wavelengths under 530 nm will activate it, which leaves only a
few wavelengths free for imaging. Commonly, we keep CRY2
fused to mCherry to quantify the optogenetic perturbation.
Additional biological readouts make use of fluorophores in the
“far-red” domain (excitation ~640 nm, emission ~670 nm)
such as iFP1 and upgraded iFP2 proteins [24].
However, in order to bypass this limitation regarding to
wavelengths, two alternative strategies can be used. First, once
the optogenetic effect is characterized, one can remove the
mCherry protein fused to CRY2 and use it for another molecular reporter. Using this strategy, we managed to do optogenetic experiments while looking at two extra proteins tagged
with mCherry and iFP2 respectively doing transient transfection with four plasmids. Second, it is possible to use the GFP
channel to look at an extra readout. When the local activation is
strong enough for the cytoplasm to be almost completely
depleted in CRY2 protein, it is possible to acquire images in
the GFP channel and visualize a GFP-fused protein without
significantly modifying the CRY2 subcellular localization. This
is at the condition that these blue illuminations are rare in time,
with small exposure time and laser power. This second
approach also requires to remove GFP from CIBN or to look
at a protein fused to GFP with a localization or expression levels
different than GFP-CIBN-CAAX.
6. Our approach to shine blue light using a FRAP module is not
the only way to induce subcellular optogenetic activation.
Other strategies are also commonly used in laboratories to
illuminate restricted areas in a field of view. A very powerful
option makes use of a Digital Micromirror Device (DMD). The
light source required is not necessarily a laser but can also be a
fluorescence lamp or LEDs, and it allows to shine patterns of
light almost instantaneously. Moreover, with a DMD one can
easily create complex patterns and gradients of light, which
would need a strong implementation in the case of a FRAP
module [14]. However, it also has drawbacks: in most cases,
even if most of the light will be shone in the region of interest, a
little amount of it will be shone everywhere in the field of view
(<5%). Finally, a simple and inexpensive way to shine local
patterns of blue light is to add an automatized square diaphragm with controlled aperture size in the light path of a
spinning disk or epifluorescence microscope. By creating a
channel for imaging at 488 nm in which the diaphragm is
kept almost closed, it allows to shine squares of blue light
only in the central part of the field of view [15]. This configuration is not versatile in terms of ROI shape but allows the
control of exposure time and laser power, and is very convenient for preliminary tests on new optogenetic constructs or
422
Leo Valon and Simon de Beco
few wavelengths free for imaging. Commonly, we keep CRY2
fused to mCherry to quantify the optogenetic perturbation.
Additional biological readouts make use of fluorophores in the
“far-red” domain (excitation ~640 nm, emission ~670 nm)
such as iFP1 and upgraded iFP2 proteins [24].
However, in order to bypass this limitation regarding to
wavelengths, two alternative strategies can be used. First, once
the optogenetic effect is characterized, one can remove the
mCherry protein fused to CRY2 and use it for another molecular reporter. Using this strategy, we managed to do optogenetic experiments while looking at two extra proteins tagged
with mCherry and iFP2 respectively doing transient transfection with four plasmids. Second, it is possible to use the GFP
channel to look at an extra readout. When the local activation is
strong enough for the cytoplasm to be almost completely
depleted in CRY2 protein, it is possible to acquire images in
the GFP channel and visualize a GFP-fused protein without
significantly modifying the CRY2 subcellular localization. This
is at the condition that these blue illuminations are rare in time,
with small exposure time and laser power. This second
approach also requires to remove GFP from CIBN or to look
at a protein fused to GFP with a localization or expression levels
different than GFP-CIBN-CAAX.
6. Our approach to shine blue light using a FRAP module is not
the only way to induce subcellular optogenetic activation.
Other strategies are also commonly used in laboratories to
illuminate restricted areas in a field of view. A very powerful
option makes use of a Digital Micromirror Device (DMD). The
light source required is not necessarily a laser but can also be a
fluorescence lamp or LEDs, and it allows to shine patterns of
light almost instantaneously. Moreover, with a DMD one can
easily create complex patterns and gradients of light, which
would need a strong implementation in the case of a FRAP
module [14]. However, it also has drawbacks: in most cases,
even if most of the light will be shone in the region of interest, a
little amount of it will be shone everywhere in the field of view
(<5%). Finally, a simple and inexpensive way to shine local
patterns of blue light is to add an automatized square diaphragm with controlled aperture size in the light path of a
spinning disk or epifluorescence microscope. By creating a
channel for imaging at 488 nm in which the diaphragm is
kept almost closed, it allows to shine squares of blue light
only in the central part of the field of view [15]. This configuration is not versatile in terms of ROI shape but allows the
control of exposure time and laser power, and is very convenient for preliminary tests on new optogenetic constructs or
422
Leo Valon and Simon de Beco