Excitation Window Research Articles

Intravital observation of high-scattering and dense-labeling hepatic tissues using multi-photon fluorescence microscopy.

Achieving high-resolution and large-depth microscopic imaging in vivo under conditions characterized by high-scattering and dense-labeling, as commonly encountered in the liver, poses a formidable challenge. Here, through the optimization of multi-photon fluorescence excitation window, tailored to the unique optical properties of the liver, intravital microscopic imaging of hepatocytes and hepatic blood vessels with high spatial resolution was attained. It's worth noting that resolution degradation caused by tissue scattering of excitation light was mitigated by accounting for moderate tissue self-absorption. Leveraging high-quality multi-photon fluorescence microscopy, we discerned structural and functional alterations in hepatocytes during drug-induced acute liver failure. Furthermore, a reduction in indocyanine green metabolism rates associated with acute liver failure was observed using NIR-II fluorescence macroscopic imaging.

SAM-SFM: High-Efficiency and High-Resolution Tandem Mass Spectrometry Enabled by Sinusoidal Amplitude Modulation of Multiple Sinusoidal Frequency-Modulated Waveforms.

In miniature ion trap mass spectrometry, achieving a balance between isolation resolution and efficiency is a formidable challenge. The presence of absorption curves causes target ions to inadvertently absorb energy from AC signal components near their resonant frequencies. To mitigate this issue, SAM-SFM waveforms introduce a parameter known as the decreasing factor. Unlike SWIFT waveforms, SAM-SFM's spectral profile intentionally departs from a rectangular window, adopting an arch-shaped excitation window to minimize the impact on target ions and improve ion isolation efficiency. SAM-SFM waveforms have the advantage of low computational complexity, enabling real-time computation using an embedded FPGA technology. Regardless of any parameter changes, the FPGA can consistently guarantee waveform output within 1 μs. This not only enhances throughput but also eliminates the need for a PC in miniature mass spectrometry devices. The performance of SAM-SFM has been validated on an improved "Brick" miniature ion trap mass spectrometer. Comparative experiments with SWIFT waveforms confirm the lossless unit-mass isolation capabilities of SAM-SFM. This waveform has the capability to simultaneously isolate multiple target ions, even allowing for the lossless isolation of ions with lower abundance within isotopic clusters, albeit at the cost of requiring extended isolation durations.

A general design of pyridinium-based fluorescent probes for enhancing two-photon microscopy

Two-photon absorbing fluorescent probes have emerged as powerful imaging tools for subcellular-level monitoring of biological substances and processes, offering advantages such as deep light penetration, minimal photodamage, low autofluorescence, and high spatial resolution. However, existing two-photon absorbing probes still face several limitations, such as small two-photon absorption cross-section, poor water solubility, low membrane permeability, and potentially high toxicity. Herein, we report three small-molecule probes, namely MSP-1arm, Lyso-2arm, and Mito-3arm, composed of a pyridinium center (electron-acceptor) and various methoxystyrene “arms” (electron-donor). These probes exhibit excellent fluorescence quantum yield and decent aqueous solubility. Leveraging the inherent intramolecular charge transfer and excitonic coupling effect, these complexes demonstrate excellent two-photon absorption in the near-infrared region. Notably, Lyso-2arm and Mito-3arm exhibit distinct targeting abilities for lysosomes and mitochondria, respectively. In two-photon microscopy experiments, Mito-3arm outperforms a commercial two-photon absorbing dye in 2D monolayer HeLa cells, delivering enhanced resolution, broader NIR light excitation window, and higher signal-to-noise ratio. Moreover, the two-photon bioimaging of 3D human forebrain organoids confirms the successful deep tissue imaging capabilities of both Lyso-2arm and Mito-3arm. Overall, this work presents a rational design strategy in developing competent two-photon-absorbing probes by varying the number of conjugated “arms” for bioimaging applications.

