Macroscopic temporally and spectrally resolved fluorescence imaging enhanced by laser-wavelength multiplexing.

We present a laser scanning system for macroscopic samples that records fully resolved decay curves in individual pixels, resolves the images in 16 wavelength channels, and records simultaneously at several laser wavelengths. By using confocal detection, the system delivers images that are virtually free of lateral scattering and out-of-focus haze. Image formats can be up to 256 × 256 pixels and up to 1024 time channels. We demonstrate the performance of the system both on model experiments with fluorescent micro-beads and on the tumor model in the living mice.

Patterns of small involuntary fixation saccades (SIFSs) in different neurodegenerative diseases: the role of noise.

During the attempt to steadily fixate at a single spot, sequences of small involuntary fixation saccades (SIFSs, known also as microsaccades οr intrusions) occur which form spatio-temporal patterns such as square wave jerks (SWJs), a pattern characterised by alternating centrifugal and centripetal movements of similar magnitude. In many neurodegenerative disorders, SIFSs exhibit elevated amplitudes and frequencies. Elevated SIFS amplitudes have been shown to favour the occurrence of SWJs ("SWJ coupling"). We analysed SIFSs in different subject groups comprising both healthy controls (CTR) and patients with amyotrophic lateral sclerosis (ALS) and progressive supranuclear palsy (PSP), i.e. two neurodegenerative diseases with completely different neuropathological basis and different clinical phenotypes. We show that, across these groups, the relations between SIFS amplitude and the relative frequency of SWJ-like patterns and other SIFS characteristics follow a common law. As an explanation, we propose that physiological and technical noise comprises a small, amplitude-independent component that has little effect on large SIFSs, but causes considerable deviations from the intended amplitude and direction of small ones. Therefore, in contrast to large SIFSs, successive small SIFSs have a lower chance to meet the SWJ similarity criteria. In principle, every measurement of SIFSs is affected by an amplitude-independent noise background. Therefore, the dependence of SWJ coupling on SIFS amplitude will probably be encountered in almost any group of subjects. In addition, we find a positive correlation between SIFS amplitude and frequency in ALS, but none in PSP, suggesting that the elevated amplitudes might arise at different sites in the two disorders.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Impact of Nanocomposite Combustion Aerosols on A549 Cells and a 3D Airway Model.

The use of nanomaterials incorporated into plastic products is increasing steadily. By using nano-scaled filling materials, thermoplastics, such as polyethylene (PE), take advantage of the unique properties of nanomaterials (NM). The life cycle of these so-called nanocomposites (NC) usually ends with energetic recovery. However, the toxicity of these aerosols, which may consist of released NM as well as combustion-generated volatile compounds, is not fully understood. Within this study, model nanocomposites consisting of a PE matrix and nano-scaled filling material (TiO2, CuO, carbon nano tubes (CNT)) were produced and subsequently incinerated using a lab-scale model burner. The combustion-generated aerosols were characterized with regard to particle release as well as compound composition. Subsequently, A549 cells and a reconstituted 3D lung cell culture model (MucilAir™, Epithelix) were exposed for 4 h to the respective aerosols. This approach enabled the parallel application of a complete aerosol, an aerosol under conditions of enhanced particle deposition using high voltage, and a filtered aerosol resulting in the sole gaseous phase. After 20 h post-incubation, cytotoxicity, inflammatory response (IL-8), transcriptional toxicity profiling, and genotoxicity were determined. Only the exposure toward combustion aerosols originated from PE-based materials induced cytotoxicity, genotoxicity, and transcriptional alterations in both cell models. In contrast, an inflammatory response in A549 cells was more evident after exposure toward aerosols of nano-scaled filler combustion, whereas the thermal decomposition of PE-based materials revealed an impaired IL-8 secretion. MucilAir™ tissue showed a pronounced inflammatory response after exposure to either combustion aerosols, except for nanocomposite combustion. In conclusion, this study supports the present knowledge on the release of nanomaterials after incineration of nano-enabled thermoplastics. Since in the case of PE-based combustion aerosols no major differences were evident between exposure to the complete aerosol and to the gaseous phase, adverse cellular effects could be deduced to the volatile organic compounds that are generated during incomplete combustion of NC.

