Optogenetics involves using optics to activate or inhibit genetics using an “opsin”.
Opsins are modified proteins that can be activated/inhibited by light,
and are artificially introduced into a specimen of interest (analogous
to GFP expression). Narrow wavelengths are important for this in order
to specifically activate opsins. The result of the activation/inhibition
is often observed via fluorescence microscopy. Use of channelrhodopsins
in living cells enables control of intracellular activity, ion flux,
and other cellular processes in response to blue light. ChR2
channelrhodopsin absorbs light at 480nm and a conformational change
opens the channel to allow ions to flow into the cell. Channelrhodopsin
(ChR, ChR2) is often used in conjunction with halorhodopsin, which is a
chloride pump that can be activated with yellow light.
Channelrhodopsin and Halorohopsin are typically used in tandem to cause
activation or inhibition of downstream activity, respectively.
Opsins can also be fused to fluorescence proteins and altered to open
and close when exposed to a specific wavelength of light. They can be
expressed in cells through several transfection techniques for
applications such as depolarization of neuronal cells, neuron
photostimulation or optogenetics.
Read how the X-Cite XLED1 was used
to activate and inhibit halorhodopsin in zebrafish.
CALI (Chromophore-Assisted Laser Inactivation)
CALI is a procedure used to inactivate specific proteins with high
spatial and temporal precision. The chromophore is illuminated with a
strong light, generating short-lived reactive oxygen species that
inactivate proteins in its vicinity. There are genetically coded
molecules that can cause protein inactivation using widefield
illumination with no lasers required.
A photoactivatable engineered variant of Green Fluorescent Protein
(GFP) used to study calcium levels in cells. Cameleon is a sensor
protein that contains a calmodulin (CALcium MODULated proteIN)
component. When calcium binds to this calmodulin component, it undergoes
a conformational change leading to emission of light at an altered
wavelength. The first cameleon was created using Blue Fluorescent
Protein (BFP), Green Fluorescent Protein (GFP),
calmodulin and a calmodulin-binding peptide. When Calcium binds to this
calmodulin-binding peptide, it brings the two separated BFP and GFP in
close proximity to one another, increasing their FRET efficiency.
Kaede protein is a photoactivatable fluorescent protein naturally originated from a stony coral, Trachyphyllia geoffroyi.
When irradiated with UV or violet light (350-400 nm), Kaede undergoes
irreversible photoconversion from green to red fluorescence. Kaede’s
tetramerization makes it unlikely to aggregate when fused to other
proteins. In its unactivated state, when excited at 480nm, it emits
fluorescence with a peak at 519nm. When activated with UV light, its
green absorption peak is red shifted to 570nm. Kaede can be used as an
optical marker for protein labeling and gene expression to study cell
behavior. It is particularly useful in providing a way to view neurons
individually. Photoactivating one neuron in relation to another can help
separate the neuronal processes by color, creating a colorful network
of distinguishable neurons.
PA-GFP (Photoactivatable Green Fluorescent Protein)
Photoactivatable GFP (green fluorescent protein) is a variant of GFP. As described in the
section, GFP has a major excitation peak at a 395 nm and a minor one at
475 nm. Intense illumination of the variant PA-GFP at 400nm shifts the
peak absorbance to 475nm, causing a 100-fold increase in green
fluorescence when excited at 488 nm. PA-GFP offers a new way of studying
intracellular protein dynamics by tracking only those photoactivated
molecules that are present and visible within the cell. Following
activation, there is an increase in fluorescence intensity at the
activation site. The redistribution of these molecules can be studied
using time-lapse imaging.
Uncaging in Fluorescence Imaging
Caged compounds are biologically inactive substances that contain a
photoactivatable group. Uncaging occurs upon absorption of an
appropriate photon, causing cleavage of the caged group which releases
the active biological substance. This makes it possible to study events
that are dependent on exposure to a certain chain of events without need
of all the events. This technique enables precise spatial and temporal
control of activation of a signal molecule, providing answers to
neurological questions and even drug delivery methods. Read how the
is used in an uncaging application alongside the
to help determine how much power was delivered to the sample to allow for uncaging to occur.