Molecular Engineering of NIR‐II AIE Luminogen Excited at 1700 nm for Ultradeep Intravital Brain Two‐Photon Fluorescence Imaging

AbstractThe limited tissue penetration depth and spatial resolution are the major bottlenecks for deep‐brain imaging. In this study, molecular engineering by tailoring electron donors is conducted to develop for the first time an NIR‐II (second near‐infrared) emissive fluorescence probe, namely DCTBT, for effective deep‐brain two‐photon fluorescence imaging. Benefiting from its good biocompatibility, high photostability, bright NIR‐II emission as aggregates and large two‐photon fluorescence action cross section at the 1700 nm excitation window, DCTBT offers the imaging depths of 2180 and 1135 µm in mouse brain with removed and intact skull, respectively. These results are the record depths for brain imaging, compared to all kinds of fluorescent probes and all modalities of multiphoton microscopy at all demonstrated excitation wavelengths. Moreover, with DCTBT labeling, hemodynamic imaging of blood flow in mouse brain vessels down to a depth of 714 µm with the intact skull is achieved. Multiphoton fluorescence imaging with the NIR‐II probe DCTBT excited at the 1700 nm window may readily provide methodology for deep‐brain structural and hemodynamic research.

De Novo Design of Aggregation‐Induced Emission Luminogen for Three‐Photon Fluorescence Imaging of Subcortical Structures Excited at Both NIR‐III and NIR‐IV Windows

AbstractThree‐photon fluorescence (3PF) imaging excited at 1700 nm window is an enabling technology for visualizing deep brain structures and dynamics. Recently, the 2200 nm window has emerged as the longest excitation window suitable for deep‐brain 3PF imaging. Bright fluorescent probes lay the material basis for deep‐brain 3PF imaging. Among various fluorescent probes, aggregation‐induced emission luminogens (AIEgens) have great potential in 3PF imaging excited at the 1700 nm window in vivo. However, to the best of knowledge, there is no AIEgens applicable to 3PF imaging excited at both the 1700 and 2200 nm windows. To readily fill this gap, here this study designs and synthesizes a novel AIEgen, namely TPE‐DPTT‐ICP, which generates bright 3PF signals excited at both 1700 and 2200 nm. The accordingly fabricated TPE‐DPTT‐ICP nanoparticles (NPs) possess excellent water dispersibility, colloidal stability, biocompatibility, photostability and large 3P action cross section, key to in vivo imaging. In mouse brain in vivo, TPE‐DPTT‐ICP NPs enable deep‐brain 3PF imaging of subcortical structures excited at both the two windows, reaching depths of 1640 and 880 µm below the brain surface, respectively. TPE‐DPTT‐ICP NPs are thus a versatile material simultaneously catering to the need at two infrared optical windows with deep tissue penetration.

A small excitation window allows long-duration single-molecule imaging, with reduced background autofluorescence, in C. elegans neurons

Single-particle imaging using laser-illuminated widefield epi-fluorescence microscopy is a powerful tool to investigate molecular processes in vivo. Performing high-quality single-molecule imaging in such biological systems, however, remains a challenge due to difficulties in controlling the number of fluorescing molecules, photobleaching, and the autofluorescence background. Here, we show that by exciting only a small, 5-15 μm wide region in chemosensory neurons in live C. elegans, we can significantly improve the duration and quality of single-molecule imaging. Small-window illumination microscopy (SWIM) allows long-duration single-particle imaging since fluorescently labelled proteins are only excited upon entering the small excited area, limiting their photobleaching. Remarkably, we also find that using a small excitation window significantly improves the signal-to-background ratio of individual particles. With the help of theoretical calculations, we explain that the improved signal-to-background ratio is due to reduced background, mostly caused by out-of-focus autofluorescence. We demonstrate the potential of this approach by studying the dendritic transport of a ciliary calcium channel protein, OCR-2, in the chemosensory neurons of C. elegans. We reveal that OCR-2-associated vesicles are continuously transported back and forth along the length of the dendrite and can switch between directed and diffusive states. Furthermore, we perform single-particle tracking of OCR-2-associated vesicles to quantitatively characterize the transport dynamics. SWIM can be readily applied to other in vivo systems where intracellular transport or cytoskeletal dynamics occur in elongated protrusions, such as axons, dendrites, cilia, microvilli and extensions of fibroblasts.

In Vivo Deep-Brain 3- and 4-Photon Fluorescence Imaging of Subcortical Structures Labeled by Quantum Dots Excited at the 2200 nm Window.

