Living cells are crowded with macromolecules and organelles. As a result, there is an urgent need for molecular sensors for quantitative, site-specific assessment of the macromolecular crowding effects on a myriad of biochemical processes toward quantitative cell biology and biophysics. Here we investigate the excited-state dynamics and translational diffusion of a novel FRET sensor (mCerulean-linker-mCitrine) in a buffer (PBS, pH 7.4) at room temperature. Complementary experiments were carried out on free CFP, YFP, and the cleaved FRET probe as controls. The wavelength-dependent fluorescence lifetime measurements of the donor and acceptor in the FRET probe, using the time-correlated single-photon counting technique, indicate an energy transfer efficiency of 6.8 ± 0.9% in PBS, with distinct excited-state dynamics from the recombinant CFP and YFP. The estimated mCerulean-mCitrine distance in this FRET probe is 7.7 ± 0.2 nm. The energy transfer efficiency increases (11.5 ± 0.9%) as the concentration of Ficoll-70 increases over the range of 0-300 g/L with an estimated mCerulean-mCitrine distance of 6.1 ± 0.2 nm. Complementary time-resolved anisotropy measurements suggest that the rotational diffusion of hetero-FRET in PBS is sensitive to the energy transfer from the donor to the acceptor. The results also suggest that the linker, -(GSG)A(EAAAK)A(GSG)A(EAAAK)A(GSG)-, is rather flexible, and the observed rotational dynamics is likely to be due to a segmental mobility of the FRET pairs rather than an overall tumbling motion of a rigid probe. Comparative studies on a new construct of a FRET probe with a shorter, more flexible linker, mCerulean-(GSG)-mCitrine, reveal enhanced energy transfer efficiency. On the millisecond time scale, fluorescence fluctuation analyses of the acceptor (excited at 488 nm) provide a means to examine the translational diffusion coefficient of the FRET probe. The results also suggest that the linker is flexible in this FRET probe, and the observed diffusion coefficient is faster than predicted as compared to the cleaved FRET probe. Our results serve as a point of reference for this FRET probe in a buffer toward its full potential as a sensor for macromolecular crowding in living cells and tissues.

Living cells are crowded with macromolecules and organelles, which affect a myriad of biochemical processes. As a result, there is a need for sensitive molecular sensors for quantitative, site-specific assessment of macromolecular crowding. Here, we investigated the excited-state dynamics of recently developed hetero-FRET sensors (mCerulean3–linker–mCitrine) in homogeneous and heterogeneous environments using time-resolved fluorescence measurements, which are compatible with fluorescence lifetime imaging microscopy (FLIM). The linker in these FRET constructs, which tether the mCerulean3 (the donor) and mCitrine (the acceptor), vary in both length and flexibility. Glycerol and Ficoll-70 solutions were used for homogeneous and heterogeneous environments, respectively, at variable concentrations. The wavelength-dependent studies suggest that the 425-nm excitation and the 475-nm emission of the donor are best suited for quantitative assessment of the energy transfer efficiency and the donor-acceptor distance of these FRET probes. Under the same experimental conditions, the enzymatically cleaved counterpart of these probes was used as a control as well as a means to account for the changes in the environmental refractive indices. Our results indicate that the energy transfer efficiency of these FRET probes increases as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor a compact structure with enhanced energy transfer efficiency and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the stretched conformation of these FRET probes is more favorable in the viscous, homogeneous environment with a reduced energy transfer efficiency and relatively larger donor-acceptor distance as compared with those in pure buffer, which was attributed to a reduced structural fluctuation of the mCerulean3–mCitrine FRET pair in the viscous, more restrictive glycerol-enriched buffer. Our findings will help to advance the potential of these hetero-FRET probes using FLIM for spatio-temporal assessment of the compartmentalized crowding in living cells.

Macromolecular crowding is prevalent in all living cells due to the presence of large biomolecules and organelles. Cellular crowding is heterogeneous and is known to influence biomolecular transport, biochemical reactions, and protein folding. Emerging evidence suggests that some cell pathologies may be correlated with compartmentalized crowding. As a result, there is a need for robust biosensors that are sensitive to crowding as well as quantitative, noninvasive fluorescence methods that are compatible with living cells studies. Here, we have developed a model that describes the rotational dynamics of hetero-Förster resonance energy transfer (FRET) biosensors as a means to determine the energy-transfer efficiency and donor–acceptor distance. The model was tested on wavelength-dependent time-resolved fluorescence anisotropy of hetero-FRET probes (mCerulean3–linker–mCitrine) with variable linkers in both crowded (Ficoll-70) and viscous (glycerol) solutions at room temperature. Our results indicate that the energy-transfer efficiencies of these FRET probes increase as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor compact structures with enhanced energy-transfer efficiencies and a shorter donor–acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the extended conformation of these FRET probes is more favorable in viscous, homogeneous environments with a reduced energy-transfer efficiency compared to those in pure buffer, which we attribute to reduced structural fluctuations of the mCerulean3–mCitrine FRET pair in the glycerol-enriched buffer. Our results represent an important step toward the application of quantitative and noninvasive time-resolved fluorescence anisotropy of hetero-FRET probes to investigate compartmentalized macromolecular crowding and protein–protein interactions in living cells as well as in controlled environments.

We present a new theoretical design for diode lasers which should be capable of generating sub-100-ps pulses with pulse energies of more than 10 nJ per 100-μm-contact width by active Q-switching. We show that the carrier-induced reduction of the refractive index in the active layer, i.e. the vertical anti-index guiding effect, results in a dependence of the optical confinement factor on the carrier density which can be exploited for a further enhancement of the pulse energy and a reduction of the pulse length.

Cw-operation, gain-switched, and passively Q -switched mode locking of Cr(4+): YAG microchip lasers with output powers of several hundred milliwatts is demonstrated experimentally in the eye-safe region near 1.5microm . Requirements for cw mode locking of such lasers are investigated by numerical simulations.