Abstract: The axonal membrane is not the seat of nerve conduction: it is the boundary between two osmotic reservoirs whose asymmetry is the thermodynamic engine of the action potential. Voltage-gated ion channels are not the generators of the nerve signal -- they are its osmotic amplifiers, and their spatial distribution along the axon is a geometric necessity, not an arbitrary anatomical feature. The Ionic-Mechano-Hydraulic (IMH) model formalises this principle: intracellular K$^{+}$ adsorbed on the cytoplasmic polyelectrolyte gel triggers an ionic phase transition; extracellular Na$^{+}$ amplifies the resulting hydraulic wave via Nav channels; Kv channels close the osmotic cycle and enforce the refractory period. Conduction velocity is predicted from myelin elastic modulus, not sodium channel density. The model resolves a 75-year-old anomaly that Huxley and St\"{a}mpfli themselves described as impossible in a purely electrical system: positive current enters a node before the membrane potential at the preceding node has reached its maximum. Nine falsifiable predictions are presented -- among them, a graded reduction in conduction distance under partial tetrodotoxin block, a bell-shaped relationship between node length and conduction velocity, and an upper diameter limit for unmyelinated fibres derived from first physical principles. The Hodgkin-Huxley model is not discarded: it is explained.

Abstract: Fighting against gravity is a common challenge for all terrestrial animals, including most mammals. It means, in particular, avoiding falls on the ground while performing daily tasks, such as standing up, locomotion or foraging for food. This means that balance control in humans involves a wide variety of contexts and balance paradigms, such as upright standing, hand-standing, tightrope walking, ice skater spinning, bicycling, whole-body gesturing, and stick balancing on a finger,tip among others. From the cybernetic point of view, the underlying control problem is to keep the CoP (Center of Pressure) and the CoM (Center of Mass) aligned dynamically on the common vertical, and this means that the variety of balance strategies can be reduced to two basic paradigms: the CoP strategy (the CoP is the control variable and the CoM is the controlled variable) and the CoM strategy (the CoM is simultaneously the control and the controlled variable). The two balance strategies are implemented by combining different control paradigms: • Opportunistic control: exploitation of a physical phenomenon as the gyroscopic effect. • Stiffness control: exploiting the elastic properties of skeletal muscles. • Feedback control (continuous or intermittent): measuring an incipient fall index and closing the loop in real-time. • Feedforward control: exploiting an internal body model for generating stable whole-body synergies in an anticipatory manner. Such control paradigms are illustrated with experimental and simulated experiments.

Abstract: Background: genistein and resveratrol are bioactive compounds isolated from plants, recognised for their diverse biological activities including anti-cancer properties. Both compounds are also known as modulators of potassium channels, including the Kv1.3 ones. These channels are expressed in both normal and cancerous tissues. Their activity is crucial in regulating cell proliferation and apoptosis in cells that express Kv1.3 channels. The potential clinical application of channel inhibitors may extend to treating cancers characterized by an over expression of Kv1.3 channels. Methods: this study investigates the inhibitory effects of genistein and resveratrol on Kv1.3 channels in cancer cells – human leukemic Jurkat T cells, applying the whole-cell patch-clamp technique. Results: applying both compounds at concentrations ranging from 3 μM to 90 μM leads to a dose-dependent inhibition of the channel activity, reducing it to approximately 50% of control level. This inhibitory effect was reversible and associated with a significant reduction of the activation rate. When combined with simvastatin, the inhibitory effect exhibited synergy; however, it was additive when co-applied with mevastatin. Conclusion: the inhibition of Kv1.3 channels is likely linked to the anti-cancer activities of these compounds on Kv1.3 channel- expressing cancer cells, especially when co-applied with the statins.

Abstract: Molecular dynamics (MD) simulation is a fundamental technique for resolving biomolecular structures and functions at atomic resolution. Accelerated by GPU computing and machine learning-integrated force fields (FF), modern MD simulation facilitates the study of large-scale systems and rare biological events, such as protein folding, allosteric transitions, etc. While advanced sampling methods and AI integration have significantly enhanced efficiency in drug discovery and protein engineering, the field still faces challenges regarding FF accuracy, timescale constraints, and quantum effects. Continued development of hybrid quantum and molecular mechanics methods and standardized workflows is essential to further improve the predictive power and reproducibility of MD in biotechnological research. In this review, we attempted to provide the latest developments in the MD simulations.