Multiphoton microscopy (MPM) is an enabling technology for visualizing deep-brain structures at high spatial resolution in vivo. Within the low tissue absorption window, shifting to longer excitation wavelengths reduces tissue scattering and boosts penetration depth. Recently, the 2200 nm excitation window has emerged as the last and longest window suitable for deep-brain MPM. However, multiphoton fluorescence imaging at this window has not been demonstrated, due to the lack of characterization of multiphoton properties of fluorescent labels. Here we demonstrate technologies for measuring both the multiphoton excitation and emission properties of fluorescent labels at the 2200 nm window, using (1) 3-photon (ησ3) and 4-photon action cross sections (ησ4) and (2) 3-photon and 4-photon emission spectra both ex vivo and in vivo of quantum dots. Our results show that quantum dots have exceptionally large ησ3 and ησ4 for efficient generation of multiphoton fluorescence. Besides, the 3-photon and 4-photon emission spectra of quantum dots are essentially identical to those of one-photon emission, which change negligibly subject to the local environment of circulating blood. Based on these characterization results, we further demonstrate deep-brain vasculature imaging in vivo. Due to the superb multiphoton properties of quantum dots, 3-photon and 4-photon fluorescence imaging reaches a maximum brain imaging depth of 1060 and 940 μm below the surface of a mouse brain, respectively, which enables the imaging of subcortical structures. We thus fill the last gap in multiphoton fluorescence imaging in terms of wavelength selection.

X-ray excited Mn2+-doped persistent luminescence materials with biological window emission for in vivo bioimaging

In recent years, persistent luminescence materials (PLMs) excited by X-rays and emitting in biological windows have received extensive attention in the field of high-sensitivity bioimaging. Transition metal Mn2+ is an ideal emission center, but few studies focus on Mn2+-doped PLMs with X-ray excitation and biological window emission. Here, we report a Mn2+-doped PLM, LiYGeO4:Mn2+ (LYGM), with excellent biological window persistent luminescence emission. After excitation by UV, LYGM produces a durable biological window of persistent luminescence emission at 660 nm for up to 20 h. More importantly, LYGM can be repeatedly excited by X-rays, resulting in long-term biological window persistent luminescence emission. In addition, we obtain LYGM around 200 nm in diameter by ball milling and centrifugation and improve its biocompatibility by surface modification to apply it to in vivo imaging in mice. After LYGM are injected into mice through the tail vein, in situ excitation of X-rays can be achieved. After the persistent luminescence decays, LYGM can be re-excited for repeated imaging. Therefore, LYGM shows potential prospects for in vivo deep tissue and long-term bioimaging.

Deep-skin third-harmonic generation (THG) imagingin vivoexcited at the 2200 nm window

The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy (MPM). Shifting to longer excitation wavelengths may help reduce skin scattering and aberration, potentially enabling larger imaging depths. However, previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700[Formula: see text]nm window, leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM. Here, in order to explore the longer-wavelength territory, first, we demonstrate characterization of the broadband transmittance of excised mouse skin, revealing a high transmittance window at 2200[Formula: see text]nm. Then, we demonstrate third-harmonic generation (THG) imaging in mouse skin in vivo excited at this window. With 9[Formula: see text]mW optical power on the skin surface operating at 1[Formula: see text]MHz repetition rate, we can get THG signals of 250[Formula: see text][Formula: see text]m below the skin surface. Comparative THG imaging excited at the 1700[Formula: see text]nm window shows that as imaging depth increases, THG signals decay even faster than those excited at 2200[Formula: see text]nm. Our results thus uncover the 2200[Formula: see text]nm window as a new, promising excitation window potential for deep-skin MPM.

An exciplex-type thermally activated delayed fluorescence material with wide up-conversion fluorescence excitation window

Exciplexes play an important role in the field of light-emitting materials due to the effective enhancement on fluorescence efficiency through thermally activated delayed fluorescence (TADF) mechanism. This mechanism is also expected to contribute to nonlinear optical applications. Here, we report an exciplex-type TADF material with significant two-photon excited fluorescence (TPEF) properties. It consists of Di-[4-(N,N-di-p-tolyl-amino)-phenyl]cyclohexane (TAPC) as the donor and 1,3,5-Tris(1-phenyl-1H-benzimidazol-2-yl)benzene (TPBi) as the acceptor. The TAPC:TPBi film emits blue fluorescence (peaked at ∼470 nm) under near-infrared excitation. A very wide long-wavelength excitation window of at least 200 nm (660 nm–860 nm) was observed. The reported exciplex material may have the potential to play an important role in the field of one- and two-photon excited fluorescence.

Stereocontrolled Self-Assembly of Ln(III)-Pt(II) Heterometallic Cages with Temperature-Dependent Luminescence.

