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Home » Courses » Microscopy Series » Fluorescence Microscopy

Fluorescence Lifetime Imaging Microscopy

[00:00:12;21] We're going to talk about
[00:00:17;19] how we use polarized light or polarizing microscopes
[00:00:21;17] for studying biological subjects,
[00:00:23;25] especially living cells.
[00:00:26;21] And polarizing microscopes
[00:00:30;05] have been used by crystallographers
[00:00:33;09] and mineralogists for many many years
[00:00:39;07] for looking at crystals and natural minerals.
[00:00:42;19] But what we need for biology
[00:00:45;20] is slightly different from what the
[00:00:50;20] standard commercial polarizing microscope is.
[00:00:56;27] And therefore, I'm going to make some demonstrations here
[00:01:01;25] to explain how the polarized light microscope works.
[00:01:08;24] And these demonstrations partly use crystalline material
[00:01:11;21] and partly biological examples.
[00:01:19;06] So what we have here is the word "birefringence,"
[00:01:22;05] I hope you can see on your screen,
[00:01:25;12] and what I'm going to do is,
[00:01:27;25] in front of this word,
[00:01:31;21] I'm going to place a crystal of calcite
[00:01:35;04] Calcite is Iceland spar,
[00:01:40;20] and Iceland spar is a crystal calcium carbonate.
[00:01:45;08] And it's well-known because it introduces
[00:01:48;14] birefringence, or double refraction.
[00:01:51;17] Double refraction is the property
[00:01:54;08] that we use very often in biology.
[00:01:58;13] And one of the properties of this double refraction
[00:02:05;05] is that the light that comes through the crystal
[00:02:10;23] before it's just a single image becomes double.
[00:02:14;08] And because it's double, it's called birefringence.
[00:02:20;07] And now if we put a pair of sunglasses in front of it.
[00:02:23;29] one or the other of the image disappears.
[00:02:28;18] What this is telling us is that light can come through
[00:02:33;26] or the birefringent material itself is being polarized.
[00:02:36;10] Now I'll explain polarization in a minute.
[00:02:39;18] But in any event, birefringence,
[00:02:46;08] or birefringent material takes ordinary light
[00:02:48;16] and then splits it into two
[00:02:54;11] and that split light itself becomes polarized.
[00:02:57;10] So now what we're going to do
[00:03:00;22] is take two birefringent materials,
[00:03:08;08] two polarizing materials, here is polarizing material,
[00:03:09;29] and putting it under the birefringent
[00:03:16;07] Under the crystal, and then depending
[00:03:17;29] on how I set the crystal,
[00:03:22;18] we should be able to see two images.
[00:03:31;27] Now then, when I put a second polarizing material above
[00:03:33;16] and cross the two,
[00:03:38;17] then we'll still see the birefringent material.
[00:03:40;08] As I turn the crystal,
[00:03:46;06] then the image becomes brighter or darker.
[00:03:52;00] And this is the property of birefringence.
[00:03:56;26] Calcite is not only interesting as a special mineral
[00:03:59;21] it has strong birefringence,
[00:04:04;04] but also it occurs in many of our body's structures.
[00:04:07;26] And for example, in bone, and teeth, and so on,
[00:04:11;08] one of the mineral components is calcite.
[00:04:15;26] And when we look at a sea urchin embryo
[00:04:18;24] for example, we see even in the young embryo
[00:04:23;06] tiny little specules which are made of pure calcite
[00:04:25;23] and those show as birefringence.
[00:04:28;08] Instead of the calcite crystal
[00:04:32;16] I'm going to put in a birefringent biological sample here
[00:04:38;16] which you may just be able to see or not be able to see
[00:04:43;09] because the contrast is terribly weak between cross polarizers.
[00:04:46;24] Now I'm going to put another birefringent material
[00:04:50;16] and it should become brighter or darker.
[00:05:00;13] So this is a model of a mitotic spindle,
[00:05:02;09] which is weakly birefringent
[00:05:08;06] in fact the birefringence is only a few hundredths
[00:05:12;17] or thousandths of the birefringence of the calcite crystal you saw.
[00:05:15;27] So we need this compensator
[00:05:19;26] in order to see which way the molecules are lined up.
[00:05:29;20] But the birefringent material itself is just like the calcite crystal,
[00:05:36;25] separating,depending on the orientation of the material.
[00:05:46;18] What the birefringence is showing us
[00:05:51;05] is that molecules are lined up and what I'm doing
[00:05:55;17] is taking this piece of plastic and pulling it so that
[00:05:58;12] the molecules line up.
[00:06:04;13] It becomes bright and even shows color
[00:06:11;29] and then this is birefringent just like the other material.
[00:06:14;03] But let's just see here
[00:06:15;20] lining up the molecule
[00:06:20;16] made this birefringent material change
[00:06:23;00] into a birefringent material.
[00:06:27;06] I'll demonstrate the same effect by using sound waves.
[00:06:30;20] Although sound is still somewhat different from light waves.
[00:06:34;01] I cut a piece of wood here
[00:06:39;04] so that one block is running parallel to the grain
[00:06:41;06] and the other is across the grain.
[00:06:46;01] And sound travels must faster along the grain
[00:06:47;12] than across the grain,
[00:06:50;13] so when I tap on the edge of this
[00:06:54;28] then you'll find the resonance sound.
[00:06:57;13] This is parallel to the grain (fast tapping sound),
[00:06:59;08] and this is across the grain (slow tapping sound).
[00:07:04;06] And you see there's almost an octave difference
[00:07:07;10] because sound travels twice as fast
[00:07:09;17] along the grain than across the grain.
[00:07:12;26] So this is acoustic anisotropy,
[00:07:17;14] somewhat similar to optical anisotropy.
[00:07:21;11] Except there is a fundamental difference
[00:07:23;12] between sound and light.
[00:07:26;27] Sound travels faster in denser medium,
[00:07:30;23] whereas light travels slower in denser medium.
[00:07:33;15] So this distinction you should remember.
[00:07:39;17] But anyhow, this is double refraction of a kind.

