💡Biophotonics Unit 4 – Fluorescence Microscopy and Imaging

Fundamentals of Fluorescence

• Fluorescence occurs when a molecule absorbs light at one wavelength and emits light at a longer wavelength
• Involves three main processes: excitation, vibrational relaxation, and emission
• Excitation happens when a photon is absorbed by a fluorophore, causing an electron to jump to a higher energy state
• Vibrational relaxation is the non-radiative transition of the electron to the lowest vibrational level of the excited state
  • Occurs on a picosecond timescale
• Emission is the radiative transition of the electron back to the ground state, releasing a photon of lower energy than the excitation photon
• Stokes shift is the difference between the excitation and emission wavelengths
  • Allows for efficient separation of excitation and emission light using filters
• Fluorescence lifetime is the average time a fluorophore spends in the excited state before returning to the ground state (typically nanoseconds)
• Quantum yield is the ratio of emitted photons to absorbed photons, indicating the efficiency of the fluorescence process

Light-Matter Interactions

• Light-matter interactions form the basis of fluorescence microscopy
• Absorption occurs when a photon's energy matches the energy difference between the ground state and an excited state of a molecule
• Scattering is the redirection of light by particles or molecules
  • Rayleigh scattering is elastic (no energy loss) and occurs when particles are much smaller than the wavelength of light
  • Raman scattering is inelastic (energy exchange) and provides information about molecular vibrations
• Refraction is the bending of light as it passes through materials with different refractive indices (e.g., air and glass)
• Reflection is the bouncing of light off surfaces, which can cause image artifacts in microscopy
• Polarization describes the orientation of the electric field vector of light
  • Can be used to control contrast and reduce glare in microscopy
• Interference is the superposition of light waves, which can be constructive or destructive
  • Utilized in techniques like fluorescence interference contrast microscopy (FLIC)

Fluorophores and Labeling Techniques

• Fluorophores are molecules that exhibit fluorescence and are used to label specific structures or molecules in a sample
• Intrinsic fluorophores are naturally occurring in biological samples (e.g., tryptophan, NADH, flavins)
  • Provide information about the native environment and function of biomolecules
• Extrinsic fluorophores are added to the sample to label specific targets (e.g., fluorescent dyes, quantum dots)
  • Can be conjugated to antibodies, nucleic acid probes, or other targeting moieties
• Immunofluorescence uses antibodies conjugated to fluorophores to label specific proteins
  • Primary antibodies bind directly to the target, while secondary antibodies, conjugated to fluorophores, bind to the primary antibodies
• Fluorescent proteins (e.g., GFP) can be genetically encoded and expressed in living cells
  • Allow for dynamic imaging of protein localization and interactions
• FISH (fluorescence in situ hybridization) uses fluorescently labeled nucleic acid probes to detect specific DNA or RNA sequences
• Fluorescent small molecules (e.g., DAPI) can label specific cellular structures or report on the local environment (pH, ion concentration)

Microscope Components and Setup

• Fluorescence microscopes consist of an excitation light source, filters, objective lens, and a detector
• Light sources can be lamps (mercury, xenon) or lasers (gas, solid-state, diode)
  • Lasers provide high intensity, narrow bandwidth, and better control over illumination
• Filters select specific wavelengths of light for excitation and emission
  • Excitation filters select the wavelengths that excite the fluorophore
  • Emission filters select the wavelengths emitted by the fluorophore, blocking the excitation light
  • Dichroic mirrors reflect excitation light and transmit emission light, separating the two
• Objective lenses focus the excitation light on the sample and collect the emitted fluorescence
  • Characterized by magnification, numerical aperture (NA), and working distance
  • High NA objectives provide better resolution and light collection but have shorter working distances
• Detectors convert the emitted fluorescence into an electrical signal
  • Photomultiplier tubes (PMTs) and charge-coupled devices (CCDs) are common detectors in fluorescence microscopy
• Proper alignment and calibration of the microscope components are crucial for optimal performance and image quality

Image Acquisition and Processing

• Image acquisition involves capturing the fluorescence signal from the sample
• Exposure time, gain, and binning are key parameters that affect signal-to-noise ratio and temporal resolution
  • Longer exposure times and higher gain increase signal but also increase noise
  • Binning combines adjacent pixels to improve signal-to-noise ratio at the cost of spatial resolution
• Z-stacks are series of images acquired at different focal planes, allowing for 3D reconstruction of the sample
• Time-lapse imaging captures dynamic processes by acquiring images at regular intervals
• Image processing improves the quality and interpretability of the acquired data
  • Background subtraction removes unwanted background signal
  • Flat-field correction compensates for uneven illumination across the field of view
  • Deconvolution reduces out-of-focus blur and improves contrast and resolution
• Quantitative analysis extracts numerical data from images, such as fluorescence intensity, colocalization, or object size and shape
• Image display and visualization techniques (e.g., color mapping, intensity scaling) enhance the presentation of the data

