We systematically engineered fluorescent protein-nanobody probes with different coupling rigidities and binding geometries to specific targets and analyzed the diffusion properties of their complexes by polarization-dependent fluorescence correlation spectroscopy (Pol-FCS). The results demonstrate that probe architecture critically affects Pol-FCS readouts, suggesting practical design principles for structure-sensitive probes. Polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) simultaneously measures translational and rotational diffusion of fluorescently labeled molecules and provides information on molecular size and shape. Rotational diffusion is, in principle, expected to provide even higher sensitivity than translational diffusion, which is routinely used in conventional FCS to detect binding of fluorescent protein (FP)-fused binders to their targets. However, when FPs are fused to binders via flexible linkers, local wobbling of the FP can decouple its rotational diffusion from that of the binding complex. Quantitative evaluation of this decoupling effect remains lacking. Here, leveraging our expertise from polarization-dependent orientation probes, POLArIS, we designed and compared two classes of probes, termed "Rigid" and "Flex," that differ in how tightly the FP is linked to the anti-ALFAtag nanobody, to evaluate how probe architecture affects Pol-FCS readouts. Using ALFAtag and the rod-like repeat protein DHR10 as a structurally anisotropic binding target, we varied probe binding geometry while keeping molecular weight nearly constant and measured rotational and translational diffusion. Flex probes exhibited shorter rotational diffusion times than Rigid probes when bound to the same targets, consistent with the expected local reorientation. Notably, translational diffusion times also showed architecture-dependent differences between Rigid and Flex probes. These results demonstrate that probe architecture is a key determinant of diffusion readouts in FP-based Pol-FCS and suggest practical probe design principles for sensitive reporters of intermolecular binding accompanied by changes in complex shape.

Mechanical stress on cells is transmitted through many biological processes, for example, cell shape control, tissue patterning, and axonal homeostasis. Microtubules, a cytoskeletal component, presumably play a significant role in the mechanoregulation of cellular processes. We investigate motor protein-driven transport of quantum dots along mechanically deformed microtubules. We found that microtubule deformation significantly slowed kinesin-driven transport, whereas we previously reported dynein-driven transport was rather robust. Such dualistic modulation of transportation dynamics of the motor proteins by microtubule deformation can be attributed to the altered affinity of the motor proteins for buckled microtubules. Our results may form the basis for understanding microtubules’ role in regulating cellular processes in a mechanically adverse environment through its detection ability and response to mechanical stress.

Living cells arecharacterized by the micrometric confinementofvarious macromolecules at high concentrations. Using droplets containingbinary polymer blends as artificial cells, we previously showed thatcell-sized confinement causes phase separation of the binary polymersolutions because of the length-dependent wetting of the polymers.Here, we demonstrate that the confinement-induced heterogeneity ofpolymers also emerges in single-component polymer solutions. The resultingstructural heterogeneity also leads to a slower transport of smallmolecules at the center of cell-sized droplets than that in bulk solutions.Coarse-grained molecular simulations support this confinement-inducedheterogeneous distribution by polymer length and demonstrate thatthe effective wetting of the shorter chains at the droplet surfaceoriginates from the length-dependent conformational entropy. Our resultssuggest that cell-sized confinement functions as a structural regulatorfor polydisperse polymer solutions that specifically manipulates thediffusion of molecules, particularly those with sizes close to thecorrelation length of the polymer chains.

We evaluated the dispersion and diffusion of fluorescent-labeled lipophilic vitamin E (VE) in microemulsions (MEs) including water-in-oil (W/O) type ME, oil-in-water (O/W) type ME, and bicontinuous ME (BME), using fluorescence correlation spectroscopy (FCS). We prepared a fluorescent ATTO 488 or BODIPY group labeled VE (VE-ATTO or VE-BODIPY). VE-ATTO possesses lipophilic and hydrophilic parts, while VE-BODIPY consists solely of the lipophilic part. The VE-ATTO dissolved in heptane solution as an oil phase appeared hot pink in color due to the solvatochromism effect under room light and almost no fluorescent signal, which was unlike the VE-ATTO dissolved in ME solutions and all the VE-BODIPY solutions (typical fluorescent green color). The FCS measurement proved that VE-BODIPY diffuses faster than VE-ATTO. This is presumably because the "surfactant-like" VE-ATTO is localized and trapped at the micro-water/micro-oil interface of the MEs, while the VE-BODIPY exists in the ME phase and macro-oil phase with good dispersion. These results demonstrate that FCS is a powerful tool for the rapid evaluation of the lipophilic probe behavior in heterogeneous ME solutions.

In this study, the concentration and brightness measured by fluorescence correlation spectroscopy (FCS) in samples containing multiple species with different brightness levels was demonstrated. FCS measurements of such samples are generally difficult. However, the calculation we introduced here can provide the measurement results of the FCS. The effectiveness of the calculation was investigated based on simulations and experiments in the case of a mixture of fluorescent beads with known brightness and other fluorescent beads with unknown brightness. The results show that the concentration of the known brightness agrees well with the expected values. The obtained concentration and brightness of the species with unknown brightness is possible, and it worked well in the simulation; however, the accuracy for the species was lower than that of the species with known brightness. As a result, the calculation is useful in measuring the concentration of species with known brightness in samples containing undesired bright species, such as aggregation. The calculation for the species with unknown brightness may also be useful if good protocols or instruments are established in the future.

