Deformable microlaser force sensing.

Mechanical forces are key regulators of cellular behavior and function, affecting many fundamental biological processes such as cell migration, embryogenesis, immunological responses, and pathological states. Specialized force sensors and imaging techniques have been developed to quantify these otherwise invisible forces in single cells and in vivo. However, current techniques rely heavily on high-resolution microscopy and do not allow interrogation of optically dense tissue, reducing their application to 2D cell cultures and highly transparent biological tissue. Here, we introduce DEFORM, deformable microlaser force sensing, a spectroscopic technique that detects sub-nanonewton forces with unprecedented spatio-temporal resolution. DEFORM is based on the spectral analysis of laser emission from dye-doped oil microdroplets and uses the force-induced lifting of laser mode degeneracy in these droplets to detect nanometer deformations. Following validation by atomic force microscopy and development of a model that links changes in laser spectrum to applied force, DEFORM is used to measure forces in 3D and at depths of hundreds of microns within tumor spheroids and late-stage Drosophila larva. We furthermore show continuous force sensing with single-cell spatial and millisecond temporal resolution, thus paving the way for non-invasive studies of biomechanical forces in advanced stages of embryogenesis, tissue remodeling, and tumor invasion.

Hyperspectral confocal imaging for high-throughput readout and analysis of bio-integrated microlasers.

Integrating micro- and nanolasers into live cells, tissue cultures and small animals is an emerging and rapidly evolving technique that offers noninvasive interrogation and labeling with unprecedented information density. The bright and distinct spectra of such lasers make this approach particularly attractive for high-throughput applications requiring single-cell specificity, such as multiplexed cell tracking and intracellular biosensing. The implementation of these applications requires high-resolution, high-speed spectral readout and advanced analysis routines, which leads to unique technical challenges. Here, we present a modular approach consisting of two separate procedures. The first procedure instructs users on how to efficiently integrate different types of lasers into living cells, and the second procedure presents a workflow for obtaining intracellular lasing spectra with high spectral resolution and up to 125-kHz readout rate and starts from the construction of a custom hyperspectral confocal microscope. We provide guidance on running hyperspectral imaging routines for various experimental designs and recommend specific workflows for processing the resulting large data sets along with an open-source Python library of functions covering the analysis pipeline. We illustrate three applications including the rapid, large-volume mapping of absolute refractive index by using polystyrene microbead lasers, the intracellular sensing of cardiac contractility with polystyrene microbead lasers and long-term cell tracking by using semiconductor nanodisk lasers. Our sample preparation and imaging procedures require 2 days, and setting up the hyperspectral confocal microscope for microlaser characterization requires <2 weeks to complete for users with limited experience in optical and software engineering.

Red-Shifted Excitation and Two-Photon Pumping of Biointegrated GaInP/AlGaInP Quantum Well Microlasers.

Biointegrated intracellular microlasers have emerged as an attractive and versatile tool in biophotonics. Different inorganic semiconductor materials have been used for the fabrication of such biocompatible microlasers but often operate at visible wavelengths ill-suited for imaging through tissue. Here, we report on whispering gallery mode microdisk lasers made from a range of GaInP/AlGaInP multi-quantum well structures with compositions tailored to red-shifted excitation and emission. The selected semiconductor alloys show minimal toxicity and allow the fabrication of lasers with stable single-mode emission in the NIR (675-720 nm) and sub-pJ thresholds. The microlasers operate in the first therapeutic window under direct excitation by a conventional diode laser and can also be pumped in the second therapeutic window using two-photon excitation at pulse energies compatible with standard multiphoton microscopy. Stable performance is observed under cell culturing conditions for 5 days without any device encapsulation. With their bio-optimized spectral characteristics, low lasing threshold, and compatibility with two-photon pumping, AlGaInP-based microlasers are ideally suited for novel cell tagging and in vivo sensing applications.

Narrowband Organic Light-Emitting Diodes for Fluorescence Microscopy and Calcium Imaging.

Fluorescence imaging is an indispensable tool in biology, with applications ranging from single-cell to whole-animal studies and with live mapping of neuronal activity currently receiving particular attention. To enable fluorescence imaging at cellular scale in freely moving animals, miniaturized microscopes and lensless imagers are developed that can be implanted in a minimally invasive fashion; but the rigidity, size, and potential toxicity of the involved light sources remain a challenge. Here, narrowband organic light-emitting diodes (OLEDs) are developed and used for fluorescence imaging of live cells and for mapping of neuronal activity in Drosophila melanogaster via genetically encoded Ca2+ indicators. In order to avoid spectral overlap with fluorescence from the sample, distributed Bragg reflectors are integrated onto the OLEDs to block their long-wavelength emission tail, which enables an image contrast comparable to conventional, much bulkier mercury light sources. As OLEDs can be fabricated on mechanically flexible substrates and structured into arrays of cell-sized pixels, this work opens a new pathway for the development of implantable light sources that enable functional imaging and sensing in freely moving animals.

