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[Maximilian Lozano]

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The National Bureau of Statistics of China on Tuesday announced disappointing GDP growth figures. According to official yet preliminary data, the Chinese economy grew by just 3.0 percent in 2022. The reading was the lowest annual result since modern Chinese GDP records began in the 1970s with the exception of the 2020 Covid crisis year. Yet, China was the only major world economy to post positive annual GDP growth in 2020 and last year was arguably also struggling less economically than others despite the invasion of Ukraine and domestic coronavirus lockdowns that continued into 2022 in the country.

China had set a goal of at least 6 percent GDP growth in 2021 and surpassed that when the country's economy grew by 8.4 percent that year. Just a decade ago, any result in this vicinity would have been seen as rather average, but after weak domestic demand paired with the cooling effects of the trade war with the United States had contributed to a significant GDP slowdown in the later half of the 2010s, China's economic glory days seemed far away. As U.S. tariffs persist, pandemic effects linger and the war in Ukraine poses new challenges, China is cut down to an even more modest growth outlook.

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Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.

Bioanalytical, diagnostic, and security applications require the fast determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats. Among the most popular approaches is the use of multiparametric fluorescence techniques for sample analysis due to their versatility and often straightforward use1,2,3. Many fluorescence techniques rely on a toolbox of luminescent labels for encoding and multiplexing2,3,4,5,6. An approved and versatile technique for bioanalytical high-throughput single-particle measurements is flow cytometry (FCM). FCM with optical detection is routinely exploited in the life sciences e.g., for single cell analysis for blood count, in diagnostics as well as for next generation sequencing, here in conjunction with intensity or colour encoded surface-functionalised polymer particles acting as carriers for different DNA sequences. State-of-the-art instruments are capable of reading almost 20 different colour codes7,8,9, but that still does not satisfy the requirements of complex research, e.g. in cell biology and immunology10.

Depending on the specific purpose, method development for FCM either faces increasing complexity of analytical problems or the demand for low-cost and robust techniques for routine analyses11. Commonly applied spectral multiplexing approaches based on organic fluorophores are limited in both directions: on the one hand, spectral overlap of labels restricts the number of codes3,12 and makes elaborate correction schemes necessary. On the other hand, even lower degrees of multiplexing often require sophisticated optical setups with multiple excitation light sources and detection channels which increases instrument complexity and costs. Particularly, II/VI semiconductor quantum dots (QDs) with their characteristic spectral properties allow for the use of a larger number of distinguishable color codes compared to dyes13,14 but even their spectral distinction is limited. Furthermore, they are not widely accepted due to possible environmental concerns15,16,17 and their toxicity is still a subject of ongoing discussion18.

A promising alternative to spectral multiplexing and intensity encoding is offered by luminescence lifetime as an additional encoding parameter3,12,18,19. This provides that luminescence codes could be distinguished based on their characteristic decay kinetics. In combination with spectral encoding, it can increase the number of accessible parameters20. Furthermore, due to the availability of fast electronics, miniaturized or portable lifetime measurement setups21 can be assembled which allow to complement routine techniques based on spectral encoding by lifetime-encoded ones at reduced cost, especially in countries of the developing world. Lifetime encoding in flow cytometry can find applications ranging from alternative staining strategies for cell analysis to the use of stains or labels whose emission properties are sensitive to their microenvironment or respond to binding events by a change in luminescence decay kinetics. Thereby, it helps to increase the information content from bioassays by efficient detection of multiple analytes or targets in a single measurement.

Lifetime encoding in flow cytometry has been discussed for decades now and increasingly draws interest for high-throughput methods based on time-resolved measurements4. However, only very specialized applications22,23,24,25 have been reported so far and lifetime multiplexing in flow is still not accepted in routine applications due to the limited measurement time per object and the resulting issue of reduced photon count numbers26,27. Moreover, reported lifetime detection in flow cytometry mostly relied on phase-domain techniques24,28,29,30,31,32, which requires higher signal intensities and might face problems analysing multi-exponential decays. Even though time- and frequency-domain methods are theoretically equivalent in terms of resolution33, time-domain methods can be superior for low signal intensities. This can be the case for systems which contain a limited number of emitters like the labelling of biomolecules with low label concentrations or when the excitation intensity has to be limited to avoid photobleaching in multi-step analyses of the same sample. Moreover, time-domain measurements directly visualise the decay kinetics.

In this study, the issues of time-domain cytometry with very short interaction times and limited number of detected photons are addressed. We present a lifetime flow cytometry (LT-FCM) platform based on a compact setup and straightforward time-domain measurements utilizing LT-encoded luminescent beads. These polymer microbeads are loaded with different organic fluorophores exhibiting lifetimes in the nanosecond range or semiconductor quantum dots to extend the accessible lifetime range. The beads serve as a model system in our studies but further applications will not be limited to distinguishing luminescent beads with well-defined properties. We discuss the concept of this platform and address its application potential including achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.

As lifetime encoded systems, we employed commercially available PMMA beads stained with organic dyes from PolyAn GmbH, Germany, and tailor-made melamine beads with a polyelectrolyte-based layer-by-layer (LbL) coating, loaded with CdSe/CdS/ZnS core/shell quantum dots (QDs) and a final poly(sodium 4-styrensulfonate) (PSS) layer provided by the research group of Sukhanova and Nabiev34,35. As emitters we chose organic dyes expected to reveal lifetimes in the low nanoseconds range and II/VI semiconductor QDs with lifetimes up to tens of nanoseconds. Table 1 gives a brief overview over the corresponding luminophores of the lifetime-encoded beads as well as the mean bead diameters.

Overview and concept of LT-FCM. (a) Schematic drawing of the optical setup of the LT-FCM. Optical components are indicated: (D)M refers to (dichroic) mirrors, BP/LP to band pass/long pass filters, ND to the neutral density filter, and BS to the beam splitter. The detection channels are LT (lifetime), SSC (side scatter), FL1/2 (fluorescence). Details on the optical components can be found in the Supplementary Information, Table S1. (b) Block diagram of the novel signal processing unit for single photon lifetime analysis for LT-FCM. (c) Visualisation of lifetime encoding with beads: Each code should exhibit similar spectral properties but differ in the decay kinetics from the other codes.

Numerical simulations based on synthetic decay curves were performed. We studied the impact of measurement parameters and conditions such as the integration time range θ, the bin width of the decay histograms, the number of acquired photon counts, and the background level. Simulations were carried out with custom-made Octave37 scripts. Synthetic decay curves were obtained from random number distributions following exponential decay laws. The generation of these random number distributions was based on computational physics textbook methods38. Details are given in the Supplementary Information. Repeated simulations were carried out for each parameter set under study to obtain a reasonable statistical validation of the results. The respective numbers of repetitions are given throughout the discussion and resemble the number of analysed objects in an FCM experiment.

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