Abstract: Accurate assessment of blood viscosity and red blood cell (RBC) aggregation under continuous flow is important for hemorheological analysis. However, simultaneous measurement remains challenging because both properties are influenced by flow conditions and RBC sedimentation. In this study, a microfluidic method is developed for the simultaneous measurement of blood viscosity and RBC aggregation index (AI) during continuous blood delivery from a driving syringe. The proposed device consists of a viscosity-sensing channel for viscosity measurement and aggregation-sensing channel for AI evaluation. The effects of flow rate, hematocrit, suspension medium, and syringe on-off operation are systematically investigated. Blood viscosity and AI are strongly affected by these factors and transient flow interruption enhances RBC sedimentation in the syringe, thereby altering hemorheological properties. The pro-posed method is further used to thermally shocked RBCs which reduce RBC aggregation and suppress RBC sedimentation when compared with control blood. At higher exposure temperatures and longer exposure times, blood viscosity and AI remain nearly constant over time, indicating minimal contribution of damaged RBCs to RBCs sedimentation. These results demonstrate that the proposed method enables reliable simultaneous evaluation of blood viscosity and RBC aggregation and could be regarded as useful for detecting functional alterations of RBCs under continuous-flow conditions.

Abstract: Blood viscosity is strongly dependent on hematocrit, and the hematocrit–viscosity relationship is an important determinant of blood rheology under physiological and pathological conditions. However, obtaining a full hematocrit–viscosity curve requires multiple measurements over a wide hematocrit range. In this study, a simple method is proposed to reconstruct the full hematocrit–viscosity curve using only three-dataset Krieger–Dougherty (K–D) regression as μ=μ0(1-ϕϕm)-α ϕm. Based on suspended blood, RBC-rich blood and RBC-depleted blood are prepared after centrifugation. Hematocrit of each blood is measured using a micro hemocytometer. Simultaneously, blood viscosity of each blood is measured using coflowing streams method. The proposed method is evaluated sequentially using reference datasets and hematocrit-viscosity datasets of control blood. According to results, full hematocrit–viscosity curve obtained from selected three datasets is in well agreement with the experimental data and yields lower root-mean-square error than conventional method using all datasets. The exponent of K–D model is strongly influenced by the midpoint dataset whereas μ0 is mainly affected by suspending medium (dextran solution). In contrast, GA-induced rigidified RBCs do not significantly affect μ0. In conclusion, the proposed method provides simple, efficient, and reliable approach for estimating the full hematocrit–viscosity curve.

Abstract: Non‑thermal millimeter‑wave (MMW) irradiation (75–110 GHz) represents a promising non‑invasive strategy for cancer therapy. Lung cancer remains the leading cause ‎of cancer‑related mortality worldwide, highlighting the need for alternative therapeutic modalities that can overcome resistance and minimize toxicity. Yet the effects ‎of MMW exposure in physiologically relevant 3D systems remain insufficiently ‎characterized. Here, we evaluated the anti‑cancer efficacy of MMW exposure in 3D ‎lung cancer spheroids (NCI‑H1299, A549) alongside noncancerous WI‑38 fibroblasts. ‎Cells were irradiated using two antenna types—a waveguide (WG; localized, ‎high-power density) and a pyramidal horn (PH; broader coverage, lower power ‎density)—with or without a frequency multiplier to modulate local energy delivery. ‎Acute responses were assessed by XTT viability assays (day 2) and apoptosis (flow ‎cytometry), while chronic effects were evaluated using clonogenic survival (day 10) ‎and senescence markers. ‎ MMW exposure reduced cancer cell survival in a time‑ and power‑dependent manner ‎and induced sustained growth inhibition. Apoptosis was markedly higher in cancer ‎cells than in non‑cancerous WI‑38 cells and was further amplified under power-enhanced conditions. WG irradiation produced strong localized antiproliferative ‎effects, whereas the PH antenna enabled broader coverage while maintaining selective cytotoxicity toward NCI‑H1299 cells. Notably, p53‑deficient NCI‑H1299 cells ‎exhibited up to ~64% apoptosis after 60 min of exposure, whereas WI‑38 fibroblasts ‎remained below ~20%, demonstrating robust cancer selectivity. These findings high-‎light the selective, non‑thermal anticancer potential of MMW irradiation in 3D tumor ‎models and provide a mechanistic and experimental foundation for further preclinical ‎optimization of MMW‑based therapeutic strategies.

