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The light digital microscope can be used to visualize structures as small as ~1 micron

Total Internal Reflection Fluorescence Microscopy

 

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Introduction

Total internal reflection fluorescence microscopy (TIRFM) is a cutting-edge optical technique that excites the fluorophores in a fragile axial region to visualize the cellular events occurring close to the cell surface. The evanescent field, a near-field wave which decays in intensity over a sub-wavelength distance, is used as the basis for total internal reflection fluorescence microscopy (TIRFM). The evanescent field occurs when the incident light is totally reflected at the interface of two transparent media having different refractive indices. The TIRFM has a wide range of applications in visualizing the biological events and quantifying their kinetic rates. The total internal reflection fluorescence microscopy is used to study the protein-protein and protein-nucleic acid biochemical interactions. The total internal reflection fluorescence microscopy has made it easier to understand the mechanism and function of cellular components including signaling cascades, membrane proteins, and molecular motors. The relative ease of use of TIRF and the high sensitivity in single-molecule detection makes it an indispensable technique in biomedical science to tackle a wide array of cellular questions.

Principle

The total internal reflection fluorescence microscopy is based on the evanescent field, which exclusively illuminates a thin plane just below the glass coverslip. The evanescent field occurs as a result of the total internal reflection of the light rays at the interface of the imaging surface and an aqueous medium. The refractive index of the optical medium tells about the propagation of the electromagnetic waves through it relative to the propagation of the wave through the vacuum. When light rays traveling through one medium strike at the interface of another medium with a different refractive index, the subsequent direction of the light rays is changed depending on the angle at which the light meets the interface. The energy of the evanescent field decreases as it travels to the interface, only the fluorophores close to the coverslip are excited. This creates the images with an outstanding signal-to-noise ratio, as the rest of the fluorophores in the cell are hardly excited. Therefore, the membrane-associated processes like cell adhesion, molecule transport, hormone binding, and exocytotic and endocytotic processes are observed (Yildiz. & Vale., 2015).

Apparatus

The total internal reflection fluorescence microscopy consists of an objective lens, an excitation beam path which passes the light through the objective lens to the sample, and a coupling element arranged in the back focal plane of the objective lens. The coupling element consists of two areas; one for relaying light to the objective lens for total internal reflection illumination and the second for separating the light emitted by the sample and passing it through the excitation beam path in reverse direction. The laser beams are joined with a dichroic mirror and expanded via Gaussian beam expander. These laser beams are focused on the back focal plane of the objective with an achromatic doublet lens. A set of multiband dichroic and emission filters reflect the laser beams on the objective and transmit the fluorescence simultaneously. The fluorescence is then separated by a Dual View instrument which is equipped with a dichroic mirror to split the fluorescence, and band-pass emission filters to reduce the cross talk between the two fluorescence channels (Fish, 2009).

Protocol
Procedure for observing exocytosis in the endocrine cells (Trexler. & Taraska., 2017)

Coverslip preparation

  1. Place the coverslips in a ceramic or Teflon coverslip holder.
  2. Place the coverslip holder in the bottom of a 2-liter glass beaker and add 300 mL water in it.
  3. Add 60 mL of the 30 % hydrogen peroxide (H2O2) to the beaker and place it in a fume hood.
  4. Add 60 mL of 27 % ammonium hydroxide to the beaker. And place the beaker on a hotplate and turn it to high. Wait for 5 minutes and check for gentle bubbling in the beaker. After the gentle bubbling begins, incubate the coverslips for 15 minutes. After the incubation, make sure that the solution is vigorously bubbling at 80–90 °C.
  5. Remove the beaker from the hotplate. With the help of wire tongs, transfer the coverslip holder to a 2-liter beaker filled with 1 liter of water. Then transfer the coverslip holders to smaller containers of 100 % ethanol for long-term storage.
  6. Coat the coverslips according to the cell type. Remove the coverslips from ethanol inside the safety cabinet. Air-dry the coverslips and transfer one coverslip to each well of a six-well plate. Add 100–200 μL of poly-l-lysine (PLL) solution to each coverslip.
  7. Incubate them for 10 minutes at room temperature and then aspirate the PLL solution from the coverslip. Wash the coverslips twice with 2 mL media and then cover it with 2 mL media for cell addition. Rinse away the unbound PLL.

