow deeper imaging than two-photon microscopy (2PM) in scattering biological tissues, such as the mouse brain, since the longer excitation wavelength reduces tissue scattering and the higher-order non-linear excitation suppresses out-of-focus background fluorescence. Imaging depth and resolution can further be improved by aberration correction using adaptive optics (AO) techniques where a spatial light modulator (SLM) is used to correct wavefront aberrations. Here, we present and analyze a 3PM AO system for in vivo mouse brain imaging. We use a femtosecond source at 1300 nm to generate three-photon (3P) fluorescence in yellow fluorescent protein (YFP) labeled mouse brain and a microelectromechanical (MEMS) SLM to apply different Zernike phase patterns. The 3P fluorescence signal is used as feedback to calculate the amount of phase correction without direct phase measurement. We show signal improvement in the cortex and the hippocampus at greater than 1 mm depth and demonstrate close to diffraction-limited imaging in the cortical layers of the brain, including imaging of dendritic spines. In addition, we characterize the effective volume for AO correction within brain tissues, and discuss the limitations of AO correction in 3PM of mouse brain.

Spatially resolved measurements of ballistic and total transmission in microscale tissue samples from 450†nm to 1624†nm.

We built a simple and versatile setup to measure tissue ballistic and total transmission with customizable wavelength range, spatial resolution, and sample sizes. We performed ballistic transmission and total transmission measurements of overlying structures from biological samples ex vivo. We obtained spatially resolved transmission maps to reveal transmission heterogeneity from five microscale tissue samples: Danionella skin, mouse skull bone, mosquito cuticle, wasp cuticle, and rat dura over a wide spectral range from 450 nm to 1624 nm at a spatial resolution of ∼25 µm for ballistic transmission measurements and ∼50 µm for total transmission measurements. We expect our method can be straightforwardly applied to measuring the transmission of other samples. The measurement results will be valuable for multiphoton microscopy. The total transmission of a sample is important for the collection of multiphoton excited fluorescence and the assessment of laser-induced sample heating. The ballistic transmission determines the excitation power at the focus and hence the fluorescence signal generation. Therefore, knowledge of ballistic transmission, total transmission, and transmission heterogeneity of overlying structures of animals and organs are essential to determine the optimal excitation wavelength and fluorophores for non-invasive multiphoton microscopy.

Imaging deeper than the transport mean free path with multiphoton microscopy.

Multiphoton fluorescence microscopy enables deep in vivo imaging by using long excitation wavelengths to increase the penetration depth of ballistic photons and nonlinear excitation to suppress the out-of-focus fluorescence. However, the imaging depth of multiphoton microscopy is limited by tissue scattering and absorption. This fundamental depth limit for two-photon microscopy has been studied theoretically and experimentally. Long wavelength three-photon fluorescence microscopy was developed to image beyond the depth limit of two-photon microscopy and has achieved unprecedented in vivo imaging depth. Here we extend the theoretical framework for characterizing the depth limit of two-photon microscopy to three-photon microscopy. We further verify the theoretical predictions with experimental results from tissue phantoms. We demonstrate experimentally that high spatial resolution diffraction-limited imaging at a depth of 10 scattering mean free paths, which is nearly twice the transport mean free path, is possible with multiphoton microscopy. Our results indicate that the depth limit of three-photon microscopy is significantly beyond what has been achieved in biological tissues so far, and further technological development is required to reach the full potential of three-photon microscopy.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Intravital Imaging of the Murine Subventricular Zone with Three Photon Microscopy.

The mouse subventricular zone (SVZ) produces neurons throughout life. It is useful for mechanism discovery and is relevant for regeneration. However, the SVZ is deep, significantly restricting live imaging since current methods do not extend beyond a few hundred microns. We developed and adapted three-photon microscopy (3PM) for non-invasive deep brain imaging in live mice, but its utility in imaging the SVZ niche was unknown. Here, with fluorescent dyes and genetic labeling, we show successful 3PM imaging in the whole SVZ, extending to a maximum depth of 1.5 mm ventral to the dura mater. 3PM imaging distinguished multiple SVZ cell types in postnatal and juvenile mice. We also detected fine processes on neural stem cells interacting with the vasculature. Previous live imaging removed overlying cortical tissue or lowered lenses into the brain, which could cause inflammation and alter neurogenesis. We found that neither astrocytes nor microglia become activated in the SVZ, suggesting 3PM does not induce major damage in the niche. Thus, we show for the first time 3PM imaging of the SVZ in live mice. This strategy could be useful for intravital visualization of cell dynamics, molecular, and pathological perturbation and regenerative events.

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle.

We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticle. Our measurements showed that the fly head cuticle has surprisingly high transmission at wavelengths >900nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, we performed multiphoton imaging of the fly brain through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body γ-lobes in behaving flies short term and long term. The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.

Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes.

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 µm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 µm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.

Shot noise limits on binary detection in multiphoton imaging.

Much of fluorescence-based microscopy involves detection of if an object is present or absent (i.e., binary detection). The imaging depth of three-dimensionally resolved imaging, such as multiphoton imaging, is fundamentally limited by out-of-focus background fluorescence, which when compared to the in-focus fluorescence makes detecting objects in the presence of noise difficult. Here, we use detection theory to present a statistical framework and metric to quantify the quality of an image when binary detection is of interest. Our treatment does not require acquired or reference images, and thus allows for a theoretical comparison of different imaging modalities and systems.