Abstract: This article addresses a current point of contention in the field of single molecule/single particle tracking, as well as relevant literature, and supplements it with some published cell-based experiments to illustrate our conclusions and known theorems. We attempt to explain the controversy surrounding the differing biophysical and cell biological results of studies on the individual molecule and those “at the single-molecule level” as well as at the level of many molecules in such a way that even readers who are unfamiliar with the subject can understand it without having to read all the mathematical, physical, and biophysical references. Given this abundance of studies in the literature, it is obvious that genuine single-molecule studies are urgently needed, i.e., single-molecule studies that focus on increasing the sensitivity of the temporal resolution of single-molecule measurements and not just on spatial resolution.

Abstract: One of the most puzzling and unsolved challenges in molecular biology is understanding how proteins fold. Despite having advanced predictive tools that can accurately estimate the native structures of proteins, we still lack a comprehensive model that explains how amino acid sequences dictate folding pathways and trajectories. This manuscript introduces a novel treatment for the issue by employing the “principle of least action.” This approach enables us to explore an intriguing question: how does a protein achieve its native state at a constant folding rate and within a biologically plausible time frame? A response to this inquiry will help us understand why proteins must fold along specific pathways and identify the boundary conditions that limit their availability. Furthermore, the principle of least action—together with the effective trajectory conjecture—enables us to explain why different proteins could exhibit the same folding rate. Finally, it will enable us to provide an in-depth description of the genesis and solution of Levinthal's paradox. Our results are expected to pave the way for a more profound understanding of how proteins fold, shedding light on how the amino acid sequence and its surrounding environment encode the protein's folding pathways and, consequently, the protein's three-dimensional structure.

Abstract: Current efforts in improving photodynamic therapy focus on nanomaterials that integrate deep-tissue imaging with efficient reactive oxygen species generation. Gold nanoclusters (Au NCs) are promising alternatives to conventional photosensitizers due to their effective ROS production and enhanced biocompatibility when stabilized by protein corona. However, both photosensitizers and Au NCs are typically activated by ultraviolet or visible light, which cannot penetrate deeper into tissues and is limited to superficial applications. Here, we report a near-infrared (NIR)-activated photodynamic nanoplatform based on core-shell upconverting nanoparticles (UCNPs; NaGdF₄:Yb³⁺,Er³⁺@NaGdF₄:Yb³⁺,Nd³⁺), functionalized with a protein corona containing bovine serum albumin-stabilized Au NCs (BSA-Au NCs) and photosensitizer chlorin e6 (Ce6). Spectroscopic data confirmed the formation of the UCNP-BSA-Au-Ce6 nanoplatform and demonstrated 32% energy transfer efficiency from UCNPs to Ce6, resulting in efficient reactive oxygen species generation under 808 nm irradiation. Cellular experiments confirmed effective internalization and optimal biocompatibility of the nanoplatform in human breast cancer and healthy cells. Upon 808 nm irradiation, the nanoplatform significantly reduced viability of MDA-MB-231 cancer cells. These findings indicate that the UCNP-BSA-Au-Ce6 nanoplatform couples NIR activation with enhanced singlet oxygen production, providing a multifunctional platform for deep-tissue imaging and NIR-activated photodynamic therapy.

Abstract: The method of X-ray Footprinting and Mass Spectrometry (XFMS) using high brightness synchrotron X-ray sources has become an established method in structural biology and is based on the radiolytic production of hydroxyl radicals which oxidatively modify protein sidechains. While other methods of producing hydroxyl radicals are available, one benefit of using high flux density sources is that hydroxyl radical scavenging reactions can be minimized, and exposure times kept short to minimize secondary reactions. Here we present an application of the XFMS method using low dose rate X-rays from a commercial instrument. We demonstrate the feasibility of the approach using short peptides, characterizing the oxidative modifications +14, +16, and +32 Da under both aerated and low-oxygen conditions, and we additionally quantify the hydrogen peroxide production for various doses using the low dose rate source. These results provide fundamental information on the oxidative damage to peptides due to hydroxyl radicals using a low dose rate X-ray source.

