Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

Two-photon microscopy has had an enormous influence on biological studies providing a tool for high-resolution imaging hundreds of microns within live tissues. Yet, penetrating deeper into tissue remains a challenge due to scattering and absorption of light. Here we review the results of our recent work and previous findings on the advantages of non-degenerate two-photon microscopy (ND-TPM) for deep-tissue imaging. In ND-TPM the required energy for transition from ground state to excited state is delivered via absorption of two photons of different energies from two pulsed lasers. This is in contrast with degenerate two-photon microscopy (D-TPM), commonly referred to as two-photon microscopy (TPM), in which both absorbed photons have identical energies. The obtained freedom in wavelength selection has several important advantages including: fluorophores have larger absorption cross section and are brighter under non-degenerate two-photon excitation, and out-of-focus excitation can be significantly decreased by selecting the wavelengths outside the fluorophores excitation range and spatially displacing the beams. Lower background and higher signal leads to higher penetration depth for ND-TPM compared to conventional TPM.

In non-degenerate two-photon microscopy (ND-TPM), the required energy for fluorescence excitation occurs via absorption of two photons of different energies derived from two synchronized pulsed laser beams. ND-TPM is a promising imaging technology offering flexibility in the choice of the photon energy for each beam. However, a formalism to quantify the efficiency of two-photon absorption (TPA) under non-degenerate excitation, relative to the resonant degenerate excitation, is missing. Here, we derive this formalism and experimentally validate our prediction for a common fluorophore, fluorescein. An accurate quantification of non-degenerate TPA is important to optimize the choice of photon energies for each fluorophore.

BACKGROUND: Functional magnetic resonance imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available owing to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, though microscopic (e.g., two-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a challenge. Owing to variability in behavior among animals, performing all types of measurements within the same subject is highly desirable and can lead to higher scientific rigor. METHODS: We demonstrated blood oxygenation level-dependent fMRI in awake mice implanted with long-term cranial windows that allowed optical access for microscopic imaging modalities and optogenetic stimulation. We started with two-photon imaging of single-vessel diameter changes (n = 1). Next, we implemented intrinsic optical imaging of blood oxygenation and flow combined with laser speckle imaging of blood flow obtaining a mesoscopic picture of the hemodynamic response (n = 16). Then we obtained corresponding blood oxygenation level-dependent fMRI data (n = 5). All measurements could be performed in the same mice in response to identical sensory and optogenetic stimuli. RESULTS: The cranial window did not deteriorate the quality of fMRI and allowed alternation between imaging modalities in each subject. CONCLUSIONS: This report provides a proof of feasibility for multiscale imaging approaches in awake mice. In the future, this protocol could be extended to include complex cognitive behaviors translatable to humans, such as sensory discrimination or attention.

In non-degenerate two-photon excitation (ND-TPE), electronic transition of fluorophores happens via absorption of two photons with different energies. This contrasts with conventional - or degenerate - two-photon excitation (D-TPE), where two photons with identical energies are absorbed. ND-TPE can improve performance of two-photon microscopy by extending the excitation wavelength range, reducing out-of-focus excitation, and increasing resolution and penetration depth. However, a systematic study of fluorophore performance under ND-TPE is missing, which is critical for the selection of optimal excitation wavelength combinations. It is a well-known fact that degenerate two-photon absorption spectra often deviate from theoretical predictions based on one-photon absorption spectra. Therefore, it is not clear whether non-degenerate two-photon absorption spectra are predictable from the corresponding degenerate spectra. Using our sensitive fluorescence-based spectroscopy technique, we measured non-degenerate two-photon absorption cross-sections (ND-TPACS) of several commonly used fluorophores and generated 2-dimensional ND-TPACS maps. We observed that the shape of the measured ND-TPACS spectra follows the spectra of the degenerate two-photon absorption cross-sections (D-TPACS). However, ND-TPACS are higher in magnitude, which is predicted by the “resonant enhancement” phenomenon. Therefore, we show that ND-TPACS spectra are predictable from the corresponding degenerate D-TPACS spectra under consideration of resonant enhancement. Predictability of ND-TPACS spectra is an important finding that helps choosing the optimal combination of wavelengths for ND-TPE of a given fluorophore without prior experimental measurement of ND-TPACS.

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.

We demonstrate that non-degenerate 2-photon excitation offers a ~3-fold increase in excitation efficiency compared to degenerate 2-photon excitation and realize this advantage for in vivo brain imaging, trading the excitation efficiency for laser power.

In non-degenerate 2-photon excitation (ND-2PE) microscopy, a fluorophore simultaneously absorbs two photons of different energies. We performed a ND-2PE study of fluorescent proteins and synthetic dyes (eg.eGFP, FITC, and etc.) continuously varying energies and numbers of both photons to create two-dimensional map of fluorescence landscapes. By using the best photon energy combination from our two-dimensional map, we found an increase in detected fluorescent image brightness with ND-2PE as we imaged cortical neurons labeled with enhanced green fluorescent protein (eGFP). It should be noted that the photons corresponding to longer wavelength will penetrate deeper into the tissue at reduced scattering. Additionally, using non-overlapping spatial modes carrying the photons at different energies will significantly reduce out of focus fluorescence from the large number of low energy photons, and by a proper choice of the number of high energy photons the ND-2PE fluorescence can be obtained from deep tissue. Experimentally, we strategically displaced two laser beams until they reached the sample plane such that the unwanted background in the excitation beam path was suppressed. In contrast, these two pump beams were well overlapped at focus which still produced sufficient number fluorescence photons for detection. In our experiment, the temporal alignment was achieved with optical delay line in the optical path of IR beam. With this technique we demonstrated experimentally that ND-2PE with side-by-side beams provided a better signal to background ratio in the scattering phantom as compared with D-2PE. The excitation volume of ND-2PE with side-by-side beam was also investigated and determined to be comparable in size with that of the D-2PE.