Label-Free Macroscopic Fluorescence Lifetime Imaging of Brain Tumors.

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients' glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas.

Singlet oxygen phosphorescence imaging by superconducting single-photon detector and time-correlated single-photon counting: publisher's note.

This publisher's note contains corrections to Opt. Lett.46, 1217 (2021) OPLEDP0146-959210.1364/OL.415229.

Singlet oxygen phosphorescence imaging by superconducting single-photon detector and time-correlated single-photon counting.

This Letter presents, to the best of our knowledge, a novel optical configuration for direct time-resolved measurements of luminescence from singlet oxygen, both in solutions and from cultured cells on photodynamic therapy. The system is based on the superconducting single-photon detector, coupled to the confocal scanner that is modified for the near-infrared measurements. The recording of a phosphorescence signal from singlet oxygen at 1270 nm has been done using time-correlated single-photon counting. The performance of the system is verified by measuring phosphorescence from singlet oxygen generated by the photosensitizers commonly used in photodynamic therapy: methylene blue and chlorin e6. The described system can be easily upgraded to the configuration when both phosphorescence from singlet oxygen and fluorescence from the cells can be detected in the imaging mode. Thus, co-localization of the signal from singlet oxygen with the areas inside the cells can be done.

Increased Insulin Concentrations During Growth Hormone Treatment in Girls With Turner Syndrome Are Ameliorated by Hormone Replacement Therapy.

Objective: Turner syndrome (TS) is characterized by complete or partial loss of one sex chromosome and is commonly associated with short stature, metabolic changes (such as central obesity, abnormal glucose tolerance and high triglycerides) and premature ovarian insufficiency (POI). Primary management of TS during childhood and adolescence comprises treatment with human growth hormone (hGH) and, in cases with early loss of ovarian function, hormone replacement therapy (HRT). Given that metabolic parameters are altered when HRT is applied during menopause, we analyzed whether metabolic changes might be positively or negatively affected within 10 years after HRT and/or hGH in girls with TS. Design: Observational study. Methods: Data were collected from the medical records of 31 girls with TS attending two endocrinologic centers in Germany between 2000 and 2020. Descriptive statistics are reported as the mean ± SEM or percentages. Results: The mean age at first presentation was 99.06 ± 8.07 months, the mean height was 115.8 ± 3.94 cm, and the mean BMI 19.0 ± 0.99 was kg/m2. Treatment with hGH was given to 96.8% of the girls, starting at an average age of 99.06 ± 8.70 months, and was continued for 67.53 ± 6.28 months. HRT was administered to 80.6% of all patients and was started at a mean age of 164.4 ± 4.54 months. During the follow-up, we did not observe any significant absolute changes in lipid parameters, but we detected beneficial effects of childhood hGH: significantly lower cholesterol (-0.206/month; p = 0.006), lower low density lipoprotein cholesterol (-0.216/month; p = 0.004), and higher high density lipoprotein cholesterol (+0.095/month; p = 0.048). Insulin concentrations, showed a significant increase attributable to hGH treatment (+0.206/month; p = 0.003), which was ameliorated by concomitant or subsequent HRT (-0.143/month; p = 0.039). Conclusion: Treatment with hGH and HRT is provided to most girls with TS. Metabolic effects are associated with both modalities. Monitoring of metabolic changes appears to be important to detect unfavorable effects, and could guide treatment adjustment and duration.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy.

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

Saccadic intrusions in amyotrophic lateral sclerosis (ALS).

The attempt to quietly fixate at a small visual object is continuously interrupted by a variety of fixational eye movements comprising, among others, a continuum of saccadic intrusions (SI) which range in size from microsaccades with amplitudes ≤0.25° to larger refixation saccades of up to about 2°. The size and frequency of SI varies considerably among individuals and is known to increase in neurodegenerative diseases such as progressive supranuclear palsy (PSP), and amyotrophic lateral sclerosis (ALS). However, studies of ALS disagree whether also the frequency of SI increases. We undertook an analysis of SI in 119 ALS patients and 47 age-matched healthy controls whose eye movements during fixation and tests of executive functions (e.g antisaccades) had been recorded by video-oculography according to standardised procedures. SI were categorised according to their spatio-temporal patterns as stair case, back-and-forth and square wave jerks (a subcategory of back-and-forth). The SI of patients and controls were qualitatively similar (same direction preferences, similar differences between patterns), but were enlarged in ALS. Notably however, no increase of SI frequency could be demonstrated. Yet, there were clear correlations with parameters such as eye blink rate or errors in a delayed saccade task that suggest an impairment of inhibitory mechanisms, in keeping with the notion of a frontal dysfunction in ALS. However, it remains unclear how the impairment of inhibitory mechanisms in ALS could selectively increase the amplitude of intrusions without changing their frequency of occurrence.