Abstract: Biological systems display phenomena—particularly in enzymatic catalysis, excitonic coherence, and protein folding—that appear to exploit selective stabilisation of microstates beyond what standard quantum mechanics typically predicts for warm, noisy environments. We propose that these deviations can be interpreted as signatures of an informational reservoir: a hidden, aperiodic layer of structured information accessible only to sufficiently complex biological systems. Standard quantum mechanics then emerges as a limiting, coarse‑grained description in which the reservoir term vanishes. The proposed reservoir is not reducible to any finite set of underlying parameters; instead, it functions as a high‑complexity information landscape that can be “read” only by finely organised biomolecular architectures. We outline empirically testable predictions and discuss implications for biological stability, functional directionality, and the physical foundations of living systems.

Abstract: Is an ant colony conscious? What about a group of people talking, a cloud-hosted language model, or even a galaxy? Can a conscious mind only get so big? Does consciousness depend only on what is computed, or when and where? I see two possibilities affecting the answers to these questions. I name them Chord and Arpeggio, and formalize the distinction mathematically. If the ingredients of a subjective experience must be simultaneously true at one objective instant and causally exchange influence within a time window θ, then the system diameter D satisfies D ≤ κvθ, where v is the signal speed ceiling and κ depends on exchange architecture. I call this requirement Chord, because it is like a musical chord whose notes sound together. The alternative is Arpeggio. It asks only that each ingredient occur somewhere in the window. I prove that Arpeggio is strictly weaker than Chord, and that architectures with limited concurrency can satisfy Arpeggio while structurally forbidding Chord. I argue for Chord on formal, neuroscientific, and architectural grounds. A mechanistic model confirms a fragmentation transition at the theoretical threshold. I examine primate corpus callosum data to estimate empirical lower bounds on θ. I provide case studies showing that under Chord, ant colonies and human populations are ruled out as single conscious entities, cloud-hosted AI is constrained by co-instantiation rather than diameter, and brain-computer interface hybrids face latency-dependent limits. A mind can only get so big. Arpeggio is far more permissive, implying consciousness seemingly everywhere.

Abstract: Background: Tumor microenvironments (TMEs) frequently exhibit extracellular acidity (pH ~6.5), a biophysical feature known to play a critical role in cellular behavior, tumor progression, immune suppression, and altered therapeutic response. While synthetic regulatory circuits capable of sensing acidity have been proposed, quantitative frameworks describing how microenvironmental pH dynamics interact with tumor–immune systems remain limited. Methods: We developed a computational modeling framework describing acidity-mediated regulatory dynamics in coupled tumor–immune systems. The model integrates interacting processes including tumor population dynamics, effector T-cell activity under acid-dependent suppression, regulatory vector dynamics, pH-responsive promoter activation, buffering or alkalinization mechanisms, cytokine-mediated feedback, and proton concentration kinetics calibrated to physiological pH ranges (6.0–7.4). Alternative acidity-modulating strategies, including substrate-dependent and substrate-independent buffering mechanisms, were examined through parameter sweeps, sensitivity analysis, and spatial reaction–diffusion extensions. System behavior was analyzed using stability and regime characterization methods. Results: The model exhibits distinct dynamical regimes in which acidity modulation reshapes tumor–immune interactions. Simulation of the acidity-responsive regulatory module demonstrated that promoter-driven therapeutic activation reduces tumor burden through two mechanistically distinct pathways. The alkalinization strategy elevated steady-state pH (ΔpH ≈ 0.2–0.6), partially restoring immune activity and reducing tumor persistence via microenvironmental feedback. In contrast, immune reactivation enhanced cytotoxic pressure directly, producing more rapid tumor suppression without substantially normalizing extracellular pH. In both architectures, therapeutic output increased under acidic conditions and diminished as pH approached physiologic levels, demonstrating dynamically coupled and self-limiting behavior. Sensitivity and scaling analyses further revealed hierarchical parameter control and architectural differences between substrate-dependent and substrate-independent buffering mechanisms. Conclusions: This study provides a quantitative theoretical framework for understanding how microenvironmental acidity functions as a regulatory variable in tumor–immune dynamics. The results highlight generalizable principles governing acidity-mediated feedback, system stability, and scaling behavior, offering mechanistic insights relevant to microenvironment-responsive regulatory systems. These findings emphasize the importance of biophysical microenvironmental factors in shaping cellular system dynamics and provide a basis for future experimental investigation of acidity-responsive biological regulation.