Number and brightness (N&B) analysis helps to visualize protein oligomer and its localization in a living cell. N&B analysis provides apparent brightness, which reflects the oligomeric state of a fluorescently labeled protein, by analyzing the temporal intensity fluctuation at each pixel. N&B analysis is useful in understanding the dynamic oligomerization in signal transduction and neurodegenerative diseases. Furthermore, it also helps in gaining useful insights regarding the controlling mechanisms in protein function. In this chapter, we describe the basic theory and notations of N&B analysis implemented with confocal laser scanning microscopy for quantitative analyses.

We studied the diffusion properties of lipophilic vitamin E (VE) through bicontinuous microemulsions (BME) using both electrochemical and fluorescence correlation spectroscopy (FCS) measurements. We investigated the effect of different composition ratios of micro-water and micro-oil phases in BMEs (W/OBME). When we employed the BME with a lower W/OBME value of 40/60 (oil-rich BME) as an electrolyte solution, we obtained a larger current response from VE at a fluorinated nanocarbon film electrode. Further voltammetric studies revealed that a higher VE diffusion coefficient was observed in the oil-rich BME. The FCS results also exhibited faster diffusion through the oil-rich BME, which played a significant role in accelerating the VE diffusion probably due to the widening of the micro-oil phase pathway in the BME. Moreover, the effect of increasing the VE diffusion was pronounced at the interface between the electrode surface and the BME solution. These results indicate that controlling the conditions of the BME as the measurement electrolyte is very effective for achieving superior electrochemical measurements in a BME.

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.

A robust fluorescence correlation spectroscopy system called fiber-optic based fluorescence correlation spectroscopy (FB-FCS) was developed; this system enables the measurement of diffusion dynamics and concentration of fluorescent molecules based on the principle of fluorescence correlation spectroscopy without any mechanical adjustment of the experimental setup. The system consisted of fiber optics and a water-immersion objective lens. The hydrodynamic diameters and concentrations of organic fluorescent dyes and fluorescently labeled proteins were successfully measured. Because of the fiber-optic-based setup, the FB-FCS system is compact and inexpensive. We expect FB-FCS to be suitable for use in laboratories, medical diagnosis, and environmental measurements.

Number and brightness (N&B) analysis is useful for monitoring the spatial distribution of the concentration and oligomeric state of fluorescently labeled proteins in cells. N&B analysis is based on the statistical analysis of fluorescence images by using the method of moments (MoM). Furthermore, N&B analysis can determine the particle number and particle brightness, which indicate the concentration and oligomeric state, respectively. However, the statistical accuracy and precision are limited in actual experiments with fluorescent proteins, owing to low excitation and the limited number of images. In this study, we applied maximum likelihood (ML) estimation and maximum a posteriori (MAP) estimation coupled with the empirical Bayes (EB) method (referred to as EB-MAP). In EB-MAP, we constructed a simple prior distribution for a pixel to utilize the information of the surrounding pixels. To evaluate the accuracy and precision of our method, we conducted simulations and experiments and compared the results of MoM, ML, and EB-MAP. The results showed that MoM estimated the particle number with many outliers. The outliers hampered the visibility of the spatial distribution and cellular structure. In contrast, EB-MAP suppressed the number of outliers and improved the visibility notably. The precision of EB-MAP was better by an order of magnitude in terms of particle number and 1.5 times better in terms of particle brightness compared with those of MoM. The proposed method (EB-MAP-N&B) is applicable to studies on fluorescence imaging and would aid in accurately recognizing changes in the concentration and oligomeric state in cells. Our results hold significant importance because quantifying the concentration and oligomeric state would contribute to the understanding of dynamic processes in molecular mechanism in cells.

Molecular behaviors in small liquid droplets (picoliter scale), such as phase transitions and chemical reactions, are essential for the industrial application of small droplets and their use as artificial cells. However, the droplets often differ from those in bulk solutions (milliliter scale). Since the droplet size is much larger than the molecular size, the so-called size effect that draws these differences has attracted attention as a target to be solved. Although the small volume and the membrane surface surrounding the droplet are thought to be the origin of the size effect, there were little attempts to separate and quantify them. To solve the problem, we develop a series of systems for the evaluation. Using these systems, we have evaluated the size effect of concentrated polymer solutions on molecular diffusion by dividing it into small volume and membrane surface contributions. Our results demonstrate that the size effect on the molecular diffusion originates from the long-range interaction with the surface enhanced with decreasing volume. The quantitative size effect revealed by the systems provides novel insights in the biophysical understanding of molecular behaviors in cells and to the regulation and design of micrometer-sized materials.

