2. What is Photoactivation?
Photoactivation is any change in the activity of a
protein brought about by irradiating it.
In the context of Biophysics, protein
photoactivation mostly refers to the change
in fluorescent profiles of a protein upon
irradiation with light of a certain frequency.
This property of certain proteins serve as
useful probing tools in Biophysics.
4. Fluorescent Proteins
• Fluorescent proteins exhibit fluorescence due to the
presence of a fluorophore in the structure, which is
responsible for fluorescing.
• One can artificially attach a fluorophore to a protein to
make it fluoresce.
• Suppose our fluorophore itself is a protein, then it can be
genetically encoded alongside the protein we want it to
get attached to, so that every time the protein under
study is synthesized, fused to it, the fluorophore will be
synthesized too.
6. Green Fluorescent Proteins
Green Fluorescent Proteins
(GFPs) were first discovered in
the jellyfish Aequorea victoria.
A protein called aequorin
interacts with Ca2+ ions, inducing
a blue glow.
Some of this luminescent energy
is transferred to the GFP which
fluoresces green light upon
absorbing blue light.
Overall color is shifted towards
green.
Fluorophore
Beta-
can
A slight change in the structure of Beta-can can give a
new fluorescent protein- green, yellow, orange, red
8. Photoactivation
• Kaede protein was discovered to emit red fluorescence after being left on
the bench and exposed to sunlight. Kaede, which is originally green
fluorescent, after exposure to UV light is photoconverted, becoming red
fluorescent.
• EosFP : Gene encoding a fluorescent protein from the stony coral
Lobophyllia hemprichii was cloned a in Escherichia. It was found to emit
strong green fluorescence (516 nm) that changes to red (581 nm) upon
near-UV irradiation at ~390 nm due to a photoinduced break in the peptide
backbone next to the fluorophore.
• A red-emitting, green-absorbing fluorescent state of GFP can be generated
by photoactivation with blue light easily with a laser or fluorescence
microscope lamp under conditions of low oxygen concentration.
• These processes are irreversible.
9. Photoactivation of GFP
Photoactivation of GFP in vivo. GFP-labelled E. coli cells appear as viewed through fluorescein (left) and rhodamine
(right) filter sets. GFP in cells on the lower half of the images has been photoactivated by exposure to 475–495 nm
light. Photoactivated cells appear yellow through the fluorescein filters, because they emit continuously from green to red
with local maxima at 510, 560, 590 and 600 nm.
11. Application: Protein Marker
• We can specifically mark proteins of interest within a living cell.
Fluorescent proteins are now available that allow a pool of
molecules to be “turned on” by photoactivation.
• Suppose we want to track the fate of a particular set of protein
molecules as compared to all the other molecules of that protein in
the cell, we can genetically tag the proteins with photoactivatable
fluorescent proteins. Then, we can photoactivate a small patch using
a laser, and then view the progress of the molecules that were
present in that patch since now they appear against the background
in a different colour.
• This process is mostly used to find protein diffusion rates.
• Protein photoactivation is also generally used to obtain red
fluorescence in fluorescence spectroscopy/sorting techniques.
13. Application: Protein Marker
(A) Circular region of interest selected with an Olympus FV1000
tornado scanner is illuminated at 405 nm for 5 seconds, t=0
minutes.
(B) The photoactivated actin chimera first translocates to the
ruffles at the cellular margins as fluorescence intensity
decreases in the activated region, t=5 minutes.
(C) Ruffles, cytoplasmic actin pools and the filamentous actin
network gain more intensity at t=60 Minutes.
(D) Photoconversion of a single mitochondrion (red) in a
selected region at 405 nm illumination, t=0 minutes.
(E) Close approach of a non-converted (green) mitochondrion
(arrow), t=10 minutes.
(F) Cargo exchange between mitochondria (arrow), t=20
minutes. (G-I) Examination of lamellipodia
with Dendra2-actin-C-7 in OK cells.
(J) Actin network imaged with a 488-nm laser, t=0 minutes. (K)
After completely photoswitching the labeled actin ‘off’ at 488 nm,
the region spelling FV10 was activated with a 405 nm laser, t=3
minutes.
(L) FV10 region photobleached while imaging the actin network
at 488 nm.
14. Application: Two-colour super-
resolution microscopy using PALM
Stochastic activation of
fluorescence to intermittently
photoswitch individual
photoactivatable molecules to a
bright state, which are then
imaged and photobleached.
Thus, very closely spaced
molecules that reside in the same
diffraction-limited volume (and
would otherwise be spatially
indistinguishable are temporally
separated.
We merge all of the single-
molecule positions obtained by
repeated cycles of
photoactivation, follow by imaging
and bleaching to produce the final
super-resolution.
15. Application: Two-colour super-
resolution microscopy using PALM
The prior knowledge that the diffraction-
limited image of a molecule originates from
a single source enables the estimation of the
location (center) of that molecule with a
precision well beyond that of the diffraction
limit.
