Overview of Photon Microscopes

Overview of Photon Microscopes

Introduction to Photon Microscopes

  • Study of fluorescence microscopes and their applications for fluorescence imaging.

  • Focus on confocal microscopes for precision imaging, especially for thick specimens or light mouse brain samples.

  • Confocal microscopes allow for the removal of outer distance for better imaging, but they still have limitations.

Light Propagation in Biological Tissues

Issues Encountered

  • As light travels deeper into tissue, it faces:

    • Absorption

    • Scattering

Absorption

  • Example: When placing a hand over a phone flashlight in darkness, only red light is visible due to absorption.

  • Common absorbers in biological tissues:

    • Melanin: Absorbs wavelengths closer to UV in skin.

    • Heme: Found in hemoglobin, absorbs similar wavelengths.

Scattering

  • Scattering Basics: The blue color of the sky is due to scattering.

    • Scattering depends on the size of the particle relative to light's wavelength.

    • Rayleigh Scattering: Occurs when the particle size is much smaller than the wavelength of light; inversely proportional to the fourth power of the wavelength (shorter wavelengths lead to more scattering).

    • Mie Scattering: Dominant type in biological tissues; occurs when particle size is comparable to light wavelength (100 nm to microns).

Refractive Index Matching

  • Different refractive indices between intracellular and extracellular components lead to scattering.

  • Recent techniques utilize chemicals to match refractive indices and remove lipids from brain tissue.

    • Limitation: Tissue shrinkage and loss of structural integrity.

Alternative Solutions

  • Using Longer Wavelengths:

    • Example: Siren 7.5 dye with an excitation wavelength of 810 nm and emission also around 800 nm; good depth penetration, but biological dyes typically operate in the visible range.

  • Two-Photon Excitation:

    • Instead of one short-wavelength photon to excite a fluorophore, two longer-wavelength photons are used simultaneously (within femtoseconds).

    • Increases the chance of excitation at a focal point with minimal scattering.

Mechanism of Two-Photon Excitation

  • One-photon excitation uses shorter wavelength photons to move a fluorophore from the ground to excited state, emitting light as it returns.

  • Two-Photon Process: Requires:

    • Higher wavelength photons (matching energy of shorter wavelength photons when combined).

    • Simultaneous Arrival: Two photons must reach the fluorophore within less than one femtosecond (10^{-15} seconds).

    • This creates a significant intensity of emission proportional to the product of the two intensities reaching the fluorophore.

Excitation Intensity

  • Power Requirements:

    • One-photon: approx. 1 milliwatt continuous laser.

    • Two-photon: requires approximately 10^{14} watts/m^2; cannot be continuous due to risk of burning samples.

    • Use of pulsed lasers (e.g., titanium-doped sapphire lasers) with repetition rates of 80 MHz.

Spatial and Temporal Concentration

  • Selecting high numerical aperture objectives concentrates photon excitation at focal points, allowing for refined imaging.

  • Comparison Between One-Photon and Two-Photon:

    • One-photon shows excitation both below and above focal points, leading to excess scattering.

    • Two-photon excitation restricts excitation to the focal point, enhancing imaging clarity.

    • Signal collection is critical; direct detection of light without descanning systems is possible in two-photon setups.

Scanning Mechanisms

Scanner Types

  • Galvo Scanners: Standard mirrors driven by motors; allow arbitrary scan geometries.

    • Limitations: Speed restricted by mechanical inertia—only 15 frames per second for large images.

  • Resonant Scanners:

    • Utilize mirrors in oscillatory motion for high frame rates up to 30 frames per second.

    • Ideal for live imaging due to rapid scanning capabilities.

    • Equally spaced pixel detection achieved with motion sensors in newer systems.

Dwell Time Variation

  • Differences in dwell time across scanners affect bleaching and imaging quality:

    • Galvo scanners may bleach excess areas due to slower adjustments between lines.

    • Resonant scanners maintain sharper edges due to faster scanning.

Advantages of Two-Photon Excitation

  • Increased tissue penetration due to lower interaction with cellular materials.

  • Reduced photo-damage and autofluorescence compared to one-photon techniques.

  • Higher spatial localization and precision in photomanipulation.

Disadvantages of Two-Photon Excitation

  • More significant limitations in high-resolution imaging due to larger diffraction spots and lower resolution.

  • Higher costs for specialized lasers and detection equipment.

  • Limited functionality in very thick specimens (beyond 400 microns) due to high energy requirements leading to potential damage.

Practical Applications

  • Imaging techniques involve strategies to enhance clarity and minimize artifacts caused by scattering.

  • Specific examples include imaging behaviors in mouse models by tracking neurological activities in the hippocampus while minimizing motion artifacts.

  • Application of two-photon microscopy in live organisms can track cell activities for extensive studies in neurobiology or related fields.