High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
March 26th, 2018
9.6K views
Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.
Overview
This article presents a novel fluorescent exclusion method for the full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution. The technique is essential for analyzing neurites and dynamic structures associated with neuronal growth, allowing for a deep understanding of cellular physiology and pathology.
Key Study Components
Area of Science
- Cell Biology
- Neuroscience
- Neurological Research
Background
- Neuronal volume is critical for understanding cellular characteristics.
- Volume changes at the subcellular scale are important during physiological processes.
- The method aims to improve resolution in monitoring neurite dynamics.
- It addresses the need for high-precision volume measurements in neuronal studies.
Purpose of Study
- To enable full-field imaging of neuronal volume with high resolution.
- To facilitate the analysis of neurites and their structural dynamics.
- To provide insights into neuronal growth and degeneration processes.
Methods Used
- This study utilized a PDMS microfluidic chip for volume measurements.
- The biological model mainly focuses on neurons in culture for detailed observation.
- No multiomics workflows were employed in this study.
- The method involves careful fabrication and assembly of the microfluidic device, followed by imaging under an epifluorescence microscope.
- Important steps include coating, bonding, and cell seeding processes.
Main Results
- The method provides submicrometric axial resolution, essential for observing neuronal structures.
- Dynamic changes of neurites were effectively monitored over time.
- High-resolution imaging revealed nuanced details of neuronal growth cones and structural shifts.
- The approach enables precise volume computations of neurite slices.
Conclusions
- The study demonstrates a robust technique for high-resolution neuronal volume measurements.
- This approach is pivotal for advancing knowledge of neuronal mechanisms and plasticity.
- It has significant implications for future research on cellular dynamics and neurobiology.
Frequently Asked Questions
What are the advantages of using a PDMS microfluidic chip?
The PDMS chip allows for precise control of fluid dynamics and excellent optical properties for imaging. Its flexibility also facilitates the bonding process for the assembly.
How is the neuronal model implemented in this study?
Neurons are cultured within the microfluidic chip, enabling direct observation of their growth and structural changes over time.
What kind of data do you obtain from this method?
The method allows for imaging and quantifying the volume changes of neuronal structures, particularly neurites and growth cones, under various conditions.
How can this method be adapted for other studies?
The technique can be tailored for different cell types and applications by modifying the culture conditions or the microfluidic chip design.
Are there any limitations to this technique?
The method requires specialized equipment and skilled handling during fabrication, which may limit accessibility for some labs.
What types of imaging are performed during this procedure?
The procedure involves using an epifluorescence microscope to capture high-resolution images of the cells at specific time points, allowing for dynamic analysis.
Executive Industry Relevance
Accurate quantification of neuronal volume dynamics is critical for evaluating neuroactive compounds in early discovery, particularly for assessing neurite integrity and growth cone behavior. The Fluorescence eXclusion method (FXm) combined with microfluidic patterning provides sub-micrometric axial resolution and full-field imaging, enabling mechanistic de-risking of compounds targeting neuronal structural plasticity. This approach supports predictive confidence in lead identification by linking subcellular volume changes to functional outcomes in disease-relevant in vitro models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by measuring local volume changes in neurites and growth cones as functional readouts of neuronal health.
- Operational Value: Provides quantitative, reproducible morphological data to de-risk targets involved in neurite degeneration or regeneration pathways.
Screening & Assay Development
- Scientific Value: Generates standardized, high-resolution volumetric readouts suitable for assay miniaturization and compound screening in microfluidic formats.
- Operational Value: Supports assay readiness through precise, label-free volume measurements that reduce variability in compound response assessments.
Translational & Preclinical Research
- Scientific Value: Offers disease-relevant structural metrics that bridge in vitro findings to preclinical models of neuronal injury or neurodegeneration.
- Operational Value: Facilitates longitudinal tracking of neurite dynamics, supporting risk-adjusted advancement decisions in preclinical pipelines.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by providing quantitative structural endpoints that inform lead optimization and preclinical continuity, particularly for compounds modulating neuronal morphology.
- Discovery Biology: Supports hypothesis testing of neurite integrity and growth cone dynamics through direct volumetric measurement of subcellular structures.
- Screening: Enables assay standardization via reproducible, full-field volume imaging with sub-micrometric resolution for compound evaluation.
- Analytics: Delivers quantitative volumetric outputs that allow comparison of compound-induced structural changes across experimental conditions.
- Translational Research: Connects early discovery observations to preclinical relevance by quantifying structural biomarkers of neuronal growth and degeneration.
- Enterprise Reuse: Establishes a reusable microfluidic imaging platform applicable across multiple neuronal models and compound classes.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence by reducing mechanistic ambiguity in neuronal structural responses to pharmacological perturbation.
- Operational Value: Ensures standardization and scalability through microfluidic device fabrication and epifluorescence-compatible imaging workflows.
- Strategic Value: Improves go/no-go decisions by providing early, morphologically grounded biomarkers of compound efficacy and neurotoxicity.
- Portfolio Impact: Enables risk-adjusted prioritization of compounds based on validated effects on neurite volume and growth cone dynamics.
Implementation Considerations
- Requires expertise in microfluidic fabrication, photolithography, and surface bonding techniques for device assembly.
- Depends on access to mask aligners, plasma treatment systems, and precision fluid handling equipment for chip preparation.
- Necessitates standardization of cell seeding density and imaging protocols across teams to ensure data comparability.
- Involves adaptation considerations for different neuronal subtypes or disease models via microfluidic geometry and surface patterning adjustments.
- Limited by the need for specialized cleanroom access and technical skill in PDMS handling and microfluidic integration, which may affect broad deployment.
Biopharma FAQs
Why does volumetric measurement of neurites matter for target validation?
Quantifying neurite volume changes provides a direct, structural readout of compound effects on neuronal integrity, enabling mechanistic de-risking of targets involved in neurite outgrowth or degeneration pathways.
How does isolating the independent variable (e.g., compound treatment) support discovery pipeline decisions?
By controlling compound exposure in microfluidic chambers and measuring resulting volume changes in neurites, researchers can attribute phenotypic responses specifically to the test agent, improving confidence in lead selection.
What do quantitative dependent variable measurements of neuronal volume enable in screening?
Precise volumetric readouts of neurites and growth cones allow for objective comparison of compound effects, supporting assay standardization and hit-to-lead progression based on structural biomarkers.
Why are replication requirements important for cross-functional collaboration in neuronal volume studies?
Consistent volumetric measurements across replicates ensure data reliability, enabling translational teams to confidently advance compounds based on reproducible structural phenotypes observed in neurites and growth cones.
What statistical analysis capabilities are required before implementing this method in compound screening?
The method requires capability to analyze volumetric data distributions, assess significance of neurite volume changes between conditions, and correlate structural readouts with functional outcomes to support decision-making.
Chapters in this video
0:05
Title
0:47
Fabrication of PDMS Chip
1:51
Fabrication of Patterned Coverslips
3:13
Chip Assembling and Final Implementation
6:03
Neuron Culture and Imaging
7:14
Results: Calibration of Fluorescence eXclusion Method Using a Photoresist Stripe as the Object of Interest and Neuronal Structures Volume Imaging
8:17
Conclusion