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system.

The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition the amount of purified protein is usually in the µg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. In addition, a one-step purification procedure is often not sufficient to obtain pure protein preparations. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. The first chromatographic step is the capture step followed by a desalting step. The final purification was done using size exclusion chromatography. This set-up reduces the overall-time needed for protein production, needs minimal operator invention, is easy to handle and thus increases the throughput.

Fluorescence time-resolved macroimaging.

While laser scanning fluorescence lifetime imaging (FLIM) is a powerful approach for cell biology, its small field of view (typically less than 1 mm) makes it impractical for the imaging of large biological samples that is often required for biomedical applications. Here we present a system that allows performing FLIM on macroscopic samples as large as 18 mm with a lateral resolution of 15 µm. The performance of the system is verified with FLIM of endogenous metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and genetically encoded fluorescent protein mKate2 in a mouse tumor in vivo.

Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation.

TCSPC FLIM/PLIM is based on a multi-dimensional time-correlated single-photon counting process. The sample is scanned by a high-frequency-pulsed laser beam which is additionally modulated on/off synchronously with the pixels of the scan. FLIM is obtained by building up the distribution of the photons over the scanning coordinates and the times of the photons in the excitation pulse sequence, PLIM is obtained by building up the photon distribution over the scanning coordinates and the photon times in the modulation period. FLIM and PLIM data are thus obtained simultaneously within the same imaging process. Since the technique uses not only one but many excitation pulses for every phosphorescence signal period the sensitivity is much higher than for techniques that excite with a single pulse only. TCSPC FLIM/PLIM works both with one-photon and two-photon excitation, does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.

Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy.

The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for tumor development and provides background for the relevant tumor's treatment. Here, we show that a specially designed implantable fiber-optic probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of a nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on an iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions and in living mice.

Detection of Black Plastics in the Middle Infrared Spectrum (MIR) Using Photon Up-Conversion Technique for Polymer Recycling Purposes.

The identification of black polymers which contain about 0.5 to 3 mass percent soot or black master batch is still an essential problem in recycling sorting processes. Near infrared spectroscopy (NIRS) of non-black polymers offers a reliable and fast identification, and is therefore suitable for industrial application. NIRS is consequently widely used in polymer sorting plants. However, this method cannot be used for black polymers because small amounts of carbon black or soot absorb all light in the NIR spectral region. Spectroscopy in the mid infrared spectral region (MIR) offers a possibility to identify black polymers. MIR spectral measurements carried out with Fourier-transform infrared spectrometers (FTIR) are not fast enough to meet economic requirements in sorting plants. By contrast, spectrometer systems based on the photon up-conversion technique are fast and sensitive enough and can be applied to sort black polymer parts. Such a system is able to measure several thousand spectra per second hence is suitable for industrial applications. The results of spectral measurements of black polymers are presented.

Turbidimetric method for the determination of particle sizes in polypropylene/clay-composites during extrusion.

Nanocomposites with polypropylene as matrix material and nanoclay as filler were produced in a double twin screw extruder. The extrusion was monitored with a spectrometer in the visible and near-infrared spectral region with a diode array spectrometer. Two probes were installed at the end at the extruder die and the transmission spectra were measured during the extrusion. After measuring the transmission spectra and converting into turbidity units, the particle distribution density was calculated via numerical linear equation system. The distribution density function shows either a bimodal or mono modal shape in dependence of the processing parameters like screw speed, dosage, and concentration of the nanoclays. The method was verified with SEM measurements which yield comparable results. The method is suitable for industrial in-line processing monitoring of particle radii and dispersion process, respectively.

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode.

We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the "start" input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the "stop" input. The time between start and stop is directly converted into position, with a resolution of 200-250 µm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.