Cell culture and transfection

  1. Rinse the cells with Dulbecco’s phosphate buffered saline (DPBS). After rinsing the cells in DPBS, perform trypsinizing and pelleting, and resuspend the cells in media and add dropwise to coverslips in six-well plates.
  2. After plating, allow the cells to rest overnight in an incubator.
  3. Transfect the cells after incubation. Cells should be transfected with a vesicle cargo marker.
  4. Label the vesicles with suitable probes. If needed co-tag the vesicles with a second fluorescent probe.
  5. After transfection, allow the cells to rest overnight. Visualize the cells 1–2 days post-transfection.

Microscope and sample preparation

  1. Place a 25 mm coverslip in the coverslip chamber. Add the buffer on the coverslip and rub the coverslip with the forefinger to stick the beads to the glass. Add 500 μL of the imaging buffer to the coverslip followed by the addition of 5 μL diluted fluorescent beads. Place the bead-coated coverslip on the microscope.
  2. Focus the beads stuck to the coverslip by adjusting the microscope.
  3. Adjust the TIRF angle as needed for the shallow evanescent illumination. 

Stimulation and imaging

  1. Rinse the coverslip in imaging buffer for three times before placing them in the coverslip chamber.
  2. Place the coverslip in a coverslip chamber and cover the cells with 500 μL of imaging buffer and put on the microscope.
  3. Position the perfusion tip close to the focal plane of the cells.
  4. After positioning the perfusion tip, perfuse the cells with imaging buffer briefly.
  5. Move the stage in the x-y plane in all directions to ensure that the tip is not touching the coverslip or the cells.
  6. If required, add an aspirating pipette to the microscope stage insert, and position the tip just over the desired buffer level in the coverslip
  7. Turn on the fluorescent illumination and scan the coverslip for a cell suitable for imaging.
  8. Adjust imaging parameters to get the maximum intensity from the fluorescent channels.
  9. Perfuse the cells for 5 s.
  10. Acquire the images and rinse the coverslips with 3-5 ml of the imaging buffer to wash away the residual stimulation solution.
Protocol for live-cell imaging of vesicle trafficking (Loder., Tsuboi., & Rutter., 2013)

Cleaning and coating of dishes

  1. Place the glass-bottom dishes into a 500 mL beaker containing 300 mL of Milli-Q water and put the beaker in an ultrasound bath for 1 hour.
  2. Dip the dishes into another 500 mL beaker containing 300 mL of 70% ethanol for 1 hour.
  3. Take out the dishes from the beaker inside a cell culture clean bench, and sterilize them with the help of a UV lamp for 30 minutes.
  4. Apply 100 mL of poly-l-lysine (PLL) solution on each dish.
  5. After 30 minutes, wash the dishes with 1 ml sterile phosphate buffer solution for three times.

Cell transfection

  1. Culture the cells in a 10-cm Petri dish and incubate them in 5% CO 2 at 37°C.
  2. For TIRF microscopy, plate the cells in PLL-coated glass-bottom dishes (35 mm).
  3. Transfect the cells with 3 mg of NPY–Venus vector using the Lipofectamine.

Preparation of mouse β cells

  1. Isolate the pancreatic islets by collagenase digestion of the pancreas of female CD1 mice and select the cells by hand-picking.
  2. Culture the islets RPMI medium (10 mM glucose and 2 mM glutamine supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin) for 1 day.
  3. Incubate the cells and dissociate the islets in Ca2+ -free buffer or Trypsin ethylenediamine triacetic acid EDTA for 5 minutes.
  4. Replace the solution with RPMI medium, and disrupt the islets by pipetting through a glass pipette.
  5. Culture the cells for 1 day on PLL-coated glass-bottom dishes or coverslips.

Infection

Infect the cells with adenoviruses at the rate of 30–100 infectious particles per cells for 4 hours, and then change the culture medium.

