11.3 Cellular and molecular imaging

Cellular and molecular imaging techniques let us peek inside living cells, revealing their inner workings. From fluorescence microscopy to super-resolution methods, these tools help scientists visualize biological processes with incredible detail and precision.

Optical biosensors take things a step further, allowing us to track specific molecules and events in real-time. These clever probes can detect everything from protein interactions to signaling pathways, opening new doors for understanding diseases and discovering drugs.

Optical Imaging Principles for Cells

Fluorescence Microscopy Fundamentals

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• Optical imaging techniques exploit the interaction of light with biological samples to visualize cellular structures and molecular processes with high spatial and temporal resolution
• Fluorescence microscopy is a widely used optical imaging technique that relies on the excitation and emission of fluorescent molecules (fluorophores) to generate contrast and specificity in biological samples
  • Fluorophores can be genetically encoded (green fluorescent protein) or externally added (organic dyes, quantum dots) to label specific cellular components or molecules of interest
  • The excitation and emission spectra of fluorophores determine the wavelengths of light used for illumination and detection in fluorescence microscopy

Advanced Fluorescence Imaging Techniques

• Confocal microscopy enhances the spatial resolution and contrast of fluorescence imaging by using a pinhole aperture to reject out-of-focus light and create optical sections of the sample
• Two-photon microscopy enables deep tissue imaging by using infrared light to excite fluorophores through the simultaneous absorption of two photons, reducing scattering and phototoxicity
• Super-resolution microscopy techniques, such as STED, PALM, and STORM, overcome the diffraction limit of light to achieve nanometer-scale resolution for visualizing subcellular structures and molecular interactions
• Light sheet microscopy allows for fast and gentle imaging of large biological samples by illuminating a thin plane of the sample and detecting fluorescence perpendicular to the illumination plane

Advanced Optical Biosensing Methods

Genetically Encoded and Chemically Synthesized Biosensors

• Optical biosensors are genetically encoded or chemically synthesized probes that change their optical properties (fluorescence intensity, lifetime, or spectrum) in response to specific biological events or analytes
• Förster Resonance Energy Transfer (FRET) biosensors consist of a pair of fluorophores (donor and acceptor) that undergo non-radiative energy transfer when in close proximity, enabling the detection of protein-protein interactions, conformational changes, or enzyme activities
• Fluorescent protein-based biosensors, such as Ca2+ indicators (GCaMP) and voltage indicators (ASAP), allow for real-time monitoring of intracellular ion concentrations and membrane potential changes
• Bioluminescence resonance energy transfer (BRET) biosensors use a bioluminescent protein (Renilla luciferase) as the energy donor and a fluorescent protein as the acceptor, enabling the detection of protein-protein interactions without external illumination

Environment-Sensitive and Pathway-Specific Biosensors

• Fluorescent biosensors based on environmentally sensitive dyes (Nile Red, Laurdan) can report on changes in the local microenvironment, such as membrane fluidity, pH, or hydrophobicity
• Genetically encoded biosensors for specific signaling molecules, such as cAMP, PKA, and ERK, enable the real-time monitoring of intracellular signaling pathways and their spatiotemporal dynamics
• These biosensors allow for the investigation of signaling cascades involved in cell growth, differentiation, and response to external stimuli (growth factors, hormones)
• Multiplexing different biosensors using spectrally distinct fluorophores enables the simultaneous monitoring of multiple signaling pathways within the same cell

Resolution of Optical Biosensors

Spatial Resolution and Imaging Techniques

• The spatial resolution of optical biosensors depends on the imaging technique used and the size of the biosensor molecule or complex
  • Conventional fluorescence microscopy has a spatial resolution limited by the diffraction of light, typically around 200-300 nm laterally and 500-700 nm axially
  • Super-resolution microscopy techniques can achieve spatial resolutions down to 10-20 nm laterally and 50-100 nm axially, allowing for the visualization of individual biosensor molecules and their subcellular localization
• The expression level and cellular localization of genetically encoded biosensors can influence their spatial and temporal resolution, as well as their potential perturbation of the biological system under study
• Advances in imaging techniques, such as structured illumination microscopy (SIM) and expansion microscopy, further improve the spatial resolution of optical biosensors

Temporal Resolution and Biosensor Kinetics

• The temporal resolution of optical biosensors is determined by the kinetics of the biosensor response and the acquisition speed of the imaging system
  • Biosensors based on fluorescent proteins typically have response times in the millisecond to second range, limited by the maturation and folding of the protein
  • Biosensors based on small organic dyes or quantum dots can have faster response times, in the microsecond to millisecond range, enabling the detection of rapid biological events
• The photostability and brightness of the biosensor molecules affect the signal-to-noise ratio and the duration of imaging that can be achieved without significant photobleaching
• Optimization of biosensor design, such as the use of fast-maturing fluorescent proteins or circular permutations, can improve the temporal resolution and kinetics of the biosensor response

Optical Biosensors for Disease Study

Investigating Disease Mechanisms

• Optical biosensors can be used to investigate the molecular mechanisms underlying various diseases, such as cancer, neurodegeneration, and metabolic disorders, by monitoring specific cellular processes or signaling pathways implicated in the disease pathogenesis
• Biosensors for monitoring apoptosis, such as caspase activity sensors or mitochondrial membrane potential indicators, can provide insights into the cellular mechanisms of cell death in disease conditions or in response to therapeutic interventions
• Biosensors for detecting oxidative stress, such as reactive oxygen species (ROS) indicators, can help elucidate the role of oxidative damage in various pathological conditions and the efficacy of antioxidant therapies
• Optical biosensors can be combined with advanced cell culture models, such as organoids or microfluidic devices, to create more physiologically relevant platforms for studying disease mechanisms and drug effects in a tissue-specific context

Drug Discovery and Screening

• Optical biosensors can be employed in high-throughput screening assays to identify and characterize novel drug candidates based on their effects on specific cellular targets or pathways
• Biosensors for monitoring protein aggregation, such as those based on FRET or bimolecular fluorescence complementation (BiFC), can be used to study the formation and clearance of protein aggregates in neurodegenerative diseases and to screen for potential therapeutic compounds
• The development of multiplexed biosensor assays, using different colors or modalities of optical biosensors, can enable the simultaneous monitoring of multiple cellular processes or signaling pathways, providing a more comprehensive understanding of disease mechanisms and drug actions
• Optical biosensors can be integrated with automated imaging systems and data analysis pipelines to streamline the drug discovery process and improve the efficiency of lead compound identification and optimization