Functional cellular imaging by light microscopy MICROSCOPIES.
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Transcript of Functional cellular imaging by light microscopy MICROSCOPIES.
Functional cellular imaging by light microscopy
MICROSCOPIES
Why use Light?
Good (enough) resolution:Spatial – classically a few hundred nanometers; now tens of nmTemporal - <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 andGreat 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 microscopyOut-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. Increased penetration of infrared light allows deeper imaging.
2. No out-of-focus fluorescence.3. Photo-damage and bleaching are confined to diffraction-
limited spot.4. Multiple fluorochrome excitation allows simultaneous,
diffraction-limited, co-localization.5. Imaging of UV-excited compounds with conventional optics.
Two-photon imaging of exocytosis in pancreatic acinar cells
Exocytic events evoked by addition of acetylcholine
C25 m
5 m15 m
DC
EL
AL
M
PF
TZ
100 m
Single-cell imaging in intact lymph node
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
glass
air
glass
Total internal reflection microscopy
glass
air
Evanescent wave
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
© Molecular Expressions Microscopy Primer
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
Sidling around the diffraction limit
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
active state
Fluorescence emission
Bleached state
Activating laser405 nm
Excitation laser532 nm
Repeat thousands of times
Non-fluorescent state
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 TIRFEos-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