Abstract: Neuronal excitability manifests itself mainly in the form of non-linear, self-regenerative waves of electricity moving along the surface of neuronal axons. These waves are commonly known as action potentials (APs). Theorizing and experimental investigation of the physical and functional characteristics of APs has broadly followed along the lines of the ionic hypothesis and the associated mathematical model introduced by Hodgkin and Huxley (HH). In the current form of this bioelectrical framework, adopted in mainstream physiology and other biological sciences, the axonal membrane is conceptualized as an electronic circuit where electric current is generated and propelled as the result of time-dependent opening and closure of voltage-operated ion channel proteins allowing passive flow of specific ions across and along the membrane powered by their respective electrochemical gradients. Although representing mainstream research, the bioelectric perspective has been criticized for its narrow focus on electrical characteristics of APs, whilst ignoring other physical manifestations of the nerve signal, in particular mechanical and thermal changes coinciding with AP propagation. As an alternative, a thermodynamics-based acoustic theory has been outlined in which all, electric and non-electric, manifestations of the nerve signal are considered as the result of a single density pulse in the axonal membrane carried by a reversible lipid membrane phase transition and momentum conservation. Representing a minority view, however, this unified, thermodynamic perspective on the physical nature of neuronal excitability is largely ignored by representatives of the bioelectric perspective.Here we draw special attention to the philosophical dimension of the communication failure between the two communities of scientists. We argue that adherents of the bioelectric perspective favor a mechanist-type of explanation, whilst supporters of the thermodynamic perspective are committed to so-called covering-law types of explanation. We conclude that it is this, thus far unrecognized, philosophical rift, rather than specific scientific differences of opinion that blocks fruitful interdisciplinary cooperation necessary for building a comprehensive, fully integrated, notion of the physical nature of neuronal excitability. Suggestions of how to bridge this conceptual gap are formulated.

Abstract: Telmisartan (TEL) is a non-peptide, orally administered antihypertensive agent primarily known as angiotensin II type 1 (AT1) blocker. In this review, we provide a detailed overview of how TEL modulates voltage-gated Na⁺ current (I_Na) and affects action potential (AP) firing behavior. TEL exerts differential stimulatory effects on the peak and late components of I_Na when subjected to brief depolarizing pulses across a range of cell types, such as mHippoE-14 hippocampal neuron, cultured dorsal root ganglion neurons, and HL-1 atrial cardiomyocytes. TEL can augment the inactivating (persistent) I_Na elicited by ascending long ramp pulse in mHippoE-14 cells. By using a parvalbumin-expressing interneuron-based modeled cell combined with bifurcation analysis, it is possible to predict how applied current influences subthreshold oscillations and the generation of somatic spiking in the presence of TEL. According to the Hodgkin-Huxley model, mimicking the action of TEL—characterized by an increased peak amplitude of I_Na and a slowed inactivation time course—leads to the emergence of periodic oscillations in membrane potential. Using a Markovian process, a separate model can also be mathematically constructed, showing that changes in certain rate constants can simulate the effect of TEL on I_Na in cardiac cells. The molecular docking prediction between TEL and the NaV1.7 channel was made by expected formation of hydrophobic interactions as well as hydrogen bonding. Beyond its antagonistic action on AT1 receptor and agonistic activation of peroxisome proliferator-activator-γ, the direct stimulation of I_Na may also contribute to its modulation of AP firing in various excitable cells. Current evidence supports TEL’s modulatory impact on Na_V channel activity and cellular excitability, while also acknowledging that the mechanism—whether direct or indirect—remains under investigation.