This Talk
Speaker: Philippe Bastiaens
Audience:
  • Researcher
Recorded: May 2012
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Talk Overview

The fluorescence lifetime of a dye molecule is the amount of time that elapses between excitation of the dye and its emission of a photon. Fluorescence lifetime imaging is the technique by which this lifetime is imaged and can be done using either widefield or confocal (time correlated single photon counting) methods. The fluorescence lifetime of a dye depends both on the dye and on the environment surrounding the dye. Because of this, FLIM can be a sensitive probe for environment as well as FRET. This lecture discusses fluorescence lifetime, microscopes used to image it, and some biological applications of FLIM.

Questions

  1. The lifetime of a fluorophore usually is characterized by:
    1. A Linear distribution
    2. A single exponential decay
    3. Multiple exponential decays
    4. A Gaussian distribution
  2. To estimate the fraction of donor fluorophores undergoing FRET, one needs to:
    1. Extract from the measured decay the decay characteristic for donor alone and the decay for the donor undergoing FRET
    2. Subtract the donor alone lifetime from the measured lifetime
    3. Divide the measured lifetime by the lifetime of the donor alone
    4. Divide the measured lifetime by the lifetime of the donor undergoing FRET
  3. Fluorescence lifetime can be measured using:
    1. A gated detector that measures photon arrival time after excitation
    2. Oscillating the intensity of excitation and sensitivity of the detector
    3. Both A and B
    4. A stopwatch
  4. To use a fluorescent protein (FP) as a donor in FRET measurements using FLIM, it is important that:
    1. the FP’s lifetime shows mono-exponential decay
    2. the FP’s lifetime show multi-exponential decay
    3. the FP is the brightest available
    4. the FP matures fast
  5. In frequency domain FLIM, the phase lifetime and modulation lifetime are:
    1. identical for fluorophores with mono-exponential lifetime
    2. unrelated
    3. different for fluorophores with multi-exponentials lifetimes
    4. both A and C
  6. Homodyne detection is:
    1. use of a gated detector to measure photon arrival times
    2. Measurement of homo-FRET
    3. Using the same frequency for camera sensitivity and excitation intensity
    4. Use of a PMT with only a single dynode

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Speaker Bio

Philippe Bastiaens

Philippe Bastiaens

Philippe Bastiaens is Director of the Department of Systemic Cell Biology at the Max Planck Institute of Molecular Physiology and a Professor at the Technische Universitat Dortmund. Bastiaens and his colleagues have developed special fluorescence microscopy techniques such as FLIM and FRET, and use them to explore protein signaling pathways. Continue Reading

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