Summary Advanced Cellular Imaging Techiques

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Summary - Advanced Cellular Imaging Techiques

  • 1 General Introduction

  • Key to understanding of cells is the observation of dynamics in a system. Seeing how it changes over time.
  • 2 Microscope Construction

  • Condenser lens
    Illuminates the sample
  • Objective and eyepiece
    Magnify the sample
  • Lens
    Redirects light by reftraction on the glass-air surfaces
  • Refraction
    - Occurs at a transition in refractive index.
    - Light rays originating from a point on the subject are redirectes towards a point in the image plane.
  • Image formation
    A parallel lightbeam is focused at the focal point
  • Focal length
    The distance from the lens to the focal point
  • S
    Object and image distance = the distance from the object to the lens or from the lens to the image.
  • Y
    Object size; distance from horizontal axis to the object Q
  • Y'
    Image size; distance from the horizontal axis to the image Q'
  • Magnification
    Placing an object near the focal plane of the objective generates an image faraway. 
    - Moving it closer to the focal plane will generate an image at infinity.
    - In microscopy, objectives with short focal length are used, so the object is close to the objective lens.
  • Focal plane
    The plane that runs through the middle of the lens. The S and F lie in this plane
  • 2.1 Compound microscope

  • A compound microscope reaches higher magnification by...
    Using extra lenses, such as an eyepiece.
  • If a compound microscope has 3 lenses, these are:
    1. Condenser lens (before objective)
    2. Objective lens
    3. Eyepiece lens
  • 2.2 Infinity optical system

  • Infinity optical system
    Here there are two lenses, but no intermediate-image.
    - Object is placed in the focal plane (s=f, s'=infinity)
    - Each point of the object forms a parallel bundle
    - A tubelens forms a real image on a camera sensor.
  • Maginification infinity optical system
    Ftube / Fobj
  • 2.3 Lens Abberations

  • An ideal lens...
    Will image light originating from a single point on the sample into a single point in the image plane.
  • Lens abberations cause...
    Image blur and image distortion
  • Spherical abberation
    Parallel light rays that travel close and far away from the optical axis are not focused to the same point, resulting in a blurred image.
  • Chromatic abberation
    Lenses refract different colors differently (chromatic dispersion)
  • Chromatic dispersion
    The refactive index of a material is not constant but depends of color.
  • Achromats
    Lenses that are corrected such that blue and red light are imaged into a single common focal point.
  • Plan achromats
    Corrected for blue and red light AND corrected for spherical abberations.
  • Plan apochromats
    The most highly corrected lenses for 4-5 different colors.
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FRET efficiency of FLIM?
Ratio Tau donor-acceptor / Tau Donor
What are the two disadvantages of FLIM?
1. Measurement time
2. Expensive
In the presence of an acceptor...
The donor lifetime will decrease, because it wil partially transfer its energy to the acceptor.
What is meant with sensitive to the environment?
1. Viscosity, pH, polarity and solvation
2. Size and shape
3. Molecular interactions.
So you can test all these things?
4 Advantages of FLIM are...
1. Absolute measure, so fluorescence intensity is a relative quantity
2. Intrinsic property of fluorophore
3. Independent of probe concentration or laser excitation intensity
4. Sensitive to the environment
FLIM is based on...
Differences in excited state decay rates from a fluorescent sample, often using different excited states to calculate the average one.
With counting photons...
You excite a photon with a laser and measure how long it emits energy. Do this multiple times to get an average.
What is the fluorescence lifetime of a fluorophore?
The average time a fluorophore remains in its excited state before returning to the ground state by emitting a photon.
An advantage of APB?
It's fast and easy to do.
Name some disadvantages of APB (4)
1. Dependent on how susceptible the acceptor is for bleaching
2. Differences of local donor and acceptor concentrations
3. Limited spatial resolution
4. Not suitable for living samples --> destructiveness