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🔬Modern Optics

🔬modern optics review

14.4 Advanced microscopy techniques

4 min readLast Updated on July 22, 2024

Advanced microscopy techniques have revolutionized our ability to visualize and study complex biological systems. Confocal, two-photon, and light sheet microscopy offer unique advantages in resolution, depth penetration, and sample compatibility, enabling researchers to probe deeper into living tissues and organisms.

Each technique has its strengths and trade-offs, making them suitable for different applications. Confocal excels in thin samples, two-photon shines in deep tissue imaging, and light sheet allows for rapid, low-phototoxicity imaging of large specimens. Understanding these differences is crucial for choosing the right tool for your research needs.

Advanced Microscopy Techniques

Advanced microscopy techniques comparison

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  • Confocal microscopy
    • Uses a pinhole to block out-of-focus light improving resolution and contrast
    • Achieves optical sectioning by scanning the sample point-by-point
    • Suitable for thin samples (tissue sections, cell cultures) and fluorescence imaging
  • Two-photon microscopy
    • Uses femtosecond pulsed laser to excite fluorophores with two lower-energy photons simultaneously
    • Provides deeper tissue penetration (brain tissue) and reduced photobleaching compared to confocal microscopy
    • Ideal for thick, scattering samples (organoids, tissue explants) and in vivo imaging (animal models)
  • Light sheet microscopy
    • Illuminates the sample with a thin sheet of light reducing out-of-focus excitation and phototoxicity
    • Enables high-speed, low-phototoxicity imaging of large, transparent samples (embryos, cleared tissues)
    • Particularly useful for developmental biology (zebrafish, Drosophila) and whole-organism imaging (C. elegans, Arabidopsis)

Principles of microscopy techniques

  • Confocal microscopy principles and advantages
    • Resolution: Achieves sub-micron lateral resolution by using a pinhole to reject out-of-focus light improving contrast and signal-to-noise ratio
    • Depth penetration: Limited to relatively thin samples (up to ~100 μm) due to scattering of excitation and emission light
    • Sample preparation: Requires fluorescent labeling (immunofluorescence, genetic reporters) and mounting of fixed or live samples on glass slides or dishes
  • Two-photon microscopy principles and advantages
    • Resolution: Similar lateral resolution to confocal microscopy but improved axial resolution due to nonlinear excitation confined to the focal plane
    • Depth penetration: Can image deeper into scattering tissues (up to ~1 mm) due to longer excitation wavelengths (near-infrared) and reduced scattering
    • Sample preparation: Requires fluorescent labeling but allows for in vivo imaging of live animals with minimal invasiveness and tissue damage
  • Light sheet microscopy principles and advantages
    • Resolution: Achieves high lateral resolution with axial resolution determined by the light sheet thickness (typically a few microns)
    • Depth penetration: Can image large, transparent samples (up to several mm) with minimal scattering and aberrations
    • Sample preparation: Requires transparent, fixed, or live samples; often used with clearing techniques (CLARITY, CUBIC) to enhance transparency

Applications in scientific fields

  • Biology applications
    • Confocal microscopy: Imaging of fixed cells and tissues (histology), co-localization studies (protein interactions), and live-cell dynamics (membrane trafficking)
    • Two-photon microscopy: In vivo brain imaging (neuron activity), calcium imaging (cell signaling), and deep tissue imaging (tumor microenvironment)
    • Light sheet microscopy: Developmental biology (embryogenesis), whole-embryo imaging (organogenesis), and 3D tissue imaging (organoids, spheroids)
  • Materials science applications
    • Confocal microscopy: Characterization of surface topography (roughness), defects (cracks, voids), and phase distributions (composites) in materials
    • Two-photon microscopy: Imaging of nonlinear optical properties (second harmonic generation) and deep-level defects (fluorescence) in semiconductors (silicon, GaAs)
    • Light sheet microscopy: 3D imaging of porous materials (ceramics), polymers (hydrogels), and composites (fiber-reinforced)
  • Nanotechnology applications
    • Confocal microscopy: Imaging of nanostructures (nanowires), nanoparticles (quantum dots), and self-assembled monolayers (SAMs)
    • Two-photon microscopy: Characterization of nonlinear optical properties of nanomaterials (graphene, carbon nanotubes)
    • Light sheet microscopy: 3D imaging of nanodevices (MEMS), microfluidic systems (lab-on-a-chip), and lab-on-a-chip platforms (organ-on-a-chip)

Trade-offs in microscopy selection

  • Resolution trade-offs
    1. Confocal and two-photon microscopy offer the highest lateral resolution (sub-micron) while light sheet microscopy provides better axial resolution (a few microns)
    2. Increasing resolution often requires slower imaging speeds (longer pixel dwell times) and may be limited by sample properties (thickness, transparency)
  • Speed trade-offs
    1. Light sheet microscopy enables the fastest imaging speeds (hundreds of frames per second) followed by spinning disk confocal microscopy and two-photon microscopy
    2. Higher imaging speeds may compromise resolution (undersampling) and signal-to-noise ratio (reduced photon counts)
  • Sample compatibility trade-offs
    1. Confocal microscopy is best suited for thin, fixed, or live samples that can be fluorescently labeled and mounted on glass substrates
    2. Two-photon microscopy is ideal for thick, scattering samples and in vivo imaging of live animals with minimal invasiveness
    3. Light sheet microscopy requires transparent, fixed, or live samples and is often used with clearing techniques to enhance optical clarity
  • Selecting the appropriate technique
    1. Consider the desired resolution (lateral vs axial), imaging speed (temporal resolution), and sample properties (thickness, transparency, labeling) when choosing a microscopy technique
    2. Prioritize the most critical factors for the given application (e.g., speed for live-cell imaging, depth for in vivo studies) and optimize the imaging parameters (excitation power, exposure time, z-step size) accordingly
    3. Consult with experts, attend workshops, and review literature to gain hands-on experience and stay updated with the latest advances in microscopy techniques and applications


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AP® and SAT® are trademarks registered by the College Board, which is not affiliated with, and does not endorse this website.