Autofluorescence Imaging to Evaluate Cellular Metabolism
November 15th, 2021
This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.
Overview
This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Metabolism
Background
- ADH and FAD are endogenous molecules that exhibit autofluorescence.
- They are used as co-enzymes in metabolic reactions.
- Autofluorescence microscopy allows for assessment of cellular metabolism.
- This method does not require exogenous labels and is non-destructive.
Purpose of Study
- To assess cellular metabolism using autofluorescence imaging.
- To provide a method applicable to live samples and time course studies.
- To demonstrate the broad applicability of autofluorescent imaging in various cell types.
Methods Used
- Fluorescence imaging of NAD(P)H and FAD.
- Autofluorescence microscopy.
- Analysis of live samples.
- Time course studies on various cell types.
Main Results
- Autofluorescence imaging can be used on neurons, cancer cells, and stem cells.
- The method provides subcellular resolution.
- It allows for non-destructive assessment of cellular metabolism.
- Applicable to both in-vivo and ex-vivo studies.
Conclusions
- Autofluorescence imaging is a valuable tool for studying cellular metabolism.
- The technique is versatile and applicable to a wide range of biological samples.
- It enables researchers to conduct time course studies without damaging samples.
Frequently Asked Questions
Executive Industry Relevance
Autofluorescence imaging of NAD(P)H and FAD enables label-free, quantitative assessment of cellular metabolism, supporting early discovery and mechanistic de-risking in biopharma R&D. This non-destructive, high-resolution approach facilitates real-time metabolic profiling across diverse cell types, enhancing predictive confidence in disease-relevant models. Integration of these metabolic readouts strengthens target validation and informs risk-adjusted portfolio decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of metabolic pathways in live cells without exogenous labels.
- Supports functional target validation by quantifying metabolic shifts in response to perturbations.
- Facilitates mechanistic de-risking through real-time, quantitative metabolic measurements.
- Improves predictive confidence for early-stage portfolio triage.
Screening & Assay Development
- Provides standardized, reproducible metabolic assays for compound screening.
- Delivers quantitative fluorescence lifetime and intensity outputs for robust assay development.
- Enables high-content, label-free screening across multiple cell types and conditions.
- Supports scalability and platform reuse for diverse discovery programs.
Translational & Preclinical Research
- Aligns metabolic phenotyping with disease-relevant cellular models for translational continuity.
- Enables longitudinal studies and time-course analyses in live samples.
- Supports risk-adjusted advancement decisions by linking metabolic changes to functional outcomes.
- Facilitates integration of metabolic biomarkers into preclinical validation workflows.
Pipeline & Workflow Integration
Autofluorescence imaging positions within the discovery continuum from early hypothesis testing through lead identification and preclinical validation, providing a reusable capability for metabolic assessment.
- Discovery Biology: Quantifies metabolic responses to perturbations, clarifying pathway involvement and biological risk.
- Screening: Delivers reproducible, quantitative metabolic readouts for compound evaluation and assay standardization.
- Analytics: Provides fluorescence lifetime and intensity data to compare metabolic states across conditions.
- Translational Research: Bridges discovery and preclinical phases by enabling metabolic biomarker alignment in disease models.
- Enterprise Reuse: Offers a non-destructive, label-free platform adaptable to multiple cell types and research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in metabolic studies.
- Operational Value: Enhances standardization, reproducibility, and scalability of metabolic assays.
- Strategic Value: Informs go/no-go decisions and improves capital efficiency by de-risking early-stage programs.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of discovery assets.
Implementation Considerations
- Requires expertise in fluorescence microscopy and metabolic assay design.
- Needs access to multi-photon fluorescence lifetime imaging instrumentation and analytical software.
- Demands cross-team standardization of imaging parameters and data analysis workflows.
- Adaptable across various live cell and tissue models with appropriate optimization.
- Laser power and acquisition settings must be carefully managed to avoid sample damage while ensuring sufficient signal.
Biopharma FAQs
Chapters in this video
0:04
Introduction
1:04
Multiphoton Fluorescence Lifetime Imaging (FLIM) of Nicotinamide Adenine (Phosphate) Dinucleotide (NAD(P)H) and Flavin Adenine Dinucleotide (FAD)
2:13
Nicotinamide Adenine (Phosphate) Dinucleotide (NAD(P)H) Imaging
3:35
Flavin Adenine Dinucleotide (FAD) Imaging
4:45
Cyanide Experiment Preparation
5:28
Results: The Fluorescence Imaging of MCF-7 Cells Before and After Cyanide Treatment
7:05
Conclusion