Structurally well-defined discrete d/f heterometallic complexes show diverse application potential in electrooptic and magnetic materials. However, precise control of the component and topology of such heterometallic compounds with fine-tuned photophysical properties is still challenging. Herein, we report the stereocontrolled syntheses of a series of LnIII-PtII heterometallic cages through coordination-driven self-assembly of enantiopure alkynylplatinum-based metalloligands (L1R/S, L2R/S) with lanthanide ions (Ln = EuIII, YbIII, NdIII, LuIII). Taking advantage of the metal-to-ligand charge transfer (MLCT) excited state on the designed alkynylplatinum ligands, the excitation window for the sensitized near-infrared (NIR) luminescence on the YbIII- and NdIII-containing cages can be extended to the visible region (up to 500 nm). Linear temperature-dependent red and NIR emissions observed on the Ln4(L2R/S)6 (LnIII = EuIII and YbIII, respectively) complexes suggest their potential applications as luminescent temperature sensors, with sensitivities of -0.54% (LnIII = EuIII, 77-250 K) and -0.17% (LnIII = YbIII, 77-300 K) per K achieved. This work not only offers a good strategy to prepare new d/f heterometallic supramolecular cages but also paves the way for the design of stimuli-responsive luminescent materials.

N-doped carbon dots: triple mode excitation–emission features for dual channel sensing and logic gate application

Carbon dots (CDs) have congregated massive opportunities in basic and applied research streams owing to their versatile properties. Herein, a unique N-doped CD (NCD) was synthesized via the hydrothermal technique using 3,5-diaminobenzoic acid and hydrazine hydrate as precursors. The NCDs exhibit exceptional optical properties; they emit very different colors under different excitation windows. The fluorescence maxima were at 404 nm (blue), 506 nm (green), and 605 nm (red), respectively, when the excitation wavelengths fall within 300–340 nm, 380–460 nm, and 500–540 nm. The emission features were drastically different from more straightforward CDs (CDBlank) prepared using 3,5-diaminobenzoic acid only via the same hydrothermal route without hydrazine hydrate. We found that the 506 nm and 605 nm emissions originate from various surface states, while the 404 nm emission comes from the NCD core states. The triple emissive NCDs were implemented for sensing; Fe3+ effectively quenches the green and red emissions through turn-off quenching, while ascorbic acid (AA) successfully recovers the quenched emission in a turn-on manner. Both static and dynamic mechanisms were responsible for quenching green and red emissions and were accounted for by complexation-induced aggregation and electron transition from the electron-rich NCDs to the vacant d orbital of Fe3+, respectively. The detection limits for the green and red regions were 7.9 nM and 12.2 nM, respectively. The AA restores both the emission signals by reducing Fe3+ to Fe2+. The calculated detection limits for AA recognition were 0.55 μM and 1.05 μM, respectively, for the green and red emissions. For real-life practicability, some actual samples were examined under the excitation wavelengths (420 nm and 500 nm) with satisfactory outcomes. Furthermore, a multi-input logic gate was constructed to convert the molecular information as fluorescence signal outputs.

Table tennis bat cypress baseplate derived fluorescence carbon dots: In vivo bioimaging applications

Inspired by sports, this is the first study where waste table tennis bat cypress baseplates have been used as a carbon source in order to sustainably synthesize fluorescent carbon dots (FCDs). The prepared FCDs were suitable for bioimaging of the C2C12 muscle myoblasts, as well as in vivo bioimaging. The average particle size of the amorphous FCDs was ∼7.45 nm, whereas nitrogen-containing and oxygen-containing functional groups on the surface of the carbon structure were observed. The fabricated FCDs exhibit excellent water solubility, biocompatibility and photoluminescent (PL) properties, while the maximum PL intensity was found at 401 nm on the excitation wavelength at 334 nm with a fluorescence lifetime of 6.43 ns. On top of that, the FCDs displayed a wavelength-dependent emission over the 315–355 nm excitation window with an emission range of 385–405 nm, resulting in blue fluorescence emission. Furthermore, the synthesis mechanism of the FCDs was thoroughly explored. These attractive results indicate that sporting goods waste can also be an ideal alternative raw material for the development of fluorescence carbon dots. • Novel carbon dots were easily obtained from table tennis bat cypress baseplates. • The fundamental synthesis mechanism of FCDs was discussed. • FCDs display good fluorescence properties and biocompatibility. • They are suitable for bioimaging both in C2C12 muscle myoblasts and in vivo.

Interleaved fluid-attenuated inversion recovery (FLAIR) MRI and deuterium metabolic imaging (DMI) on human brain in vivo.