Imaging of exocytosis

  1. Culture the cells in Krebs Ringer buffer (KRB) for 30 minutes at 37°C.
  2. Place the glass-bottom dishes to the thermostat-controlled heating stage (37°C) of the TIRF microscope.
  3. Locate the fluorescent NPY–Venus-expressing cells or infected dispersed primary β-cells.
  4. Adjust the incident angle of the laser to obtain total internal reflection.
  5. Focus the beam on the cell surface.
  6. Obtain the images at 30- to 300-ms intervals.
  7. Change the buffer from KRB to either high glucose containing KRB and continue imaging for 20 minutes.

 

Applications
Assessment of GLUT4 trafficking across the membrane (Wasserstrom., Morén., & Stenkula., 2018)

Insulin-responsive GLUT4 storage vesicles (GSV) helps in the glucose uptake by translocating to the cell surface. In the study, the total internal reflection fluorescence microscopy was used to understand the events involved in glucose uptake and insulin-regulated GLUT4 translocation in both cultured 3T3-L1 adipocytes and primary adipocytes isolated from the rodents and humans. The cells were prepared, transfected, and imaged under the TIRF microscope. It was found that the GSV traffic is decreased as the vesicles lead to the plasma membrane followed by fusion. It was observed that the insulin-induced GSV fusion is followed by the release of GLUT4 monomers into the plasma membrane. The total internal reflection fluorescence microscopy has been found as a powerful tool to observe the vesicle translocation across the plasma membrane.

Quantification of receptor pharmacology (Fang, 2015)

The total internal reflection fluorescence (TIRF) microscopy has been widely used to visualize single molecules in the cells to unveil fundamental aspects of cell biology as it selectively excites a very thin fluorescent volume close to the substrate on which the cells are grown. TIRFM has been used to track single receptors having a SNAP-tag, and to compare their arrangement, mobility, and supramolecular organization. The studies presented that the G-protein coupled receptors (GPCRs) possess varying degrees of di-/oligomerization. Whereas β1- or β2-Andregenic receptors are freely diffusive on the cell surface. These results suggest that GPCRs are located on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be stimulated or targeted, in a ligand-regulated manner, to different cell-surface micro-domains. The total internal reflection fluorescence microscopy has become a promising technique in profiling the receptor pharmacology in clinical applications. 

Visualizing the cell-substrate contact regions (Thompson., Pearce., & Hsieh., 1993)

Cell-substrate contact regions demonstration is one of the applications of total internal reflection fluorescence microscopy. The evanescent excitation has been used to image the arrangement of fluorescent probes for different membrane components in cell-substrate contact regions. The fluorescent reporters attached to cytoskeletal elements are visualized in rat myotube membranes adjacent to glass substrates. TIR-FPPR was used to probe the lateral mobility of fluorescent antibodies which linked the rat basophil leukemia cells to supported planar membranes. It was found that the fluorescence is intense if the cell-to-substrate distance is large and is weak if the distance is small. The TIRFM has also been used to measure the spatial distribution of fluorescence intensities that provides a two-dimensional map of cell-to-substrate contact distances and the binding kinetics of the cell-substrate contact.

Live-cell imaging of the estrogen receptor (Kisler. & Dominguez., 2016)

The total internal reflection fluorescence microscopy has been used to visualize the trafficking of plasma membrane-localized intracellular estrogen receptors following estradiol stimulation in living cells. To visualize estrogen receptor trafficking N-38 neurons were used as a model for membrane-initiated estradiol signaling. The TIRFM permits observation of live, intact cells while allowing visualization of the receptor activation cascade following estradiol activation. The TIRFM yielded high-contrast real-time images of fluorescently labeled E6BSA molecules on and just below the cell surface and was found a powerful tool for studying estrogen receptor trafficking in living cells.

Evaluation of intracellular signaling (Mattheyses., Simon., & Rappoport., 2010)

The total internal reflection fluorescence microscopy has also been used to demonstrate different steps of intracellular signaling. The TIRF has been instrumental in delineating plasma membrane recruitment and spatial distributions of signaling molecules. The plasma-membrane-targeted biosensor enabled the imaging of temporal oscillations of cAMP (cyclic Adenosine monophosphate) signaling, instigating the research in the regulation of upstream targets. The single plasma membrane Ca2+ channels have also been imaged with spatial and temporal resolution revealing uneven molecular kinetics. The TIRF and patch-clamp methods have successfully demonstrated the localization and signaling of open calcium channels and calcium-sensing molecules, explaining the spatial dynamics of intracellular calcium signaling.

