Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Förster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.

We present the first demonstration of fluorescence correlation spectroscopy (FCS) using superconducting nanowire single-photon detectors (SSPDs) which are free of afterpulses unlike the avalanche photodiode (APD). Multimode-fiber-coupled SSPDs with high system detection efficiency for visible wavelengths were developed and implemented in the FCS system. We performed FCS measurements for Rhodamine B and 6G as fluorescent samples, and found that autocorrelation functions obtained by the SSPDs showed a noise-free curve in the short correlation time region of sub microseconds where the afterpulse effect was dominant using the APD. The obtained results clearly indicate the advantage of SSPDs for the FCS system.

Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (K_d) of mCherry₂-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, K_d of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. K_d of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.

Macromolecular crowding (MMC) in cells is a hot topic in biology; therefore, well-characterized measurement standards for the evaluation of the nano-environment in MMC solutions are necessary. We propose to use polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) for evaluation of macromolecular crowding in solutions. Pol-FCS can simultaneously measure the relaxation times of rotational and translational diffusion of fluorescent molecules at the same position, even in living cells with low damage. In this report, the differences in the nano-environment among solutions of small molecules, gels, and MMC solutions were evaluated by comparing their rotational and translational diffusion using Pol-FCS. Moreover, this method could distinguish the phase shift in the polyethylene glycol solution. Finally, we separately evaluated the nano-environment in the cytosol and nucleus of living cells in different cell lines and cell cycles. We expect this evaluation method to be useful in characterizing the nano-environment in MMC studies. In addition, the proposed method may be useful for other nano-environments such as liquid–liquid phase separation.

Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins.

Conventional polarization-dependent fluorescence correlation spectroscopy (pol-FCS) requires two sets of photon detectors to eliminate after-pulse noises (dual-channel pol-FCS; DC-pol-FCS) in the sub-microsecond range. In this study, we successfully realized pol-FCS using a visible-wavelength superconductive nanowire single-photon detector (single-channel pol-FCS; SC-pol-FCS). The detector used is free of after-pulse noises and thus eliminates the need for dual channels in pol-FCS. Further, the optics in the SC-pol-FCS system are easier to adjust than those in the conventional system. Consequently, we obtained higher signal-to-noise ratios compared with conventional DC-pol-FCS systems. Thus, SC-pol-FCS is a potentially useful system for obtaining pol-FCS measurements, and can facilitate improved rotational diffusion studies.