Advanced Fluorescence Techniques

• Confocal microscopy uses a pinhole to reject out-of-focus light, improving contrast and resolution
  • Allows for optical sectioning and 3D reconstruction of the sample
• Two-photon microscopy uses pulsed infrared lasers to excite fluorophores via the simultaneous absorption of two photons
  • Provides deeper tissue penetration, reduced photobleaching, and improved signal-to-noise ratio
• Super-resolution techniques overcome the diffraction limit of light, achieving resolutions below 200 nm
  • STED (stimulated emission depletion) uses a donut-shaped depletion beam to narrow the excitation volume
  • STORM/PALM (stochastic optical reconstruction microscopy/photoactivated localization microscopy) relies on the sequential activation and localization of single fluorophores
  • SIM (structured illumination microscopy) uses patterned illumination to extract high-frequency information
• FRET (Förster resonance energy transfer) measures the distance-dependent energy transfer between two fluorophores
  • Provides information about molecular interactions and conformational changes
• FLIM (fluorescence lifetime imaging microscopy) maps the spatial distribution of fluorescence lifetimes
  • Sensitive to the local environment and can report on pH, ion concentration, or protein interactions
• Light sheet microscopy illuminates the sample with a thin sheet of light, reducing out-of-focus excitation and photobleaching
  • Enables high-speed, low-phototoxicity imaging of living samples

Applications in Biomedical Research

• Fluorescence microscopy is widely used in cell biology to study the localization, dynamics, and interactions of proteins and organelles
• Neuroscience applications include imaging neuronal activity (calcium imaging), mapping neural circuits, and studying synaptic plasticity
• Developmental biology uses fluorescence microscopy to track cell lineages, monitor gene expression, and visualize morphogenetic processes
• Cancer research employs fluorescence techniques to study tumor progression, metastasis, and drug response
  • Fluorescent reporters can be used to monitor tumor growth and spread in animal models
• Infectious disease research uses fluorescence microscopy to visualize host-pathogen interactions, track pathogen invasion and spread, and assess therapeutic interventions
• High-content screening combines fluorescence microscopy with automated image analysis to screen large libraries of compounds for their effects on cellular processes
• Tissue engineering and regenerative medicine use fluorescence imaging to monitor cell differentiation, tissue organization, and biomaterial integration
• Diagnostic applications include the detection of biomarkers, pathogens, or genetic abnormalities in clinical samples

Limitations and Future Developments

• Photobleaching is the irreversible loss of fluorescence due to prolonged or intense illumination
  • Can be mitigated by optimizing imaging conditions, using more photostable fluorophores, or employing specialized techniques (e.g., FRAP, PAINT)
• Phototoxicity is the damage caused to living samples by the excitation light
  • Can be reduced by minimizing exposure time, using lower excitation intensities, or employing gentler techniques (e.g., light sheet microscopy)
• Spectral overlap between fluorophores can lead to crosstalk and complicate multi-color imaging
  • Spectral unmixing algorithms can help separate the signals from different fluorophores
• Tissue scattering and absorption limit the penetration depth of light, particularly in thick samples
  • Clearing techniques (e.g., CLARITY, iDISCO) can render tissues transparent and improve imaging depth
• Quantitative analysis can be challenging due to variations in fluorophore concentration, labeling efficiency, and imaging conditions
  • Standardized protocols and appropriate controls are essential for reliable quantification
• Advances in fluorophore design aim to create brighter, more photostable, and spectrally distinct probes
  • Genetically encoded fluorescent sensors (e.g., GCaMP) enable the monitoring of specific cellular processes or analytes
• Computational methods, such as machine learning and artificial intelligence, are increasingly used for image analysis and interpretation
  • Deep learning algorithms can automate tasks such as cell segmentation, object detection, and pattern recognition
• Integration of fluorescence microscopy with other techniques (e.g., optogenetics, electrophysiology) provides a more comprehensive understanding of biological systems
  • Correlative light and electron microscopy (CLEM) combines the specificity of fluorescence with the high resolution of electron microscopy