Precautions
  • The cells used for TIRF microscopy must be adherent because TIRF illuminates only the region close to the coverslip and cannot be used to image non-adherent cells.
  • It is essential to coat the coverslips with extracellular matrix molecules to ensure cell adherence.
  • If fixed cells are used, then they must be mounted in a low refractive index media.
  • Maintain the live cells at 37°C as the temperature gradients can lead to focal drift.
  • Keep the tagged samples in the dark.
  • Carefully adjust the incident angle to produce the desired effects.

 

Strengths and limitations
  • The total internal reflection fluorescence microscopy provides the imaging of even 100-nanometer sections.
  • The TIRFM allows restricted illumination which is best suited for the visualization of membrane receptors and events.
  • The TIRFM is much more economical because the technique does not require complex scanning galvanometer systems.
  • The total internal reflection fluorescence microscopy is a powerful technique used for the imaging of cellular events occurring close to the plasma membrane.
  • The total internal reflection fluorescence microscopy is limited to analyze small samples of 10-300 nm sections only.
  • The TIRFM is limited to the specimen regions having an appropriate refractive index.

 

References
  1. Trexler., & Taraska., J. W. (2017). Two-Color Total Internal Reflection Fluorescence Microscopy of Exocytosis in Endocrine Cells. Methods Mol Biol, 1563, 151-165.
  2. Mattheyses., S. M. Simon., & Rappoport., J. Z. (2010). Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci, 3621-8.
  3. , & Vale., R. D. (2015). Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc, 9.
  4. Fang, Y. (2015). Total Internal Reflection Fluorescence Quantification of Receptor Pharmacology. Biosensors (Basel)., 5(2), 223-40.
  5. Fish, K. N. (2009). Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr Protoc Cytom.
  6. Kisler., & Dominguez., R. (2016). Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy. Methods Mol Biol, 1366, 175-187.
  7. K. Loder., T. Tsuboi., & Rutter., G. A. (2013). Live-cell imaging of vesicle trafficking and divalent metal ions by total internal reflection fluorescence (TIRF) microscopy. Methods Mol Biol, 950, 13-26.
  8. L. Thompson., K. H. Pearce., & Hsieh., H. V. (1993). Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes. Eur Biophys J, 22(5), 367-78.
  9. Wasserstrom., B. Morén., & Stenkula., K. G. (2018). Total Internal Reflection Fluorescence Microscopy to Study GLUT4 Trafficking. Methods Mol Biol, 1713, 151-159.

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Introduction

In behavioral neuroscience, the Open Field Test (OFT) remains one of the most widely used assays to evaluate rodent models of affect, cognition, and motivation. It provides a non-invasive framework for examining how animals respond to novelty, stress, and pharmacological or environmental manipulations. Among the test’s core metrics, the percentage of time spent in the center zone offers a uniquely normalized and sensitive measure of an animal’s emotional reactivity and willingness to engage with a potentially risky environment.

This metric is calculated as the proportion of time spent in the central area of the arena—typically the inner 25%—relative to the entire session duration. By normalizing this value, researchers gain a behaviorally informative variable that is resilient to fluctuations in session length or overall movement levels. This makes it especially valuable in comparative analyses, longitudinal monitoring, and cross-model validation.

Unlike raw center duration, which can be affected by trial design inconsistencies, the percentage-based measure enables clearer comparisons across animals, treatments, and conditions. It plays a key role in identifying trait anxiety, avoidance behavior, risk-taking tendencies, and environmental adaptation, making it indispensable in both basic and translational research contexts.

Whereas simple center duration provides absolute time, the percentage-based metric introduces greater interpretability and reproducibility, especially when comparing different animal models, treatment conditions, or experimental setups. It is particularly effective for quantifying avoidance behaviors, risk assessment strategies, and trait anxiety profiles in both acute and longitudinal designs.