Non-obstructive intracellular nanolasers.

Molecular dyes, plasmonic nanoparticles and colloidal quantum dots are widely used in biomedical optics. Their operation is usually governed by spontaneous processes, which results in broad spectral features and limited signal-to-noise ratio, thus restricting opportunities for spectral multiplexing and sensing. Lasers provide the ultimate spectral definition and background suppression, and their integration with cells has recently been demonstrated. However, laser size and threshold remain problematic. Here, we report on the design, high-throughput fabrication and intracellular integration of semiconductor nanodisk lasers. By exploiting the large optical gain and high refractive index of GaInP/AlGaInP quantum wells, we obtain lasers with volumes 1000-fold smaller than the eukaryotic nucleus (Vlaser < 0.1 µm3), lasing thresholds 500-fold below the pulse energies typically used in two-photon microscopy (Eth ≈ 0.13 pJ), and excellent spectral stability (<50 pm wavelength shift). Multiplexed labeling with these lasers allows cell-tracking through micro-pores, thus providing a powerful tool to study cell migration and cancer invasion.

Flexible and ultra-lightweight polymer membrane lasers.

Organic semiconductors enable the fabrication of a range of lightweight and mechanically flexible optoelectronic devices. Most organic semiconductor lasers, however, have remained rigid until now, predominantly due to the need for a support substrate. Here, we use a simple fabrication process to make membrane-based, substrate-less and extremely thin (<500 nm) organic distributed feedback lasers that offer ultralow-weight (m/A<0.5 gm-2) and excellent mechanical flexibility. We show operation of the lasers as free-standing membranes and transfer them onto other substrates, e.g. a banknote, where the unique lasing spectrum is readily read out and used as security feature. The pump thresholds and emission intensity of our membrane lasers are well within the permissible exposures for ocular safety and we demonstrate integration on contact lenses as wearable security tags.

Lasing in Live Mitotic and Non-Phagocytic Cells by Efficient Delivery of Microresonators.

Reliable methods to individually track large numbers of cells in real time are urgently needed to advance our understanding of important biological processes like cancer metastasis, neuronal network development and wound healing. It has recently been suggested to introduce microscopic whispering gallery mode lasers into the cytoplasm of cells and to use their characteristic, size-dependent emission spectrum as optical barcode but so far there is no evidence that this approach is generally applicable. Here, we describe a method that drastically improves intracellular delivery of resonators for several cell types, including mitotic and non-phagocytic cells. In addition, we characterize the influence of resonator size on the spectral characteristics of the emitted laser light and identify an optimum size range that facilitates tagging and tracking of thousands of cells simultaneously. Finally, we observe that the microresonators remain internalized by cells during cell division, which enables tagging several generations of cells.

An exciton-polariton laser based on biologically produced fluorescent protein.

Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing.

Lasing within Live Cells Containing Intracellular Optical Microresonators for Barcode-Type Cell Tagging and Tracking.

We report on a laser that is fully embedded within a single live cell. By harnessing natural endocytosis of the cell, we introduce a fluorescent whispering gallery mode (WGM) microresonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells. Our approach can be applied to different cell types, and cells with microresonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing, and adaptive imaging.

The role of regioregularity, crystallinity, and chain orientation on electron transport in a high-mobility n-type copolymer.

We investigated the correlation between the polymer backbone structural regularity and the charge transport properties of poly{[N,N'-bis(2-octyldodecyl)-1,4,5,8-naphthalenediimide-2,6-diyl]-alt-5,5'-(2,2'-bithiophene)} [P(NDI2OD-T2)], a widely studied semiconducting polymer exhibiting high electron mobility and an unconventional micromorphology. To understand the influence of the chemical structure and crystal packing of conventional regioregular P(NDI2OD-T2) [RR-P(NDI2OD-T2)] on the charge transport, the corresponding regioirregular polymer RI-P(NDI2OD-T2) was synthesized. By combining optical, X-ray, and transmission electron microscopy data, we quantitatively characterized the aggregation, crystallization, and backbone orientation of all of the polymer films, which were then correlated to the electron mobilities in electron-only diodes. By carefully selecting the preparation conditions, we were able to obtain RR-P(NDI2OD-T2) films with similar crystalline structure along the three crystallographic axes but with different orientations of the polymer chains with respect to the substrate surface. RI-P(NDI2OD-T2), though exhibiting a rather similar LUMO structure and energy compared with the regioregular counterpart, displayed a very different packing structure characterized by the formation of ordered stacks along the lamellar direction without detectible π-stacking. Vertical electron mobilities were extracted from the space-charge-limited currents in unipolar devices. We demonstrate the anisotropy of the charge transport along the different crystallographic directions and how the mobility depends on π-stacking but is insensitive to the degree or coherence of lamellar stacking. The comparison between the regioregular and regioirregular polymers also shows how the use of large planar functional groups leads to improved charge transport, with mobilities that are less affected by chemical and structural disorder with respect to classic semicrystalline polymers such as poly(3-hexylthiophene).