Abstract: Background: Intraoperative radiotherapy with low-energy X-rays (IOXRT) is an increasingly utilized modality during breast conserving therapy (BCT). However, the molecular mechanisms by which it affects the postoperative microenvironment remain to be fully elucidated. Surgical wound fluid (WF) has been demonstrated to modulate cancer cell behavior; however, its metabolomic composition has not been previously characterized in the context of breast cancer. The objective of this study was to evaluate metabolic alterations in postoperative WF and to determine whether IOXRT induces distinct metabolic signatures compared with mastectomy (AMP).Methods: Postoperative WF was collected from 54 breast cancer patients (38 BCT IOXRT; 16 AMP) at two time points: day 1 (A) and day 5 (B) after surgery. The samples were then subjected to analysis using ¹H NMR spectroscopy, encompassing NOESY, CPMG, and JRES techniques. A total of 114 spectral signals were quantified, and 42 metabolites were identified. Multivariate analyses (PCA, PLS DA, OPLS DA) and Wilcoxon signed rank tests were applied to assess temporal and intergroup differences.Results: A clear metabolic separation between time points A and B was observed in both treatment groups. However, statistical analysis revealed no significant differences between BCT IOXRT and AMP. In BCT IOXRT, on the fifth day, WF exhibited a decline in branched chain amino acids, asparagine, lysine, methionine, and glutamate, concomitant with an increase in lactate and pyruvate. AMP-specific alterations encompassed a decrease in 2-oxoglutarate and hypoxanthine on the first day, along with an increase in glucose and creatinine on the fifth day. A decline in ketone bodies (3-hydroxybutyrate, acetoacetate, acetone) was observed in both groups.Conclusions: Postoperative WF demonstrates dynamic metabolic changes reflecting early wound healing processes and treatment-related effects. IOXRT has been found to be associated with enhanced glycolytic signatures and reduced amino acid levels, suggesting altered metabolic activity in the irradiated tumor bed. The metabolomic profiling of WF has the potential to offer a novel source of biomarkers, which could facilitate the assessment of treatment response and tumor microenvironment characteristics.

Abstract: Biological coherence arises from coordinated integration of redox chemistry, hydration dynamics, electromagnetic interactions, and bioenergetic flux. Although substantial progress has been made in characterizing these processes individually, current frameworks do not fully explain how distributed biochemical events achieve stable temporal coordination across scales. In thermally noisy, dissipative environments, energy alone cannot account for sustained biological organization. A missing element is the establishment and renewal of phase reference - the temporal alignment that enables spatially distributed processes to act in synchrony. Here we propose a physical mechanism for phase reference access and anchoring based on cyclic nanodomain dynamics at a nanoscale redox-photonic interface previously termed the Redox Photonic Coupling System (RPCS). This interface supports an additional functional modality - phase breathing - a process mediated by molecular nitrogen (N₂) through which cyclic nanodomain nucleation and collapse anchors and sustains phase reference in living systems. Nitrogen-mediated oscillatory boundary dynamics create transient coherence windows that permit local access to phase reference, enabling phase-aligned oxidative-reductive resolution and anchoring of phase onto redox-generated Photonic Activation Quanta (PAQs). Absorption of phase-conditioned PAQs by adjacent hydration shells enables generation and accumulation of centropy, defined as stored organizational capacity that supports coordinated biological work.This framework identifies phase breathing as a previously unrecognized mechanism sustaining biological coherence and assigns molecular nitrogen a structural organizational role beyond respiratory dilution. By integrating nanodomain mechanics, photonic phase conditioning, and redox dynamics within a single interface, it provides a mechanistic basis for how coherent biological function is generated and maintained.

Abstract: The relationship between the thin adsorbed water layer conventionally observed on hydrophilic surfaces and the much larger "exclusion zone" described in the literature has remained unclear. In this review, we survey the evidence for both phenomena and propose that they are intimately connected: the adsorbate constitutes a structurally distinct phase that generates a magnetic field, which in turn diamagnetically orders the surrounding water over much larger distances. This model reconciles the thin adsorbate with the much larger exclusion zone, and is consistent with available data, with broader implications for water’s magnetic properties.

Abstract: Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of these molecular assemblies remains an enduring biophysical challenge, particularly in cells, where heterogeneity is the rule rather than the exception. Orthogonal signals derived from fluorescence lifetime, fluorescence fluctuations, and fluorescence polarization provide valuable metrics for probing interactions and environments, concentration and size, as well as rotational dynamics, respectively. This paper combines fluorescence lifetime imaging microscopy with image correlation analysis and polarization to determine the concentrations, brightness, lifetime, and rotational correlation time of different fluorescent states. A two-population model is examined as a prototypical example of a heterogenous system. The analysis is illustrated on a simple fluorescence model system, where cluster densities, relative brightnesses, lifetimes and rotational correlation times are extracted.