Local connectivity and synaptic dynamics in mouse and human neocortex.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Human neocortical expansion involves glutamatergic neuron diversification.

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.

Characterization of a fiber-coupled EvenField illumination system for fluorescence microscopy.

Fluorescence microscopy benefits from spatially and temporally homogeneous illumination with the illumination area matched to the shape and size of the camera sensor. Fiber-coupled illumination schemes have the added benefit of straightforward and robust alignment and ease of installation compared to free-space coupled illumination. Commercial and open-source fiber-coupled, homogenized illumination schemes have recently become available to the public; however, there have been no published comparisons of speckle reduction schemes to date. We characterize three different multimode fibers in combination with two laser speckle reduction devices and compare spatial and temporal profiles to a commercial unit. This work yields a new design, the EvenField Illuminator, which is freely available for researchers to integrate into their own imaging systems.

Integrated Morphoelectric and Transcriptomic Classification of Cortical GABAergic Cells.

Neurons are frequently classified into distinct types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 4,200 mouse visual cortical GABAergic interneurons and reconstructed the local morphologies of 517 of those neurons. We find that most transcriptomic types (t-types) occupy specific laminar positions within visual cortex, and, for most types, the cells mapping to a t-type exhibit consistent electrophysiological and morphological properties. These properties display both discrete and continuous variation among t-types. Through multimodal integrated analysis, we define 28 met-types that have congruent morphological, electrophysiological, and transcriptomic properties and robust mutual predictability. We identify layer-specific axon innervation pattern as a defining feature distinguishing different met-types. These met-types represent a unified definition of cortical GABAergic interneuron types, providing a systematic framework to capture existing knowledge and bridge future analyses across different modalities.

In Vitro Quantification of Single Red Blood Cell Oxygen Saturation by Femtosecond Transient Absorption Microscopy.

Hemoglobin, the oxygen carrying protein, ferries nearly all bodily oxygen from the lungs to cells and tissues in need. Blood oxygen saturation (sO2) thus plays an important role in maintaining energy homeostasis throughout the body. Clinical and research tools have been developed to monitor sO2 at a wide range of temporal and spatial scales. However, real-time quantification of sO2 at single red blood cell (RBC) resolution remains challenging. Such capability is critically important to study energy metabolism in heterogeneous tissues including brain and tumor tissue. In this work, we develop a ratiometric transient absorption microscopy technique to image hemoglobin sO2. By exploiting differences in transient lifetime kinetics between  oxyhemoglobin and deoxyhemoglobin, we directly quantified the sO2 of single RBCs in real-time without the need for injection of exogenous agents. This simple and high-speed nonlinear optical imaging technique is well suited for in vitro and in vivo quantification of sO2.

Label-free pathology by spectrally sliced femtosecond stimulated Raman scattering (SRS) microscopy.

Optical "virtual biopsy" is an attractive way to improve disease diagnosis and surgical guidance. Many optical microscopy techniques have been developed to provide diagnostic information without the need for tissue sectioning or staining. Among these techniques, label-free chemical imaging is the most desirable. Recently, it has been shown that narrowband, picosecond stimulated Raman scattering (SRS) can achieve comparable morphological contrast to hematoxylin and eosin staining (H&E staining), the 'gold standard' of pathology. However, to translate the technique from the bench to the bedside, optimal laser sources and parameters have yet to be identified. Here we describe an improvement to the narrowband SRS microscopy techniques for label-free tissue imaging. Through spectral slicing of broadband, femtosecond pulses, we are able to maintain the same protein/lipid contrast as narrowband SRS while achieving a higher signal-to-noise ratio (SNR). Our method draws upon the benefits of femtosecond pulses (e.g. higher peak power) while preserving those of picosecond pulses (e.g. adequate spectral resolution). We demonstrate this achievement through protein/lipid signal and contrast quantification of mouse brain tissue as a function of bandwidth, and comparison with numerical simulations. Further method validation is provided through imaging of additional mouse tissues: liver, kidney, and skin.

Label-Free Chemical Imaging of Latent Fingerprints with Stimulated Raman Scattering Microscopy.

Fingerprints have long been the gold standard for personal identification in forensic investigations. Methods for cultivating and enhancing the visualization of latent fingerprints (LFPs) are continuously evolving. One important challenge is to identify suspicious chemicals present in fingerprint residues, which requires chemical imaging capability. Recently, vibrational spectroscopy has shown that LFP analysis through tape-lift, Raman mapping, and multivariate data analysis presents a useful tool for forensic investigation. However, there are still major difficulties in terms of acquisition speed, poor spatial resolution, and lack of sensitivity. This paper demonstrates the feasibility of stimulated Raman scattering microscopy to quickly and easily extract LFP patterns from different substrates. Contrary to what has been reported, no obvious fingerprint degradation or lipid diffusion is observed with either glass or stainless steel substrate. Importantly, we demonstrate that trace exogenous chemicals can be detected in fingerprints. We further demonstrate an improvement in directly acquiring a LFP pattern lifted from tape by spectrally removing signals from tape.