Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically encoded Zn2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.

We have developed a microfluidic flow cytometry system to screen reversibly photoswitchable fluorescent proteins for contrast and stability of reversible photoconversion between high- and low-fluorescent states. A two-color array of 20 excitation and deactivation beams generated with diffractive optics was combined with a serpentine microfluidic channel geometry designed to provide five cycles of photoswitching with real-time calculation of photoconversion fluorescence contrast. The characteristics of photoswitching in-flow as a function of excitation and deactivation beam fluence, flow speed, and protein concentration were studied with droplets of the bacterial phytochrome from Deinococcus radiodurans (DrBphP), which is weakly fluorescent in the near-infrared (NIR) spectral range. In agreement with measurements on stationary droplets and HeLa S3 mammalian cells expressing DrBphP, optimized operation of the flow system provided up to 50% photoconversion contrast in-flow at a droplet rate of few hertz and a coefficient of variation (CV) of up to 2% over 10 000 events. The methods for calibrating the brightness and photoswitching measurements in microfluidic flow established here provide a basis for screening of cell-based libraries of reversibly switchable NIR fluorescent proteins.

We report a femtosecond time-resolved spectroscopic study of size-dependent dynamics in nanocrystals (NCs) of Fe(pyz)Pt(CN)4. We observe that smaller NCs (123 or 78 nm cross section and <25 nm thickness) exhibit signatures of spin crossover (SCO) with time constants of ∼5–10 ps whereas larger NCs with 375 nm cross section and 43 nm thickness exhibit a weaker SCO signature accompanied by strong spectral shifting on a ∼20 ps time scale. For the small NCs, the fast dynamics appear to result from thermal promotion of residual low-spin states to high-spin states following nonradiative decay, and the size dependence is postulated to arise from differing high-spin vs low-spin fractions in domains residing in strained surface regions. The SCO is less efficient in larger NCs owing to their larger size and hence lower residual LS/HS fractions. Our results suggest that size-dependent dynamics can be controlled by tuning surface energy in NCs with dimensions below ∼25 nm for use in energy harvesting, spin switching, and other applications.

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Fluorescent proteins with large Stokes shifted emission beyond 600 nm are actively sought for live-cell imaging applications. The mechanism of excited-state relaxation leading to the Stokes shift in the mPlum fluorescent protein, which emits at a peak wavelength of 650 nm, has been previously investigated by both ultrafast spectroscopy and theoretical methods. Here, we report that femtosecond time-resolved area-normalized emission spectra of mPlum show a clear isoemissive point. This feature can only result from a system with two emitting states, rather than a system that undergoes a continuous spectral red shift, for example, as expected from typical solvation. Global analysis of the femtosecond time-resolved fluorescence spectra reveals time constants associated with chromophore relaxation, excited-state population transfer, and an excited-state lifetime of the final state. The observations confirm the findings of recent quantum chemical calculations on mPlum.

There is a critical need for high-speed multiparameter photophysical measurements of large libraries of fluorescent probe variants for imaging and biosensor development. We present a microfluidic flow cytometer that rapidly assays 104–105 member cell-based fluorophore libraries, simultaneously measuring fluorescence lifetime and photobleaching. Together, these photophysical characteristics determine imaging performance. We demonstrate the ability to resolve the diverse photophysical characteristics of different library types and the ability to identify rare populations.

Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.

The field of bioimaging and biosensors has been revolutionized by the discovery of fluorescent proteins (FPs) and their use in live cells. FPs are characterized with rich photodynamics due to the presence of nonfluorescent or dark states which are responsible for fluorescence intermittency or “blinking”, which has been exploited in several localization-based super-resolution techniques that surpass the diffraction-limited resolution of conventional microscopy. Molecules that convert to these dark states recover to the ground states either spontaneously or upon absorption of another photon, depending on the particular FP and the structural transition that is involved. In this work, we demonstrate time- and frequency-domain methods for the measurement of the ground-state recovery (GSR) times of FPs both in live cells and in solutions. In the time-domain method, we excited the sample with millisecond pulses at varying dark times to obtain percent-recovery. In the frequency-domain method, dark-state hysteresis was employed to obtain the positive phase shift or “phase advance”. We extracted the GSR time constants from our measurements using calculations and simulations based on a three-state model system. The GSR time constants of the red FPs studied in these experiments fall in the range from μs to msec time-scales. We find that the time- and frequency-domain techniques are complementary to each other. While accurate GSR times can be extracted from the time-domain technique, frequency-domain measurements are primarily sensitive to the rates of dark-state conversion (DSC) processes. A correlation between GSR times, DSC, and photobleaching rates for the red FPs mCherry, TagRFP-T, and Kriek were observed. These time- and frequency-domain methods can be used in high-throughput screening and sorting of FPs clones based on GSR time constant and photostability and will therefore be valuable for the development of new photoswitchable or photoactivatable FPs.

Fluorescent proteins have revolutionized molecular biology research and provide a means of tracking subcellular processes with extraordinary spatial and temporal precision. Species with emission beyond 650 nm offer the potential for deeper tissue penetration and lengthened imaging times; however, the origin of their extended Stokes shift is not fully understood. We employed spectrally resolved transient grating spectroscopy and molecular dynamics simulations to investigate the relationship between the flexibility of the chromophore environment and Stokes shift in mPlum. We examined excited state solvation dynamics in a panel of strategic point mutants of residues E16 and I65 proposed to participate in a hydrogen-bonding interaction thought responsible for its red-shifted emission. We observed two characteristic relaxation constants of a few picoseconds and tens of picoseconds that were assigned to survival times of direct and water-mediated hydrogen bonds at the 16-65 position. Moreover, variants of the largest Stokes shift (mPlum, I65V) exhibited significant decay on both time scales, indicating the bathochromic shift correlates with a facile switching between a direct and water-mediated hydrogen bond. This dynamic model underscores the role of environmental flexibility in the mechanism of excited state solvation and provides a template for engineering next-generation red fluorescent proteins.

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: Sbeam3/Sbeam1) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).