Time-resolved Fluorescence Microscopy Research Articles

Simple rules for passive diffusion through the nuclear pore complex.

Passive macromolecular diffusion through nuclear pore complexes (NPCs) is thought to decrease dramatically beyond a 30-60-kD size threshold. Using thousands of independent time-resolved fluorescence microscopy measurements in vivo, we show that the NPC lacks such a firm size threshold; instead, it forms a soft barrier to passive diffusion that intensifies gradually with increasing molecular mass in both the wild-type and mutant strains with various subsets of phenylalanine-glycine (FG) domains and different levels of baseline passive permeability. Brownian dynamics simulations replicate these findings and indicate that the soft barrier results from the highly dynamic FG repeat domains and the diffusing macromolecules mutually constraining and competing for available volume in the interior of the NPC, setting up entropic repulsion forces. We found that FG domains with exceptionally high net charge and low hydropathy near the cytoplasmic end of the central channel contribute more strongly to obstruction of passive diffusion than to facilitated transport, revealing a compartmentalized functional arrangement within the NPC.

Abstract 776: Multiscale treatment response model for triple-negative breast cancer linking drug pharmacokinetics to tumor cell population dynamics

Abstract Introduction The goal of this study is to establish a predictive model of cytotoxic therapy that incorporates in vitro drug pharmacokinetics and cell-scale therapy response data, on a cell-line specific basis. We report on a series of time-resolved fluorescence microscopy experiments to characterize the uptake of doxorubicin and its effect on the population dynamics of MDA-MB-231 cells, a model of triple negative breast cancer. Experimental Design We leveraged the intrinsic fluorescence of doxorubicin to measure its uptake by MDA-MB-231 cells. Cells, labeled with a fluorescent nuclear marker, were seeded in microtiter plates and incubated with doxorubicin concentrations ranging from 10 nM to 10 μM for 6, 12, or 24 hours. These plates were imaged daily via bright field and fluorescent microscopy after addition of doxorubicin. Nuclei were segmented and automatically counted to quantify cell population size. Counts were normalized to population size at time of treatment and converted to population doublings. On a separate channel, extracellular, cytoplasmic, and nuclear doxorubicin fluorescence were quantified. A compartment model describing the movement of doxorubicin from the extracellular space into cells was fit to these data. We then constructed a cell treatment response model and fit it, coupled with the compartment model, to the population data using MATLAB. Results MDA-MB-231 cellular response to doxorubicin was tightly linked to both drug concentration and exposure time. Higher doses (> 1 μM) invariably induced rapid cell death. Smaller doses (< 1 μM) induced a concentration-dependent nonlinear response defined by an initial increase in population size that, depending on exposure time, was followed by a protracted decrease in cell number. For example, when treated with 156 nM for 6, 12, and 24 hours, we observed, respectively, an average of 2.6, 2.1, and 0.67 population doublings over the first 150 hours after treatment (p < 0.05 among groups). These populations then either held stable or receded out to 400 hours, when we observed net population doublings of 2.6, 1.7, and 0.037, respectively (p < 0.05). Untreated cells followed a logistic growth pattern, with an average total of 4.4 population doublings. Conclusion These time-resolved treatment protocols replicate clinically observed pharmacokinetics of cytotoxic therapies more closely than the constant concentrations in previous dose-response assays. By explicitly considering both drug and population dynamics, our mathematical model enables exploration, in silico, of treatment protocols intractable experimentally. Predictions from model simulations can then be tested experimentally, hopefully allowing for computationally-optimized and experimentally validated treatment regimens that maximize cytotoxic effects of doxorubicin. Citation Format: Matthew T. McKenna, Stephanie L. Barnes, Abigail Searfoss, Darren R. Tyson, Erin Rericha, Vito Quaranta, Thomas E. Yankeelov. Multiscale treatment response model for triple-negative breast cancer linking drug pharmacokinetics to tumor cell population dynamics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 776.