Michèle Desjardins, Kıvılcım Kılıç, Martin Thunemann, Celine Mateo, Dominic Holland, Christopher G.L. Ferri, Jonathan A. Cremonesi, Baoqiang Li, Qun Cheng, Kimberly L. Weldy, Payam A. Saisan, David Kleinfeld, Takaki Komiyama, Thomas T. Liu, Robert Bussell, Eric C. Wong, Miriam Scadeng, Andrew K. Dunn, David A. Boas, Sava Sakadžić, Joseph B. Mandeville, Richard B. Buxton, Anders M. Dale, Anna Devor

Simulation studies of light propagation in cerebral cortex often assume homogeneous and direction-independent scattering; however, direct measurements revealed significant directional dependence with implications for optogenetics and diffuse optical tomography.

We investigate the utility of non-degenerate 2-photon excitation (ND-2PE) as a strategy for extending the 2-photon imaging depth. For the ND-2PE scheme, two pulsed, synchronized laser sources of different wavelength each provide a photon for the 2-photon absorption process. By independently tuning their wavelengths, we are able to tune the excitation to tissue transparency windows while maintaining resonant fluorescence excitation. These transparency windows reduce excitation power loss resulting from scattering. In addition, by having two sources we are able to displace the beams in space except at their common focus; thus, reducing background fluorescence excitation. Finally, we show that ND-2PE inherently results in increased 2-photon absorption cross sections, resulting in increased fluorescence intensity. By combining beam displacement, tissue transparency and increased absorption cross sections, we achieve increased imaging depths as compared to degenerate 2-photon excitation with commonly used fluorophores.

Non-degenerate 2-photon microscopy provides deep penetration, similar to 3-photon microscopy, by offering independent tuning of the wavelengths and minimizing the background excitation, while circumventing low 3-photon excitation cross-section.

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.

Non-degenerate 2-photon excitation of a fluorophore with two laser beams of different photon energies may offer independent degree of freedom in tuning of the photon flux (i.e., the power) for each beam. Wereport a practical demonstration that the emission intensity of a fluorophore excited in the non-degenerate regime in scattering medium is more efficient than the commonly used degenerate 2-photon excitation. In our experiments we use spatially and temporally aligned Ti:Sapphiremode-locked laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, respectively. The non-degenerate 2-photon excitation mechanism takes advantage of the infrared wavelengths used in 3-photon microscopy to achieve increased penetration depth, while preserving relatively high 2-photon excitation cross section, exceeding that achievable with the 3-photon excitation. Importantly, independent control of power for each beam implies that the flux requirement for the higher photon energy NIR beam, which experiences higher scattering in biological tissue, can be relaxed at the expense of increasing the flux of the lower photon energy SWIR beam which experiences lower scattering, thus promising deeper penetration with higher efficiency of excitation.Applications for in vivo brain imaging will be also discussed.

The geometrically frustrated triangular antiferromagnet Gadolinium Gallium Garnet (Gd3Ga5O12 or GGG) exhibits a rich mix of short-range order and isolated quantum states. We investigate the effects of up to 1% neodymium substitution for gallium on the ac magnetic response at temperatures below 1 K in both the linear and nonlinear regimes. Substitutional disorder actually drives the system toward a more perfectly frustrated state, apparently compensating for the effects of imperfect gadolinium/gallium stoichiometry, while at the same time more closely demarcating the boundaries of isolated, coherent clusters composed of hundreds of spins. Optical measurements of the local Nd environment substantiate the picture of an increased frustration index with doping.

The design and development of multifunctional composite materials from artificial nano-constituents is one of the most compelling current research areas. This drive to improve over nature and produce 'meta-materials' has met with some success, but results have proven limited with regards to both the demonstration of synergistic functionalities and in the ability to manipulate the material properties post-fabrication and in situ. Here, magnetic nanoparticles (MNPs) and semiconducting quantum dots (QDs) are co-assembled in a nematic liquid crystalline (LC) matrix, forming composite structures in which the emission intensity of the quantum dots is systematically and reversibly controlled with a small applied magnetic field (<100 mT). This magnetic field-driven brightening, ranging between a two- to three-fold peak intensity increase, is a truly cooperative effect: the LC phase transition creates the co-assemblies, the clustering of the MNPs produces LC re-orientation at atypical low external field, and this re-arrangement produces compaction of the clusters, resulting in the detection of increased QD emission. These results demonstrate a synergistic, reversible, and an all-optical process to detect magnetic fields and additionally, as the clusters are self-assembled in a fluid medium, they offer the possibility for these sensors to be used in broad ranging fluid-based applications.