To integrate deuterium metabolic imaging (DMI) with clinical MRI through an interleaved MRI and DMI acquisition workflow. Interleaved MRI-DMI was enabled with hardware and pulse sequence modifications, and the performance was demonstrated using fluid-attenuated inversion recovery (FLAIR) MRI as an example. Interleaved FLAIR-DMI was developed by interleaving the 2 H excitation and acquisition time windows into the intrinsic delay periods presented in the FLAIR method. All 2 H MR signals were up-converted to the 1 H Larmor frequency using a custom-built hardware unit, which also achieved frequency and phase locking of the output signal in real-time. The interleaved measurements were compared with direct measurements both in phantom and in the human brain in vivo. The interleaved MRI-DMI acquisition strategy allowed simultaneous detection of FLAIR MRI and DMI in the same scan time as a FLAIR-only MRI acquisition. Both phantom and in vivo data showed that the MR image quality, DMI sensitivity as well as information content were preserved using interleaved MRI-DMI. The interleaved MRI-DMI technology can be used to extend clinical MRI protocols with DMI, thereby offering a metabolic component to the MR imaging contrasts without a penalty on patient comfort or scan time.

Stepwise Increase of NdIII -Based Phosphorescence by AIE-Active Sensitizer: Broadening the AIPE Family from Transition Metals to Discrete Near-Infrared Lanthanide Complexes*.

We designed two near-infrared (NIR) lanthanide complexes [(L)2 -Nd(NO3 )3 ] (L=TPE2 -BPY for 1, TPE-BPY for 2) by employing aggregation-induced emission (AIE)-active tetraphenylethylene (TPE) derivatives as sensitizers, which possessed matched energy to NdIII , prevented competitive deactivation under aggregation, even shifted the excitation window toward 600 nm by twisted intramolecular charge transfer. Furthermore, benefiting from the 4 f electron shielding effect and antenna effect, the enhanced excitation energies of the AIE-active sensitizers by structural rigidification transferred into the inert NdIII excited state through 3 LMCT, affording the first aggregation-induced phosphorescence enhancement (AIPE)-active discrete NIR-emitting lanthanide complexes. As 1 equipped with more AIE-active TPE than 2, L→Nd energy transfer efficiency in the former was higher than that in the latter under the same conditions. Consequently, the crystal of 1 exhibited one of the longest lifetimes (9.69 μs) among NdIII -based complexes containing C-H bonds.

A new auspicious scaffold for small dyes and fluorophores

An unprecedented chromophore, based on a nitrogen fused tricyclic heterocycle, is presented. This prototype molecule can be considered a “rigidified” monomethine cyanine dye and the central core is isoelectronic to pyridine-based BODIPY analogues, such as boron difluoride dipyridylmethene. The chromophore was synthesized and its photophysical properties examined. The new molecule can be considered the starting point to develop dyes, fluorophores or dual-emission fluorescent probes with excitation window in the green region of the spectrum.

Metasurface‐Enhanced Photon Upconversion upon 1550 nm Excitation

AbstractPhoton upconversion upon 1550 nm excitation is of high relevance for applications in the third biological excitation window, for photovoltaics beyond current limitations, and enables appealing options in the field of glass fiber telecommunications. Trivalent doped erbium ions (Er3+) are the material of choice for 1550 nm excited upconversion, however, they suffer from a low absorption cross‐section and a low brightness. Therefore, the ability of silicon metasurfaces to provide greatly enhanced electrical near‐fields is employed to enable efficient photon upconversion even at low external illumination conditions. Hexagonally shaped β‐NaYF4:Er3+ nanoparticles are placed on large‐area silicon metasurfaces designed to convert near‐infrared (1550 nm) to visible light. More than 2400‐fold enhanced photon upconversion luminescence is achieved by using this metasurface instead of a planar substrate. With the aid of optical simulations based on the finite‐element method, this result is attributed to the coupling of the excitation source with metasurface resonances at appropriate incident angles. Analysis of the excitation power density dependence of upconversion luminescence and red‐to‐green‐emission ratios enables the estimation of nanoscale near‐field enhancement on the metasurface. The findings permit the significant reduction of required external excitation intensities for photon upconversion of 1550 nm light, opening perspectives in biophotonics, telecommunication, and photovoltaics.