Surface Nanobubbles Studied by Time-Resolved Fluorescence Microscopy Methods Combined with AFM: The Impact of Surface Treatment on Nanobubble Nucleation.

The impact of surface treatment and modification on surface nanobubble nucleation in water has been addressed by a new combination of fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM). In this study, rhodamine 6G (Rh6G)-labeled surface nanobubbles nucleated by the ethanol-water exchange were studied on differently cleaned borosilicate glass, silanized glass as well as self-assembled monolayers on transparent gold by combined AFM-FLIM. While the AFM data confirmed earlier reports on surface nanobubble nucleation, size, and apparent contact angles in dependence of the underlying substrate, the colocalization of these elevated features with highly fluorescent features observed in confocal intensity images added new information. By analyzing the characteristic contributions to the excited state lifetime of Rh6G in decay curves obtained from time-correlated single photon counting (TCSPC) experiments, the characteristic short-lived (<600 ps) component of could be associated with an emission at the gas-water interface. Its colocalization with nanobubble-like features in the AFM height images provides evidence for the observation of gas-filled surface nanobubbles. While piranha-cleaned glass supported nanobubbles, milder UV-ozone or oxygen plasma treatment afforded glass-water interfaces, where no nanobubbles were observed by combined AFM-FLIM. Finally, the number density of nanobubbles scaled inversely with increasing surface hydrophobicity.

Stay on the right track

Cell Biology Cilia contain a well-ordered array of microtubule doublets along their length. A longstanding question in cilium structure and function is why the microtubule arrangement in cilia is so complex. Stepanek and Pigino developed a time-resolved correlative fluorescence and three-dimensional electron microscopy method to show that the doublets provide directionality to intraflagellar transport. One microtubule in the pair moves cargoes up to the ciliary tip. Meanwhile, the other microtubule moves cargoes back to the cell body. These results explain why the axoneme is built out of microtubule doublets and suggest a mechanistic picture of how the logistics of bidirectional intraflagellar transport are regulated. Science , this issue p. [721][1] [1]: /lookup/doi/10.1126/science.aaf4594

Microtubule doublets are double-track railways for intraflagellar transport trains.

The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components.

Labeling and Selective Inactivation of Gram-Positive Bacteria Employing Bimodal Photoprobes with Dual Readouts.

Carbohydrate-conjugated silicon(IV) phthalocyanines with bimodal photoactivity were developed as probes with both fluorescent labeling and photosensitizing capabilities, and the concomitant fluorescent labeling and photoinduced inactivation of Gram-positive and Gram-negative models was explored. The maltohexaose-conjugated photoprobe provides a dual readout to distinguish between both groups of pathogens, as only the Gram-positive species was inactivated, even though both appeared labeled with near-infrared luminescence. Antibiotic resistance did not hinder the phototoxic effect, as even the methicillin-resistant pathogen Staphylococcus aureus (MRSA) was completely photoinactivated. Time-resolved confocal fluorescence microscopy analysis suggests that the photoprobe sticks onto the outer rim of the microorganisms, explaining the resistance of Gram-negative species on the basis of their membrane constitution. The mannose-conjugated photoprobe yields a different readout because it is able to label and to inactivate only the Gram-positive strain.

Nanoscale Study of Polymer Dynamics.

The thermal motion of polymer chains in a crowded environment is anisotropic and highly confined. Whereas theoretical and experimental progress has been made, typically only indirect evidence of polymer dynamics is obtained either from scattering or mechanical response. Toward a complete understanding of the complicated polymer dynamics in crowded media such as biological cells, it is of great importance to unravel the role of heterogeneity and molecular individualism. In the present work, we investigate the dynamics of synthetic polymers and the tube-like motion of individual chains using time-resolved fluorescence microscopy. A single fluorescently labeled polymer molecule is observed in a sea of unlabeled polymers, giving access to not only the dynamics of the probe chain itself but also to that of the surrounding network. We demonstrate that it is possible to extract the characteristic time constants and length scales in one experiment, providing a detailed understanding of polymer dynamics at the single chain level. The quantitative agreement with bulk rheology measurements is promising for using local probes to study heterogeneity in complex, crowded systems.

Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.

Tuning the Mobility Coupling of Quaternized Polyvinylpyridine and Anionic Phospholipids in Supported Lipid Bilayers

Binding of biomacromolecules to anionic lipids in the plasma membrane is a common motif in many cell signaling pathways. Previous work has shown that macromolecules with cationic sequences can form nanodomains with sequestered anionic lipids, which alters the lateral distribution and mobility of the membrane lipids. Such sequestration is believed to result from the formation of a lipid-macromolecule complex. To date, however, the molecular structure and dynamics of the lipid-polymer interface are poorly understood. We have investigated the behavior of polycationic quaternized polyvinylpyridine (QPVP) on supported lipid bilayers doped with phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids using time-resolved fluorescence microscopy, including pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and colocalization. By labeling the polymer and lipids, we investigated the adsorption-induced translational mobility of lipids and systematically studied the influence of lipid charge density and solution ionic strength. Our results show that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid headgroup and is modulated by the ionic strength of the solution, indicating that electrostatic interactions drive the decrease in lateral mobility of anionic lipids by adsorbed QPVP. At physiological salt concentration we observe that the lipid lateral mobility is weakly influenced by QPVP and that there is no evidence of stable lipid-polymer complexes.

Living E. coli is Permeable to Propidium Iodide: A Study by Time-Resolved Second-Harmonic Scattering and Fluorescence Microscopy

Propidium iodide (PI), a hydrophobic cationic dye containing two distinct charge centers, is an indicator stain capable of assessing the viability of bacteria. Specifically, PI is known to exhibit a fluorescence enhancement upon intercalation with DNA. Given that bacterial DNA is found exclusively within the cytosol, such an interaction is feasible only if PI is able to diffuse across the cytoplasmic membrane (CM). Subsequently, as dead bacteria exhibit a breakdown-induced permeability enhancement of the CM (i.e., allowing uptake of PI), fluorescence imaging following incubation with low concentrations of PI (ca. < 4 µM) is a convenient means of deducing cell colony mortality rates following an applied stimulus (e.g., antimicrobial treatment). Our group has previously demonstrated real-time measurements of molecular uptake and membrane-specific transport in living cells using second-harmonic light scattering and optical transmission microscopy (1,2). In the current work, we extend this methodology in combination with time-resolved fluorescence imaging to quantify the concentration dependence on the uptake of PI into living E. coli. Results indicate that at low concentrations (ca. <100 µM) PI readily traverses the porin protein transport channels of the outer membrane, but is unable to cross the CM. However, as the concentration increases, PI is shown to penetrate into the cytosol. There appears to be a concentration threshold whereby PI crosses the CM. The transport kinetics and mechanism will be discussed.1. Zeng, J., et al., (2013) Biophys. J., 104:139-145.2. Wilhelm, M.J., et al., (2014) Chem. Phys. Lett., 605-606:158-163.

Bis-pyrene based reversibly multi-responsive fluorescence switches

A bis-pyrene derivativem-Py-BPdisplays multi-responsive fluorescence switching behaviors upon applying vapor, mechano, thermo and acid/base stimuli.

Surface-enhanced spectroscopy on plasmonic oligomers assembled by AFM nanoxerography.

Surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) from individual plasmonic oligomers are investigated by confocal Raman micro-spectroscopy and time-resolved fluorescence microscopy coupled to steady state micro-spectroscopy. The nanoparticle (NP) oligomers are made of either ligand protected Au or Au@SiO2 core-shell colloidal NPs, which were assembled into ordered arrays by atomic force microscopy (AFM) nanoxerography. A strong dependence of the SERS emission on the polarization of incident light relative to the specific geometry of the plasmonic oligomer was observed. The SEF studies, performed on a large collection of NP oligomers of various known configurations showed interesting fluorophore decay rate modification and red-shift of the emission spectra. The experimental results are analyzed theoretically by employing finite-difference time-domain (FDTD) simulations on equivalent realistic structures, within the local density of optical states (LDOS) framework. The presented results, together with the proven potential of the LDOS approach as a useful common tool for analyzing both SERS and SEF effects further the general understanding of plasmon-related phenomena in nanoparticle oligomers.