What Does Percentage of Time in the Centre Measure?

This metric reflects the relative amount of time an animal chooses to spend in the open, exposed portion of the arena—typically defined as the inner 25% of a square or circular enclosure. Because rodents innately prefer the periphery (thigmotaxis), time in the center is inversely associated with anxiety-like behavior. As such, this percentage is considered a sensitive, normalized index of:

  • Exploratory drive vs. risk aversion: High center time reflects an animal’s willingness to engage with uncertain or exposed environments, often indicative of lower anxiety and a stronger intrinsic drive to explore. These animals are more likely to exhibit flexible, information-gathering behaviors. On the other hand, animals that spend little time in the center display a strong bias toward the safety of the perimeter, indicative of a defensive behavioral state or trait-level risk aversion. This dichotomy helps distinguish adaptive exploration from fear-driven avoidance.

  • Emotional reactivity: Fluctuations in center time percentage serve as a sensitive behavioral proxy for changes in emotional state. In stress-prone or trauma-exposed animals, decreased center engagement may reflect hypervigilance or fear generalization, while a sudden increase might indicate emotional blunting or impaired threat appraisal. The metric is also responsive to acute stressors, environmental perturbations, or pharmacological interventions that impact affective regulation.

  • Behavioral confidence and adaptation: Repeated exposure to the same environment typically leads to reduced novelty-induced anxiety and increased behavioral flexibility. A rising trend in center time percentage across trials suggests successful habituation, reduced threat perception, and greater confidence in navigating open spaces. Conversely, a stable or declining trend may indicate behavioral rigidity or chronic stress effects.

  • Pharmacological or genetic modulation: The percentage of time in the center is widely used to evaluate the effects of pharmacological treatments and genetic modifications that influence anxiety-related circuits. Anxiolytic agents—including benzodiazepines, SSRIs, and cannabinoid agonists—reliably increase center occupancy, providing a robust behavioral endpoint in preclinical drug trials. Similarly, genetic models targeting serotonin receptors, GABAergic tone, or HPA axis function often show distinct patterns of center preference, offering translational insights into psychiatric vulnerability and resilience.

Critically, because this metric is normalized by session duration, it accommodates variability in activity levels or testing conditions. This makes it especially suitable for comparing across individuals, treatment groups, or timepoints in longitudinal studies.

A high percentage of center time indicates reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance. reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance.

Behavioral Significance and Neuroscientific Context

1. Emotional State and Trait Anxiety

The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity, where animals exhibit persistent avoidance of the center due to heightened emotional reactivity. This metric can also distinguish between acute anxiety responses and enduring trait anxiety, especially in longitudinal or developmental studies. Its normalized nature makes it ideal for comparing across cohorts with variable locomotor profiles, helping researchers detect true affective changes rather than activity-based confounds.

2. Exploration Strategies and Cognitive Engagement

Rodents that spend more time in the center zone typically exhibit broader and more flexible exploration strategies. This behavior reflects not only reduced anxiety but also cognitive engagement and environmental curiosity. High center percentage is associated with robust spatial learning, attentional scanning, and memory encoding functions, supported by coordinated activation in the prefrontal cortex, hippocampus, and basal forebrain. In contrast, reduced center engagement may signal spatial rigidity, attentional narrowing, or cognitive withdrawal, particularly in models of neurodegeneration or aging.

3. Pharmacological Responsiveness

The open field test remains one of the most widely accepted platforms for testing anxiolytic and psychotropic drugs. The percentage of center time reliably increases following administration of anxiolytic agents such as benzodiazepines, SSRIs, and GABA-A receptor agonists. This metric serves as a sensitive and reproducible endpoint in preclinical dose-finding studies, mechanistic pharmacology, and compound screening pipelines. It also aids in differentiating true anxiolytic effects from sedation or motor suppression by integrating with other behavioral parameters like distance traveled and entry count (Prut & Belzung, 2003).

