Integration of a superconducting nanowire single-photon detector into a confocal microscope for time-resolved photoluminescence (TRPL)-mapping: Sensitivity and time resolution.

This report highlights the combination of the MicroTime 100 upright confocal fluorescence lifetime microscope with a Single Quantum Eos Superconducting Nanowire Single-Photon Detector (SNSPD) system as a powerful tool for photophysical research and applications. We focus on an application in materials science, photoluminescence imaging, and lifetime characterization of Cu(InGa)Se2 (CIGS) devices intended for solar cells. We demonstrate improved sensitivity, signal-to-noise ratio, and time-resolution in combination with confocal spatial resolution in the near-infrared (NIR) range, specifically in the 1000-1300 nm range. The MicroTime 100-Single Quantum Eos system shows two orders of magnitude higher signal-to-noise ratio for CIGS devices' photoluminescence imaging compared to a standard NIR-photomultiplier tube (NIR-PMT) and a three-fold improvement in time resolution, which is now limited by the laser pulse width. Our results demonstrate the advantages in terms of image quality and time resolution of SNSPDs technology for imaging in materials science.

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing.

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

BabyLux device: a diffuse optical system integrating diffuse correlation spectroscopy and time-resolved near-infrared spectroscopy for the neuromonitoring of the premature newborn brain.

The BabyLux device is a hybrid diffuse optical neuromonitor that has been developed and built to be employed in neonatal intensive care unit for the noninvasive, cot-side monitoring of microvascular cerebral blood flow and blood oxygenation. It integrates time-resolved near-infrared and diffuse correlation spectroscopies in a user-friendly device as a prototype for a future medical grade device. We present a thorough characterization of the device performance using test measurements in laboratory settings. Tests on solid phantoms report an accuracy of optical property estimation of about 10%, which is expected when using the photon diffusion equation as the model. The measurement of the optical and dynamic properties is stable during several hours of measurements within 3% of the average value. In addition, these measurements are repeatable between different days of measurement, showing a maximal variation of 5% in the optical properties and 8% for the particle diffusion coefficient on a liquid phantom. The variability over test/retest evaluation is < 3 % . The integration of the two modalities is robust and without any cross talk between the two. We also perform in vivo measurements on the adult forearm during arterial cuff occlusion to show that the device can measure a wide range of tissue hemodynamic parameters. We suggest that this platform can form the basis of the next-generation neonatal neuromonitors to be developed for extensive, multicenter clinical testing.

Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics.

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes.

Integrated multichannel photon timing instrument with very short dead time and high throughput.

Precisely timed detection of single photons plays an important role in the field of quantum information processing and fluorescence sensing. The method of time-correlated single photon counting is therefore constantly evolving and the associated instrumentation is being improved with new ideas and technologies. Simultaneous, time tagged readout of multiple detector channels is invaluable in many applications, spanning from fluorescence lifetime imaging in biology to the measurement of quantum optical correlations in basic research. Here we present a new integrated design, providing up to three independent input channels, a very short dead time, very high throughput, and a timing resolution of 25 ps at reasonable cost and small size. Apart from design features and test results of the instrument, we show an application in quantum optics, namely, the measurement of the photon statistics of a heralded single photon source based on cavity-enhanced spontaneous parametric down-conversion.

Pulse-shape improvement during amplification and second-harmonic generation of picosecond pulses at 531 nm.

We study second-harmonic generation of picosecond pulses in bulk periodically poled lithium niobate using an all-semiconductor master oscillator-power amplifier with gain-switched seed as our fundamental source. Both during amplification and during the subsequent second-harmonic generation, the signal pulse shape improves, and the resulting pulses at 531 nm are nearly independent of the seed pump conditions. Over a wide range of repetition rates and seed settings, we obtain green pulses with a duration of less than 31 ps FWHM and a peak power of more than 5.1 W. We further investigate the influence of the fundamental's spectral dynamics on pulsed second-harmonic generation efficiency and obtain excellent agreement between our measurements and previously published theoretical treatments.

White biotechnology for cellulose manufacturing--the HoLiR concept.

A variety of approaches are available for generation of bacteria-produced nanocellulose (BNC) in different forms. BNC production under static cultivation conditions usually results in fleeces or foils, characterized by a homogeneous, three-dimensional network of nanofibers and a uniform surface. However, under static cultivation conditions in batch vessels, the widths and the lengths of the BNC sheets cultured are determined by the dimensions of the culture vessel. In this contribution, a novel, efficient process for a (semi-)continuous cultivation of planar BNC fleeces and foils with a freely selectable length and an adjustable height is presented. By means of comprehensive investigations, the comparability of the BNC harvested to that gained from static cultivation under batch conditions is demonstrated. A first estimation of the production costs further shows that this type of processing allows for significant cost reductions compared to static cultivation of BNC in Erlenmeyer flasks.

Collisional quenching of erythrosine B as a potential reference dye for impulse response function evaluation.

