Functional cellular imaging by light microscopy
MICROSCOPIES
Why use Light?
Good (enough) resolution:
Spatial – classically a few hundred nanometers; now tens of nm
Temporal - <millisecond
Compatible with live cells, tissues, organisms
Many probes available for imaging:
Fluorescent antibodies, GFP, indicators for Ca2+
membrane potential, etc.
Relatively inexpensive and simple
(vs. E.M., PET, FMRI etc)
Three decades of building
microscope systems
1977
2008
On the importance of looking
“You can observe a lot
just by watching.”
Yogi Berra
Former Yankee Catcher and
Great American Sage
And sometimes you can see beautiful things!
Chaotic and spiral calcium waves in Xenopus oocyte: Jim Lechleiter, U. Texas
Major types of light microscopy;
1. Transmitted (reflected) light. Poor contrast; paucity of specific
labels/functional probes; poor depth resolution.
2. Fluorescence. High contrast (black background); numerous
fluorescent dyes, proteins and functional probes; permits 3D
imaging (confocal, 2-photon)
A fluorescence microscope
But – conventional fluorescence imaging provides little depth discrimination, so
images are terrible because of out-of-focus fluorescence
e.g. Pollen grain imaged by conventional epifluorescence microscopy
One solution – Confocal microscopy
Out-of-focus light is rejected by blocking it with a pinhole
aperture
Confocal sections through pollen grain at 1 um intervals
3-D reconstruction of pollen grain
Another way to avoid out-of-focus
fluorescence and achieve 3D imaging –
Two-photon microscopy
Especially good for looking deep into tissues (e.g. brain)
without damaging cells
Practical theory of 2-photon microscopy
1. Near simultaneous absorption of the energy of two infrared
photons results in excitation of a fluorochrome that would
normally be excited by a single photon of twice the energy.
2. The probability of excitation depends on the square of the
infrared intensity and decreases rapidly with distance from the
focal volume.
Advantages of 2-photon microscopy
1.
2.
3.
4.
5.
Increased penetration of infrared light allows deeper
imaging.
No out-of-focus fluorescence.
Photo-damage and bleaching are confined to diffractionlimited spot.
Multiple fluorochrome excitation allows simultaneous,
diffraction-limited, co-localization.
Imaging of UV-excited compounds with conventional optics.
Two-photon imaging of exocytosis
in pancreatic acinar cells
Exocytic events evoked by addition of
acetylcholine
Single-cell imaging in intact lymph node
100 m
PF
AL
DC
TZ
M
C
25 m
EL
5 m
15 m
Miller et al., 2002. Science 296: 1869-1873
Another solution – Total Internal Reflection
(TIRF) Microscopy
Excite fluorescence in only a very thin layer right next to a
coverglass
Good for looking at things happening in or very near the plasma
membrane of a cell
Total internal reflection microscopy
air
glass
glass
Total internal reflection microscopy
Evanescent wave
air
glass
Through-the-lens total internal reflection fluorescence microscopy
(TIRFM)
© Molecular Expressions Microscopy Primer
Cultured cells expressing GFP-tagged membrane protein imaged by
conventional epifluorescence
The same cells viewed by TIRFM
TIRFM imaging of single-channel Ca2+ fluorescence signals
(SCCaFTs): Ca2+ entry through plasma membrane channels
expressed in Xenopus oocytes
Imaging single channel events with high time
resolution: SCCaFTs recorded at 500 frames s-1
The diffraction limit
© Molecular Expressions Microscopy Primer
Sidling around the diffraction limit
The position of a single point source (e.g. a fluorescent molecule) can be
localized with much higher precision, limited only by the number of
photons that can be collected.
What we then need is to have only sparse sources at any given time, so as to
avoid unresolved overlap
Photoactivation Localization Microscopy
(PALM)
(Betzig et al., Science 2006)
• Express protein of interest tagged with a photoactivatable fluorescent
protein (eg.g. EOS) in cell
• Stochastically photoactivate a low density of molecules per frame and
localize using Gaussian function
Excitation laser
532 nm
Activating laser
405 nm
Non-fluorescent state
Fluorescence
emission
active state
Bleached
state
Repeat thousands of times
Photoactivation Localization Microscopy
(PALM)
• Express protein of interest tagged with a photoactivatable fluorescent
protein (eg.g. EOS) in cell
• Stochastically photoactivate a low density of molecules per frame and
localize using Gaussian function
Imaging actin tagged with EosFP (photoactivatable protein)
Eos-actin TIRF
Eos-actin PALM
Fibroblasts expressing DsRed
“Greening” of DsRed
“greening” results from enhancement of green fluorescence
and reduction of red fluorescence
Clustered T cells after activation
Evanescent field excites Ca2+-dependent fluorescence
only in a thin layer next to cell membrane
Ca2+ indicator (fluo-4)
in cytosol