Light-responsive polymer surfaces via postpolymerization modification of grafted polymer-brush structures.

Light-induced, spatially well-defined, reversible switching of surface properties enables the creation of remote-controlled smart surfaces. We have taken advantage of the unique high-resolution structuring capabilities of extreme ultraviolet (EUV) interference lithography to produce nanostructured photoresponsive polymer brushes. Patterns of poly(glycidyl methacrylate) (PGMA) and poly(methacrylic acid) (PMAA) were grafted from two different 100 μm thick fluoropolymer substrates by means of a radiation-initiated, grafting-from approach based on free-radical polymerization (FRP). Photochromic properties were introduced via novel one- or two-step postpolymerization modifications with spiropyran (SP) derivatives, which allowed us to control the number of photochromic groups on the polymer brushes. Depending on the degree of functionalization and the local chemical environment, the SP moieties can open upon UV-light exposure to form zwitterionic, deeply colored, and fluorescent merocyanines (MCs) and reclose to the colorless SP configuration via thermal or visible light-induced relaxation. Switching kinetics were studied by means of time-resolved fluorescence microscopy and compared with kinetic measurements of the SP moiety in solution. The results indicated the importance, for the intensity of the switching, of the local chemical environment provided by both the polymer brush and added solvents, and showed the predominant influence on the ring-closing kinetics of polar solvents, which stabilize the MC form. To allow further characterization of the polymer-brush arrangements on a macroscopic scale, similar, but unstructured brush systems were grafted from fluoropolymers after large-area activation using EUV radiation or argon plasma. All steps of the postpolymerization modification were characterized in detail using attenuated total reflection infrared (ATR-IR) spectroscopy. Furthermore, a light-induced reversible static-contact-angle switch with a range of up to 15° for PGMA-SP brushes and up to 30° for PMA-SP brushes was demonstrated upon alternating UV- and visible-light irradiation.

ANG-2 for quantitative Na(+) determination in living cells by time-resolved fluorescence microscopy.

Sodium ions (Na(+)) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na(+) concentrations ([Na(+)]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na(+)-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na(+)-sensitivity appropriate for recordings in living cells. The Na(+)-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na(+)]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na(+)]i rise in cockroach salivary gland cells, which was dependent on a Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.

Thermally Activated Delayed Fluorescence of Fluorescein Derivative for Time-Resolved and Confocal Fluorescence Imaging

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

Fluorescence quenching in the lichen Peltigera aphthosa due to desiccation

Photoprotective mechanisms were studied on the tripartite lichen Peltigera aphthosa that exhibits external cephalodia. Using the methods of steady-state and time-resolved fluorescence microscopy, we studied the dynamics of the rehydration process in different parts of the lichen thalli. It was found that apical, medial and basal parts of the thallus are not only morphologically different, but also show completely different chlorophyll induction curves and other spectral characteristics. In dry state, significant contribution to the fluorescence spectrum of lichen gives a green fluorescence of hyphae forming the upper crust, which is rapidly and almost completely quenched during the rehydration process. Probably this is one of the protective mechanisms that reduce the amount of light reaching the PS II reaction centers in the dry state. In the process of rehydration, we observed an increase in the intensity of the chlorophyll fluorescence of the photobiont at 680 nm, with significant changes of the fluorescence lifetimes and the amplitude ratios of fast and slow components of fluorescence decay kinetics. While in dry state, chlorophyll fluorescence is strongly quenched (opposite to the fluorescence of the hyphae), and the fluorescence time constants recover to the typical decay times of active photosynthetic organisms during rehydration. The quantitative behavior of these changes differs largely between the apical, medial and basal parts of the thallus, probably due to the complex interactions of the fungus, algae and cyanobacteria.