Wavelength‐Dependent Anisotropic Optical Properties in Layered GaTe for Polarization‐Sensitive Applications

Anisotropic layered GaTe has emerged as a potential material for polarization‐sensitive applications. The optical anisotropy in monoclinic GaTe is triggered by its in‐plane structural anisotropy, as each layer comprises periodic chain‐like structures along its b‐axis crystal direction. However, there is ambiguity in identifying the b‐axis in GaTe in existing reports. Proper identification is vital toward the fabrication of polarization‐sensitive photodetectors and light sources. Herein, this ambiguity is addressed and a systematic structural and polarization‐resolved optical study to identify the b‐axis in GaTe is reported for a multitude of different sample thicknesses and laser excitation wavelengths. The diffraction and Kikuchi line pattern results demonstrate the relevance of the structural anisotropy on the observed anisotropic photoluminescence (PL) in GaTe. The near‐infrared PL from GaTe (≈1.66 eV) exhibits a minimum (maximum) intensity when the laser excitation is polarized parallel (perpendicular) to the b‐axis. The PL peak position behavior as a function of increasing sample temperature (77–298 K) and laser power density is characteristic of radiative recombination of free/bound excitons. Furthermore, a broadband visible excitation window is identified for which GaTe exhibits PL anisotropy. These results highlight the potential of layered GaTe in polarization‐ and wavelength‐sensitive applications.

Organotrialkoxysilane-Functionalized Prussian Blue Nanoparticles-Mediated Fluorescence Sensing of Arsenic(III).

Prussian blue nanoparticles (PBN) exhibit selective fluorescence quenching behavior with heavy metal ions; in addition, they possess characteristic oxidant properties both for liquid–liquid and liquid–solid interface catalysis. Here, we propose to study the detection and efficient removal of toxic arsenic(III) species by materializing these dual functions of PBN. A sophisticated PBN-sensitized fluorometric switching system for dosage-dependent detection of As3+ along with PBN-integrated SiO2 platforms as a column adsorbent for biphasic oxidation and elimination of As3+ have been developed. Colloidal PBN were obtained by a facile two-step process involving chemical reduction in the presence of 2-(3,4-epoxycyclohexyl)ethyl trimethoxysilane (EETMSi) and cyclohexanone as reducing agents, while heterogeneous systems were formulated via EETMSi, which triggered in situ growth of PBN inside the three-dimensional framework of silica gel and silica nanoparticles (SiO2). PBN-induced quenching of the emission signal was recorded with an As3+ concentration (0.05–1.6 ppm)-dependent fluorometric titration system, owing to the potential excitation window of PBN (at 480–500 nm), which ultimately restricts the radiative energy transfer. The detection limit for this arrangement is estimated around 0.025 ppm. Furthermore, the mesoporous and macroporous PBN-integrated SiO2 arrangements might act as stationary phase in chromatographic studies to significantly remove As3+. Besides physisorption, significant electron exchange between Fe3+/Fe2+ lattice points and As3+ ions enable complete conversion to less toxic As5+ ions with the repeated influx of mobile phase. PBN-integrated SiO2 matrices were successfully restored after segregating the target ions. This study indicates that PBN and PBN-integrated SiO2 platforms may enable straightforward and low-cost removal of arsenic from contaminated water.

Blackwater Diving: An Exciting Window Into the Planktonic Arena and Its Potential to Enhance the Quality of Larval Fish Collections

“Blackwater diving,” or nighttime SCUBA diving in epipelagic environments, has become highly popular in recent years because lay participants encounter animals that are difficult and expensive to observe through other methods. These same observations can be priceless for researchers working with these species, so an interface between the scientific communities and recreational divers would be mutually beneficial. In this paper, we describe one such interface through the photography, collection, and DNA barcoding of larval fishes from the island of Hawaii. The images and videos from this activity provide an exciting window into the epipelagic environment and the way larval fishes appear and swim within it. Blackwater diving allows us to see the often-elaborate appendages and other specializations of these larvae as they appear in situ, prior to extensive net and fixation damage. However, blackwater diving remains an almost exclusively recreational pursuit, particularly popular among underwater photographers, who have little interest in (or object to) collecting specimens for scientists. Nonetheless, a logical next step is careful hand collection of specimens for scientific study. Growing numbers of recreational divers around the world have access to an otherwise expensive-to-research habitat. Here we present, for the first time, in situ and post-fixation photos of larval fishes that were hand collected and fixed in 95% ethanol by blackwater divers operating out of Kona, Hawaii, with DNA barcode identifications congruent with morphology and pigmentation where possible. With the right motivation, blackwater diving could augment research in the pelagic ocean and significantly enhance natural history collections and our knowledge of the larvae of marine fishes.