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Organic near-infrared room temperature phosphorescence materials have unparalleled advantages in bioimaging due to their excellent penetrability. However, limited by the energy gap law, the near-infrared phosphorescence materials (>650 nm) are very rare, moreover, the phosphorescence lifetimes of these materials are very short. In this work, we have obtained organic room temperature phosphorescence materials with long wavelengths (600/657–681/732 nm) and long lifetimes (102–324 ms) for the first time through the guest-host doped strategy. The guest molecule has sufficient conjugation to reduce the lowest triplet energy level and the host assists the guest in exciton transfer and inhibits the non-radiative transition of guest excitons. These materials exhibit good tissue penetration in bioimaging. Thanks to the characteristic of long lifetime and long wavelength emissive phosphorescence materials, the tumor imaging in living mice with a signal to background ratio value as high as 43 is successfully realized. This work provides a practical solution for the construction of organic phosphorescence materials with both long wavelengths and long lifetimes. Though room-temperature phosphorescence (RTP) in organics is advantageous for bioimaging, designing materials that meet lifetime and wavelength emission requirements is challenging. Here, the authors us a guest-host doped strategy to construct RTP materials with ultralong-lifetime, NIR emission.

A simple and general chemical structure change to a panel of cell-permeable small-molecule fluorophores increases their brightness and photostability, which will enable improved single-molecule studies and super-resolution imaging. Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N , N -dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aqueous stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in organic phase. The CdS shell plays a critical role of protecting the lead sulfide (PbS) core from oxidation and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aqueous stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 hours and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per second (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke’s shift. The aqueous-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.

Stimulated Raman scattering under electronic pre-resonance conditions, combined with a new palette of probes, enables super-multiplex imaging of molecular targets in living cells with very high vibrational selectivity and sensitivity. Dyeing to look inside cells The direct visualization of a large number of different molecules inside cells could improve our understanding of many important processes, but remains challenging with existing methods. Lu Wei et al . show that stimulated Raman scattering measurements under electronic pre-resonance conditions makes it possible to image many different dye-labelled target molecules with very high selectivity and sensitivity in living cells. The team created a palette of new dyes that emit signals in the cell-silent Raman spectral window, making them readily observable, and report imaging of 16 different dye-labelled targets within live cells. Proof-of-principle experiments on neuronal co-cultures and brain tissues further underscore the potential of this super-multiplex optical imaging approach for untangling intricate interactions in complex biological systems. The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure–function relationships in nervous systems, to profile RNA in situ , to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly 1 , 2 , 3 , 4 , it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra 5 that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis) 6 , 7 , 8 . Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells 9 . Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Fluorescence imaging multiplicity of biological systems is an area of intense focus, currently limited to fluorescence channels in the visible and first near-infrared (NIR-I; ∼700–900 nm) spectral regions. The development of conjugatable fluorophores with longer wavelength emission is highly desired to afford more targeting channels, reduce background autofluorescence, and achieve deeper tissue imaging depths. We have developed NIR-II (1,000–1,700 nm) molecular imaging agents with a bright NIR-II fluorophore through high-efficiency click chemistry to specific molecular antibodies. Relying on buoyant density differences during density gradient ultracentrifugation separations, highly pure NIR-II fluorophore-antibody conjugates emitting ∼1,100 nm were obtained for use as molecular-specific NIR-II probes. This facilitated 3D staining of ∼170-μm histological brain tissues sections on a home-built confocal microscope, demonstrating multicolor molecular imaging across both the NIR-I and NIR-II windows (800–1,700 nm).

This technical report describes a method to clear fixed brain tissues while allowing for fluorescent dye tracing and retaining cellular morphology. The authors demonstrate the utility of the technique by obtaining a wiring diagram for sister mitral cells. We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

Editorial Summary A reactivity-based probe containing an iron-sensitive 1,2,4-trixolane ring conjugated to a small molecule payload combined with a high-throughput immunofluorescence assay enables the selective detection of labile intracellular ferrous iron. Improved methods for studying intracellular reactive Fe( II ) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe( II ) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe( II ) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe( II ) pools are larger in cancer cells than in nontumorigenic cells.

Super-resolution imaging of the dynamics of organelle structures in live cells is facilitated by blinking, far-red dyes. Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

Anatomic tracing is recognized as a critical source of knowledge on brain circuitry that can be used to assess the accuracy of diffusion MRI (dMRI) tractography. However, most prior studies that have performed such assessments have used dMRI and tracer data from different brains and/or have been limited in the scope of dMRI analysis methods allowed by the data. In this work, we perform a quantitative, voxel-wise comparison of dMRI tractography and anatomic tracing data in the same macaque brain. An ex vivo dMRI acquisition with high angular resolution and high maximum b-value allows us to compare a range of q-space sampling, orientation reconstruction, and tractography strategies. The availability of tracing in the same brain allows us to localize the sources of tractography errors and to identify axonal configurations that lead to such errors consistently, across dMRI acquisition and analysis strategies. We find that these common failure modes involve geometries such as branching or turning, which cannot be modeled well by crossing fibers. We also find that the default thresholds that are commonly used in tractography correspond to rather conservative, low-sensitivity operating points. While deterministic tractography tends to have higher sensitivity than probabilistic tractography in that very conservative threshold regime, the latter outperforms the former as the threshold is relaxed to avoid missing true anatomical connections. On the other hand, the q-space sampling scheme and maximum b-value have less of an impact on accuracy. Finally, using scans from a set of additional macaque brains, we show that there is enough inter-individual variability to warrant caution when dMRI and tracer data come from different animals, as is often the case in the tractography validation literature. Taken together, our results provide insights on the limitations of current tractography methods and on the critical role that anatomic tracing can play in identifying potential avenues for improvement.