Fluorescence correlation spectroscopy (FCS) is a potential tool to measure the dynamics of fluorescent molecules, either in solution or in a cell. FCS can quantify the diffusion coefficient, the target molecule concentration, and brightness of a single molecule. These parameters allow estimations of the molecular size, the molecular shape, and the affinities of molecular interactions. Moreover, varieties of FCS methods have been developed on the concept of the fluorescence lifetime, triplet state, decrease of fluorescent intensity and toward two-dimensional imaging, in order to reveal the molecular interaction in live cell and molecular crowding.

Molecular crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of molecular crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the crowding effect in the nucleus. Thus, the use of DNA sensors with variable structures can elucidate the local effects of molecular crowding in cells.

The accuracy and precision of quantification values of biomolecules, such as nucleic acids, are critical for the reliability of biomedical research and clinical examinations. To obtain an accurate quantitative value, it is necessary to use a measurement standard that has the same sequence and length as the target gene. The absence of an appropriate measurement standard leads to uncertain results. The development of a wide variety of different kinds of measurement standards, which have different sequences and lengths, is time-consuming and troublesome. We employed fluorescence correlation spectroscopy (FCS), which can be used to count the molecular number (absolute concentration) regardless of the molecular size and shape, without a standard curve. The confocal volume (i.e., the volume of excitation laser focus) of the FCS system was calibrated by measuring the primary standard of the fluorescent material. Furthermore, we investigated how to avoid artifacts originating from systematic aberrations or sample conditions. We validated the RNA concentration obtained from our FCS measurements using another primary standard RNA solution as a sample. Here, we describe an FCS calibration procedure with fluorescein solution standard reference material (SRM) 1932 as a primary standard and cross-validation of FCS values using RNA solutions certified reference material (CRM) 6204-a. The established method was applied to determine the concentrations of RNA samples that can be used as a laboratory working standards. The FCS method with a characterized SRM and CRM should serve as a universal method for absolute quantification of the number of biomolecules.

A multipoint holographic fluorescence correlation spectroscope (MP-hFCS) was successfully developed. The validity of the MP-hFCS was demonstrated using diffusion measurements of fluorescent dye solutions and of fluorescent proteins in single cells. Furthermore, the successful detection of the nuclear transport of a green fluorescent protein-tagged glucocorticoid receptor α indicates the possibility of being able to monitor directional molecular transport using the MP-hFCS. This allows multipoint analysis of the intermolecular interactions and molecular transport in living cells. Finally, the MP-hFCS can achieve multipoint diffusion measurements with high spatial and time resolution while maintaining a high photon detection sensitivity.

Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement. The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases.

Glucocorticoid receptor (GR) is a hormone-activated transcription regulatory protein involved in metabolism as well as adrenocortical responses to psychosocial stress. Ligand-activated GR localizes to the nucleus, where GR homodimers regulate gene transcription via direct binding to glucocorticoid response elements (GREs). The role of GR homodimers in transcriptional activation has not yet been elucidated. In this study, we determined the concentration of GR homodimer, and its dissociation constant (Kd), at the single-cell level, by using fluorescence correlation spectroscopy (FCS) combined with a microwell system. Results from dissociation constant analysis and diffusion analysis suggested that GR forms complexes with other proteins as well as homodimers. We determined the relationship between the concentration of GR homodimer and transcriptional activity using a triple-color FCS-microwell system-based fluorescent reporter assay. The binding affinity of GR to GREs was analyzed via fluorescence cross-correlation spectroscopy (FCCS). Our findings indicate that the GR homodimer is essential for activating target gene transcription.

The glucocorticoid receptor (GR) is a transcription factor, which interacts with DNA and other cofactors to regulate gene transcription. Binding to other partners in the cell nucleus alters the diffusion properties of GR. Raster image correlation spectroscopy (RICS) was applied to quantitatively characterize the diffusion properties of EGFP labeled human GR (EGFP-hGR) and its mutants in the cell nucleus. RICS is an image correlation technique that evaluates the spatial distribution of the diffusion coefficient as a diffusion map. Interestingly, we observed that the averaged diffusion coefficient of EGFP-hGR strongly and negatively correlated with its transcriptional activities in comparison to that of EGFP-hGR wild type and mutants with various transcriptional activities. This result suggests that the decreasing of the diffusion coefficient of hGR was reflected in the high-affinity binding to DNA. Moreover, the hyper-phosphorylation of hGR can enhance the transcriptional activity by reduction of the interaction between the hGR and the nuclear corepressors.

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 mCherry 2 -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.

Quantum dot (QD) and quantum rod (QR) nanocrystals are widely used non-organic nanocrystals. Their strong fluorescence and photostability make them suitable for biomedical imaging applications. However, their pH-dependence and antibunching properties have not been studied much, especially in aqueous conditions. In this report, we used fluorescence correlation spectroscopy (FCS) with high temporal resolution to demonstrate that the fluorescent blinking and antibunching of QDs/QRs can be changed by varying the pH of their solutions. Furthermore, herein, we reported the relationship between the aggregation and antibunching relaxation time of QDs/QRs for the first time. The findings of this study suggest that FCS can be used to discover novel environmental indicators via observing nanosecond and microsecond phenomena.