4. Sex Differences and Hormonal Modulation

Sex-based differences in emotional regulation often manifest in open field behavior, with female rodents generally exhibiting higher variability in center zone metrics due to hormonal cycling. For example, estrogen has been shown to facilitate exploratory behavior and increase center occupancy, while progesterone and stress-induced corticosterone often reduce it. Studies involving gonadectomy, hormone replacement, or sex-specific genetic knockouts use this metric to quantify the impact of endocrine factors on anxiety and exploratory behavior. As such, it remains a vital tool for dissecting sex-dependent neurobehavioral dynamics.
 The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity. Because it is normalized, this metric is especially helpful for distinguishing between genuine avoidance and low general activity.

Methodological Considerations

  • Zone Definition: Accurately defining the center zone is critical for reliable and reproducible data. In most open field arenas, the center zone constitutes approximately 25% of the total area, centrally located and evenly distanced from the walls. Software-based segmentation tools enhance precision and ensure consistency across trials and experiments. Deviations in zone parameters—whether due to arena geometry or tracking inconsistencies—can result in skewed data, especially when calculating percentages.

     

  • Trial Duration: Trials typically last between 5 to 10 minutes. The percentage of time in the center must be normalized to total trial duration to maintain comparability across animals and experimental groups. Longer trials may lead to fatigue, boredom, or habituation effects that artificially reduce exploratory behavior, while overly short trials may not capture full behavioral repertoires or response to novel stimuli.

     

  • Handling and Habituation: Variability in pre-test handling can introduce confounds, particularly through stress-induced hypoactivity or hyperactivity. Standardized handling routines—including gentle, consistent human interaction in the days leading up to testing—reduce variability. Habituation to the testing room and apparatus prior to data collection helps animals engage in more representative exploratory behavior, minimizing novelty-induced freezing or erratic movement.

     

  • Tracking Accuracy: High-resolution tracking systems should be validated for accurate, real-time detection of full-body center entries and sustained occupancy. The system should distinguish between full zone occupancy and transient overlaps or partial body entries that do not reflect true exploratory behavior. Poor tracking fidelity or lag can produce significant measurement error in percentage calculations.

     

  • Environmental Control: Uniformity in environmental conditions is essential. Lighting should be evenly diffused to avoid shadow bias, and noise should be minimized to prevent stress-induced variability. The arena must be cleaned between trials using odor-neutral solutions to eliminate scent trails or pheromone cues that may affect zone preference. Any variation in these conditions can introduce systematic bias in center zone behavior. Use consistent definitions of the center zone (commonly 25% of total area) to allow valid comparisons. Software-based segmentation enhances spatial precision.

Interpretation with Complementary Metrics

Temporal Dynamics of Center Occupancy

Evaluating how center time evolves across the duration of a session—divided into early, middle, and late thirds—provides insight into behavioral transitions and adaptive responses. Animals may begin by avoiding the center, only to gradually increase center time as they habituate to the environment. Conversely, persistently low center time across the session can signal prolonged anxiety, fear generalization, or a trait-like avoidance phenotype.

Cross-Paradigm Correlation

To validate the significance of center time percentage, it should be examined alongside results from other anxiety-related tests such as the Elevated Plus Maze, Light-Dark Box, or Novelty Suppressed Feeding. Concordance across paradigms supports the reliability of center time as a trait marker, while discordance may indicate task-specific reactivity or behavioral dissociation.

Behavioral Microstructure Analysis

When paired with high-resolution scoring of behavioral events such as rearing, grooming, defecation, or immobility, center time offers a richer view of the animal’s internal state. For example, an animal that spends substantial time in the center while grooming may be coping with mild stress, while another that remains immobile in the periphery may be experiencing more severe anxiety. Microstructure analysis aids in decoding the complexity behind spatial behavior.

Inter-individual Variability and Subgroup Classification

Animals naturally vary in their exploratory style. By analyzing percentage of center time across subjects, researchers can identify behavioral subgroups—such as consistently bold individuals who frequently explore the center versus cautious animals that remain along the periphery. These classifications can be used to examine predictors of drug response, resilience to stress, or vulnerability to neuropsychiatric disorders.

Machine Learning-Based Behavioral Clustering

In studies with large cohorts or multiple behavioral variables, machine learning techniques such as hierarchical clustering or principal component analysis can incorporate center time percentage to discover novel phenotypic groupings. These data-driven approaches help uncover latent dimensions of behavior that may not be visible through univariate analyses alone.