The molecular coordinates are not actually
represented by a single point to identify their
spatial position, rather a Gaussian intensity
distribution corresponding to the positional
uncertainty of their location is employed to
build the image map.
A cooled electron-multiplying charge-
coupled device (EMCCD) camera system.
Central to the instrumentation is a high-
speed computer system that acquires and
processes images recorded by the camera.
16. Application: Two-colour super-
resolution microscopy using PALM
There is very little direct overlap between actin and paxillin (white arrowheads),
although actin bundles densely cluster around several fibrillar paxillin adhesions (white
arrows). In widefield TIRF imaging, actin and paxillin appear to co-localize in focal
adhesions, contrary to the information afforded by super-resolution microscopy.
17. Application: Two-colour super-
resolution microscopy using PALM
• To overcome the diffraction barrier we may employ photo-switchable
fluorescent probes to resolve spatial differences in dense
populations of molecules with super-resolution.
• Pulse-Chase experiments with super-resolution
• Live-Cell Imaging with PALM
- very fast switching
- more intense light
- disadvantages of photobleaching
18. Application: In Two-photon microscopy
• If paGFP-labeled signaling molecules are visualized by means of
two-photon activation, the position of the activation volume in the
tissue can be determined precisely.
• An organelle can be marked via photoactivation.
• Two-photon microscopy allows us to image layers at various depths.
• All the local information can be combined to get the full 3D picture of
the organ.
19. Application: light-based mapping of
motor cortex
• Traditionally, mapping the motor cortex requires electrodes to stimulate the
brain and define motor output pathways. Although effective, electrode-
based methods are labour-intensive, potentially damaging to the cortex and
can have off-target effects. As an alternative method of motor mapping, we
photostimulated transgenic mice expressing the light-sensitive ion channel
channelrhodopsin-2 in predominantly layer-5 output cortical neurons. We
report that optical stimulation of these neurons in vivo using a stage
scanning laser system resulted in muscle excitation within 10-20 ms, which
can be recorded using implanted electromyogram electrodes or by a non-
invasive motion sensor. This approach allowed us to make highly
reproducible automated maps of the mouse forelimb and hind limb motor
cortex much faster than with previous methods. We anticipate that the
approach will facilitate the study of changes in the location and properties of
motor maps after skilled training or damage to the nervous system.
Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.
Ayling OG, Harrison TC, Boyd JD, Goroshkov A, Murphy TH.
20. Application: A Photoactivation ‘System’
Elizabeth Pham, Evan Mills and Kevin Truong
• Through tightly regulated Ca2+ concentration fluxes varying in location,
time, frequency, and amplitude, Ca2+ signals regulate diverse and distinct
physiological processes.
• The ability to generate local and global Ca2+ signals would allow us to
better study the Ca2+ specificity problem and to engineer synthetic proteins
that respond to specific Ca2+ signals.
• Using light to generate these Ca2+ signals is particularly desirable because
light causes minimal damage to live cells and has the potential for high
temporal and spatial resolution.
• Here, we developed LOVS1K, a genetically encoded and photoactivated
synthetic protein to reversibly generate local or global Ca2+ signals.
21. Application: A Photoactivation ‘System’
With 300 ms blue light exposure, LOVS1K translocated
to Orai1, a plasma membrane Ca2+ channel, within
seconds, generating a local Ca2+ signal on the plasma
membrane, and returning to the cytoplasm after 10sec.
A structural change produced by exposure to blue light
brings about a change in the binding ability.
22. Application: A Photoactivation ‘System’
We begin with an already
photoactivated protein and study its
interaction with the receptors under
various conditions. Whenever the
protein binds receptors, the cell
membrane becomes wholly red, since
the colour red dominates over the
greenish colour of the fluorescent tag of
the membrane.
Reversible calcium flux (false coloured)…
23. Application: Photolithography
Purified EosFP was cosslinked
by paraformaldehyd on a nitrocellulose
membrane. Based on a photography of
Albert Einstein (upper left), a mask was
created. The upper right picture shows
the unconverted green fluorescent
protein on the membrane under the
mask. After irradiation for 3 min with UV
light, the membrane was photographed
(lower left). The red fluorescence of the
photoconverted protein in the lower right
picture was excited at 540 nm and
photographed using a long pass filter
with a cut off at 590 nm.
**An interesting
prospective for data
storage on the
molecular level
24. •Wiedenmann et al.- From EosFP to mIrisFP: structure-based development of
advanced photoactivatable marker proteins of the GFP-family.
•George H. Patterson -Photoactivation and Imaging of Photoactivatable
Fluorescent Proteins
•Advances in fluorescent protein technology
Nathan C.
•Elizabeth Pham, Evan Mills and Kevin Truong- A Synthetic Photoactivated Protein
to Generate Local or Global Ca2+ Signals
•John Runions, Thorsten Brach, Sebastian Ku¨hner and Chris Hawes-
Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum
membrane.
•Fuchs, Wiedenmann, Nienhaus et al. A photoactivatable marker protein for pulse-
chase imaging with super-resolution
•www.scholarpedia.org
•www.wikipedia.org
References