Maximum entropy analysis of data simulations and practical aspects of time-resolved fluorescence measurements in the study of molecular interactions

Time-resolved fluorescence spectroscopy and microscopy is increasingly used to probe molecular interactions and dynamics events in vitro and in vivo. We point out some pitfalls to avoid in the data acquisition procedure using time correlated single photon counting. A good accuracy in fluorescence decay measurements is not only linked to the counts in the peak channel but also to the statistics at the end of the curve. A too narrow time interval between successive excitation leads to an overlap of decays, and that should be taken into account in data analysis. The counting rate in the peak channel of the excitation profile should be close to the one of the fluorescence decay.Many distributions of lifetimes can fit an incomplete or noisy data set to satisfying precisions corresponding to close values of χ2. The maximum entropy principle is appropriate to distinguish among these in a consistent way. It is also shown that encoding a prior knowledge about the system study dramatically improves the quality of the recovered distribution, particularly in case of a set of discrete components. Based on simulated noisy quantify maximum entropy method (MEM) data analysis, we propose a simple strategy for estimating the quality of information and inferences we can draw from experimental results.

Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes

The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting β-amyloid aggregates (Aβ) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aβ peptides and demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of β-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aβ1-42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in Förster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.

Fluorescence spectroscopy of individual semiconductor nanoparticles in different ethylene glycols.

The optical properties of single colloidal semiconductor nanoparticles (NPs) are considerably influenced by the direct environment of the NPs. Here, the influence of different liquid and solid glycol matrices on CdSe-based NPs is investigated. Since the fluorescence of individual NPs varies from one NP to another, it is highly desirable to study the very same individual NPs in different matrices. This was accomplished by immobilizing NPs in a liquid cell sample holder or in microfluidic devices. The samples have been investigated by space-resolved wide-field fluorescence microscopy and energy- and time-resolved confocal scanning fluorescence microscopy with respect to fluorescence intensities, emission energies, blinking behavior, and fluorescence decay dynamics of individual NPs. During the measurements the NPs were exposed to air, to liquid ethylene glycols H(OCH2CH2)nOH (also called EGn) with different chain lengths (1 ≤ n ≤ 7), to liquid 2-methylpentane-2,3-diol, or to solid polyethylene oxide. It was found that EG6-7 (also known as PEG 300) is very well suited as a liquid matrix or solvent for experiments that correlate chemical and physical modifications of the surface and of the immediate environment of individual NPs to their fluorescence properties since it leads to intense and stable fluorescence emission of the NPs.

Versatile Pulsed 560 nm Laser Source for Time-Resolved Microscopy and Spectroscopy

Fluorophores have become a standard tool in life science and biophysics as well as material sciences. Nowadays, fluorophore position and concentration are often not sufficient to answer questions in flurophore dynamics, such as diffusion behavior or molecular interactions. These aspects become accessible with the application of time-resolved fluorescence microscopy and spectroscopy when the fluorophores are excited through pulsed lasers. The large variety of fluorophores requires full spectral coverage through available pulsed lasers.Here, we present a new pulsed laser, filling the existing gap at 560 nm pulsed excitation. This freely triggerable laser source can operate in a wide range of repetition frequencies from 1 MHz up to 80 MHz which makes it easy to adapt the pulse period to different fluorescence lifetimes. Synchronization to other lasers or scanning devices is possible as well as burst operation.The wavelength around 560 nm is appropriate for a wide range of applications, especially in the life sciences where state of the art red fluorescent proteins excited at about 560 nm are essential. A pulsed 559 nm laser enables the long sought Fluorescence Lifetime studies like FLIM (Fluorescence Lifetime Imaging Microscopy) or Fluorescence Lifetime (Cross-)Correlation Spectroscopy (FL(C)CS) involving Red Fluorescent Proteins like mCherry and with that provided access to molecular interaction studies and background reduction. Implemented in a confocal microscope like the MicroTime 200 (PicoQuant) this laser becomes a versatile tool.