Total Distance Traveled

Total locomotion helps contextualize center time. Low percentage values in animals with minimal movement may reflect sedation or fatigue, while similar values in high-mobility subjects suggest deliberate avoidance. This metric helps distinguish emotional versus motor causes of low center engagement.

Number of Center Entries

This measure indicates how often the animal initiates exploration of the center zone. When combined with percentage of time, it differentiates between frequent but brief visits (indicative of anxiety or impulsivity) versus fewer but sustained center engagements (suggesting comfort and behavioral confidence).

Latency to First Center Entry

The delay before the first center entry reflects initial threat appraisal. Longer latencies may be associated with heightened fear or low motivation, while shorter latencies are typically linked to exploratory drive or low anxiety.

Thigmotaxis Time

Time spent hugging the walls offers a spatial counterbalance to center metrics. High thigmotaxis and low center time jointly support an interpretation of strong avoidance behavior. This inverse relationship helps triangulate affective and motivational states.

Applications in Translational Research

  • Drug Discovery: The percentage of center time is a key behavioral endpoint in the development and screening of anxiolytic, antidepressant, and antipsychotic medications. Its sensitivity to pharmacological modulation makes it particularly valuable in dose-response assessments and in distinguishing therapeutic effects from sedative or locomotor confounds. Repeated trials can also help assess drug tolerance and chronic efficacy over time.
  • Genetic and Neurodevelopmental Modeling: In transgenic and knockout models, altered center percentage provides a behavioral signature of neurodevelopmental abnormalities. This is particularly relevant in the study of autism spectrum disorders, ADHD, fragile X syndrome, and schizophrenia, where subjects often exhibit heightened anxiety, reduced flexibility, or altered environmental engagement.
  • Hormonal and Sex-Based Research: The metric is highly responsive to hormonal fluctuations, including estrous cycle phases, gonadectomy, and hormone replacement therapies. It supports investigations into sex differences in stress reactivity and the behavioral consequences of endocrine disorders or interventions.
  • Environmental Enrichment and Deprivation: Housing conditions significantly influence anxiety-like behavior and exploratory motivation. Animals raised in enriched environments typically show increased center time, indicative of reduced stress and greater behavioral plasticity. Conversely, socially isolated or stimulus-deprived animals often show strong center avoidance.
  • Behavioral Biomarker Development: As a robust and reproducible readout, center time percentage can serve as a behavioral biomarker in longitudinal and interventional studies. It is increasingly used to identify early signs of affective dysregulation or to track the efficacy of neuromodulatory treatments such as optogenetics, chemogenetics, or deep brain stimulation.
  • Personalized Preclinical Models: This measure supports behavioral stratification, allowing researchers to identify high-anxiety or low-anxiety phenotypes before treatment. This enables within-group comparisons and enhances statistical power by accounting for pre-existing behavioral variation. Used to screen anxiolytic agents and distinguish between compounds with sedative vs. anxiolytic profiles.

Enhancing Research Outcomes with Percentage-Based Analysis

By expressing center zone activity as a proportion of total trial time, researchers gain a metric that is resistant to session variability and more readily comparable across time, treatment, and model conditions. This normalized measure enhances reproducibility and statistical power, particularly in multi-cohort or cross-laboratory designs.

For experimental designs aimed at assessing anxiety, exploratory strategy, or affective state, the percentage of time spent in the center offers one of the most robust and interpretable measures available in the Open Field Test.

Explore high-resolution tracking solutions and open field platforms at
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References

  • Prut, L., & Belzung, C. (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. European Journal of Pharmacology, 463(1–3), 3–33.
  • Seibenhener, M. L., & Wooten, M. C. (2015). Use of the open field maze to measure locomotor and anxiety-like behavior in mice. Journal of Visualized Experiments, (96), e52434.
  • Crawley, J. N. (2007). What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. Wiley-Liss.
  • Carola, V., D’Olimpio, F., Brunamonti, E., Mangia, F., & Renzi, P. (2002). Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behavior in inbred mice. Behavioral Brain Research, 134(1–2), 49–57.

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