We studied the collisional quenching of the erythrosine B fluorophore by potassium iodide. The quenching follows a Stern-Volmer dependence up to the highest quencher concentration. The lifetime of erythrosine B decreases to 24 ps in 5.02 M of potassium iodide. The quantum yield of erythrosine B in the presence of 5.02 M KI is 0.0035. The relatively high brightness makes this compound attractive as an ultrashort reference in time-resolved measurements. In both frequency- and time-domain fluorescence techniques, there is a need for lifetime standards with extremely short decay times. Mimicking the instantaneous scattering at longer wavelengths allows color-effect-free measurements in the emission region. Another motivation is the problem of obtaining the impulse response function in the case of two-photon excitation. Time-resolved microscopy also benefits from fast-decaying dyes because the impulse response function can be evaluated at the emission wavelength of the investigated specimen without changing filters. We demonstrated that impulse response functions for commonly used detectors are practically the same for scattering as for quenched erythrosine B emission. We also analyzed a complex fluorescence decay using both elastic scattering and quenched erythrosine B emission as a response function.

Fluorescence and spin properties of defects in single digit nanodiamonds.

This article reports stable photoluminescence and high-contrast optically detected electron spin resonance (ODESR) from single nitrogen-vacancy (NV) defect centers created within ultrasmall, disperse nanodiamonds of radius less than 4 nm. Unexpectedly, the efficiency for the production of NV fluorescent defects by electron irradiation is found to be independent of the size of the nanocrystals. Fluorescence lifetime imaging shows lifetimes with a mean value of around 17 ns, only slightly longer than the bulk value of the defects. After proper surface cleaning, the dephasing times of the electron spin resonance in the nanocrystals approach values of some microseconds, which is typical for the type Ib diamond from which the nanoparticle is made. We conclude that despite the tiny size of these nanodiamonds the photoactive nitrogen-vacancy color centers retain their bulk properties to the benefit of numerous exciting potential applications in photonics, biomedical labeling, and imaging.

Scalable time-correlated photon counting system with multiple independent input channels.

Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information (time tagging), thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized to picoseconds of relative timing and yet operate independent of each other. This is achieved by means of a modular design with independent crystal-locked time digitizers and a central processing unit for sorting and processing of the timing data. The modules communicate through high speed serial links supporting the full throughput rate of the time digitizers. Event processing is implemented in programmable logic, permitting classical histogramming, as well as time tagging of individual photons and their temporally ordered streaming to the host computer. Based on the time-ordered event data, any algorithms and methods for the analysis of fluorescence dynamics can be implemented not only in postprocessing but also in real time. Results from recently emerging single molecule applications are presented to demonstrate the capabilities of the instrument.

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels.

Time-correlated single photon counting is a powerful method for sensitive time-resolved fluorescence measurements down to the single molecule level. The method is based on the precisely timed registration of single photons of a fluorescence signal. Historically, its primary goal was the determination of fluorescence lifetimes upon optical excitation by a short light pulse. This goal is still important today and therefore has a strong influence on instrument design. However, modifications and extensions of the early designs allow for the recovery of much more information from the detected photons and enable entirely new applications. Here, we present a new instrument that captures single photon events on multiple synchronized channels with picosecond resolution and over virtually unlimited time spans. This is achieved by means of crystal-locked time digitizers with high resolution and very short dead time. Subsequent event processing in programmable logic permits classical histogramming as well as time tagging of individual photons and their streaming to the host computer. Through the latter, any algorithms and methods for the analysis of fluorescence dynamics can be implemented either in real time or offline. Instrument test results from single molecule applications will be presented.

DNA-based molecular wires: multiple emission pathways of individual constructs.

The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.

Spectral identification of specific photophysics of cy5 by means of ensemble and single molecule measurements.

The triplet-state characteristics of the Cy5 molecule related to trans-cis isomerization are investigated by means of ensemble and single molecule measurements. Cy5 has been used frequently in the past 10 years in single molecule spectroscopic applications, e.g., as a probe or fluorescence resonance energy transfer acceptor in large biomolecules. However, the unknown spectral properties of the triplet state and the lack of knowledge on the photoisomerization do not allow us to interpret precisely the unexpected single molecule behaviors. This limits the application of Cy5. The laser photolysis experiments demonstrate that the trans triplet state of Cy5 absorbs about 625 nm, the cis ground state absorbs about 690 nm, and the cis triplet state also absorbs about 690 nm. In other words, the T1-Tn absorptions largely overlap the ground-state absorptions for both trans and cis isomers, respectively. Furthermore, the observation of the cis triplet state indicates an important isomerization pathway from the trans-S1 state to the cis-T1 state upon excitation. The detailed spectra presented in this article let us clearly interpret the exact mechanisms responsible for several important and unexpected photophysical behaviors of single Cy5 molecules such as reverse intersystem crossing (RISC), the observation of dim states with a lower emission intensity and slightly red-shifted fluorescence, and unusual energy transfer from donor molecules to dark Cy5 molecules acting as acceptors in single molecule fluorescence resonance energy transfer (FRET) measurements. Spectral results show that the dim state in the single molecule fluorescence intensity time traces originated from cis-Cy5 because of a lower excitation rate, resulting from the red-shifted ground-state absorption of cis-Cy5 compared to that of the trans-Cy5.

Direct observation of delayed fluorescence from a remarkable back-isomerization in Cy5.

The direct observations of delayed fluorescence and phosphorescence from the cyanine dye Cy5 are reported. The delayed fluorescence is generated from the S(1) state of trans-Cy5 through a reserve intersystem crossing from the cis-triplet state T(1) to the trans-singlet state S(1) via thermal activation. The lowest cis-triplet state is evidenced to be involved in the formation of the isomer. The back-isomerization from cis-triplet state to trans-singlet state crossing, a remarkably back-isomerization pathway that has not been reported before, plays a significant role in this unusual delayed fluorescence.

Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes.

This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.