Efficient charge generation by relaxed charge-transfer states at organic interfaces.

Interfaces between organic electron-donating (D) and electron-accepting (A) materials have the ability to generate charge carriers on illumination. Efficient organic solar cells require a high yield for this process, combined with a minimum of energy losses. Here, we investigate the role of the lowest energy emissive interfacial charge-transfer state (CT1) in the charge generation process. We measure the quantum yield and the electric field dependence of charge generation on excitation of the charge-transfer (CT) state manifold via weakly allowed, low-energy optical transitions. For a wide range of photovoltaic devices based on polymer:fullerene, small-molecule:C60 and polymer:polymer blends, our study reveals that the internal quantum efficiency (IQE) is essentially independent of whether or not D, A or CT states with an energy higher than that of CT1 are excited. The best materials systems show an IQE higher than 90% without the need for excess electronic or vibrational energy.

Fullerene-Free Polymer Solar Cells with Highly Reduced Bimolecular Recombination and Field-Independent Charge Carrier Generation.

Photogeneration, recombination, and transport of free charge carriers in all-polymer bulk heterojunction solar cells incorporating poly(3-hexylthiophene) (P3HT) as donor and poly([N,N'-bis(2-octyldodecyl)-naphthelene-1,4,5,8-bis(dicarboximide)-2,6-diyl]-alt-5,5'-(2,2'-bithiophene)) (P(NDI2OD-T2)) as acceptor polymer have been investigated by the use of time delayed collection field (TDCF) and time-of-flight (TOF) measurements. Depending on the preparation procedure used to dry the active layers, these solar cells comprise high fill factors (FFs) of up to 67%. A strongly reduced bimolecular recombination (BMR), as well as a field-independent free charge carrier generation are observed, features that are common to high performance fullerene-based solar cells. Resonant soft X-ray measurements (R-SoXS) and photoluminescence quenching experiments (PQE) reveal that the BMR is related to domain purity. Our results elucidate the similarities of this polymeric acceptor with the superior recombination properties of fullerene acceptors.

Aggregation in a high-mobility n-type low-bandgap copolymer with implications on semicrystalline morphology.

We explore the photophysics of P(NDI2OD-T2), a high-mobility and air-stable n-type donor/acceptor polymer. Detailed steady-state UV-vis and photoluminescence (PL) measurements on solutions of P(NDI2OD-T2) reveal distinct signatures of aggregation. By performing quantum chemical calculations, we can assign these spectral features to unaggregated and stacked polymer chains. NMR measurements independently confirm the aggregation phenomena of P(NDI2OD-T2) in solution. The detailed analysis of the optical spectra shows that aggregation is a two-step process with different types of aggregates, which we confirm by time-dependent PL measurements. Analytical ultracentrifugation measurements suggest that aggregation takes place within the single polymer chain upon coiling. By transferring these results to thin P(NDI2OD-T2) films, we can conclude that film formation is mainly governed by the chain collapse, leading in general to a high aggregate content of ~45%. This process also inhibits the formation of amorphous and disordered P(NDI2OD-T2) films.

Heterojunction topology versus fill factor correlations in novel hybrid small-molecular/polymeric solar cells.

The authors present organic photovoltaic (OPV) devices comprising a small molecule electron acceptor based on 2-vinyl-4,5-dicyanoimidazole (Vinazene) and a soluble poly(p-phenylenevinylene) derivative as the electron donor. A strong dependence of the fill factor (FF) and the external quantum efficiency [incident photons converted to electrons (IPCE)] on the heterojunction topology is observed. As-prepared blends provided relatively low FF and IPCE values of 26% and 4.5%, respectively, which are attributed to significant recombination of geminate pairs and free carriers in a highly intermixed blend morphology. Going to an all-solution processed bilayer device, the FF and IPCE dramatically increased to 43% and 27%, respectively. The FF increases further to 57% in devices comprising thermally deposited Vinazene layers where there is virtually no interpenetration at the donor/acceptor interface. This very high FF is comparable to values reported for OPV using fullerenes as the electron acceptor. Furthermore, the rather low electron affinity of Vinazene compound near 3.5 eV enabled a technologically important open circuit voltage (V(oc)) of 1.0 V.