Fluorescence Microscopy Study Notes

Fluorescence Microscopy Study Notes

Course Information

  • Course Title: CBS 7104 Biomedical Cell Molecular Biology

  • Instructor: Masami Yoshimura

  • Email: fukugawa@lsu.edu

  • Date: 11-21-2025

Why Live Imaging?

  • Understanding and defining the role of numerous proteins in cellular processes.

  • Advantages of examining proteins in living cells:

    • Offers insights that cannot be achieved with fixed cell techniques.

  • Advances in technology are crucial for the emergence of live cell imaging as a useful tool.

Lecture Outline

  1. What is Fluorescence?

  2. Different Microscope Systems

  3. Fluorescent Proteins as Research Tools

  4. Advanced Techniques

Fluorescence Microscopy Overview

  • Bright Field vs Fluorescence Microscopy:

    • Comparison and distinctions between the two methods.

  • Combined Use of Phase Contrast and Fluorescence Illumination:

    Figure 3

Understanding Fluorescence

  • Fluorescence Process:

    • Excitation of Electrons: Electrons are excited by energy from light sources, leading to fluorescence.

  • Stokes' Observation:

    • Phenomenon where emitted light has a longer wavelength than the absorbed light.

  • Energy Dynamics in Fluorescence:
    ![Figure 1](https://www.olympus-lifescience.com/en/microscope-resource/primer/lightandcolor/fluorointroduction/Figure 1)

Jablonski Diagram

  • Illustrates energy states in a fluorescent process:

    • S3, S2: Excited states

    • S1: Single excited state

    • Triplet States: Higher energy states that can lead to phosphorescence.

    • Transition from excited states to ground state emits light, termed fluorescence.

Spectral Properties

  • Relative Intensity:

    • Importance of spectral profiles for excitation and emission of fluorophores.

  • Stokes Shift:

    • Difference between peak excitation and peak emission wavelengths.

  • Excitation and Emission Spectral Profiles: ![Figure 2](https://www.olympus-lifescience.com/en/microscope-resource/primer/lightandcolor/fluoroexcitation/Figure 2)

    • Displays absorption and emission against wavelength (300-700nm).

Fluorochromes (Fluorophores)

  • Definition: Compounds that can re-emit light upon being excited.

  • Common Fluorophores:

    • 4'-6-diamidino-2-phenylindole (DAPI)
      ![Figure 4](https://www.olympus-lifescience.com/en/microscope-resource/primer/lightandcolor/fluorointroduction/Figure 4)

    • Texas Red

Fluorescence Microscope Systems

  1. Widefield Microscopy:

  2. Laser Scanning Confocal Microscopy:

    • Spinning disk confocal

    • Multiphoton Laser Scanning

Components of Wide-field Microscopy

  • Arc Lamp: Essential for excitation.

  • Excitation Diaphragm and Filters: Control the light used to excite the fluorophores.

Confocal Microscope Components

  • Laser Source: Provides the illumination for the sample.

  • Emission Filter and PMT: Critical for detection of emitted fluorescence.

    • Differences in focal plane imaging (Black, Red, Green lines indicating focal planes).

Light Sources

  • Relative Spectral Radiation:

    • Comparison of different light sources:

    • Mercury Arc Lamp: Wide UV and visible spectrum.

      Emission Spectrum

    • Xenon Arc Lamp: Similar functionality to mercury lamps.

    • Metal Halide Arc Lamp: Favorable spectral distribution for less bleaching.

Fluorescence Filters

  • Types of filters used in fluorescence microscopy:

    • Interference Filters: Used to ensure appropriate wavelength transmission and blockage.

    • Short Pass and Long Pass Filters: Determine which wavelengths reach the detector.

Image Observation Methods

  • Visual Observation and Digital Cameras: Techniques used to capture fluorescence images.

  • Pros and Cons of Wide-field Microscopy:

    • Advantages: Allows for multiple labels and is relatively inexpensive.

    • Disadvantages: Out-of-focus light blurs images; challenging with thick sections.

Confocal Microscopy Advantages and Disadvantages

  • Advantages: Higher resolution, stack image collection for 3D reconstructions.

  • Disadvantages: Limited excitation peaks on lasers, prone to photobleaching, expensive setup, phototoxicity effects.

Confocal Scanning Systems

  • Functional process of scanning:

    • Laser scans across the plane of interest, reconstruing the image pixel by pixel.

    • Two galvanometric-driven mirrors commonly used to achieve precise control.

Multiphoton Microscopy Fundamentals

  • Principle: Utilizing two low-energy photons to excite a fluorophore, resulting in one photon of higher energy.

    • Maximal fluorescence occurs at focal planes avoiding out-of-focus light.

  • Photon Crowd Dynamics: Explains how multiphoton absorption is enabled through tightly focused light pulses.

  • Probabilities of Absorption: Significantly lesser for two and three photon excitations compared to one.

Biological Applications of Imaging Techniques

  • Green Fluorescent Protein (GFP): Widely used in live cell imaging due to its fluorescent properties and minimal toxicity.

  • Fluorescence Recovery After Photobleaching (FRAP):

    • Measures molecular diffusion and interactions within cellular environments.

Resonance Energy Transfer (RET)

  • Fluorescence Resonance Energy Transfer (FRET): = Coupled fluorophore interactions resulting in energy transfer when in close proximity.

    • Monitors biological processes without emitted photons.

Practical Examples of FRET

  • FRET sensors allow real-time monitoring of:

    • Protein interactions, enzyme activities, and second messenger dynamics.

Conclusion

  • Overall, fluorescence microscopy provides powerful tools for observing live cells and understanding cellular dynamics across various biological contexts. Each technique discussed has unique advantages and disadvantages that affect their suitability for specific applications and experiments.