International Journal of Molecular Sciences,
Год журнала:
2024,
Номер
25(23), С. 12493 - 12493
Опубликована: Ноя. 21, 2024
The
calcium
cation
is
a
crucial
signaling
molecule
involved
in
numerous
cellular
pathways.
Beyond
its
role
as
messenger
or
modulator
intracellular
cascades,
calcium's
function
excitable
cells,
including
nerve
impulse
transmission,
remarkable.
central
of
nervous
activity
has
driven
the
rapid
development
fluorescent
techniques
for
monitoring
this
living
cells.
Specifically,
genetically
encoded
indicators
(GECIs)
are
most
in-demand
molecular
tools
their
class.
In
work,
we
address
two
issues
imaging
by
designing
based
on
successful
GCaMP6
backbone
and
protein
BrUSLEE.
first
indicator
variant
(GCaMP6s-BrUS),
with
reduced,
calcium-insensitive
fluorescence
lifetime,
potential
dynamics
high
temporal
resolution
combination
advanced
microscopy
techniques,
such
light
beads
microscopy,
where
lifetime
limits
acquisition
speed.
Conversely,
second
(GCaMP6s-BrUS-145),
flexible,
calcium-sensitive
relevant
static
measurements,
particularly
determining
absolute
concentration
values
using
(FLIM).
To
identify
structural
determinants
sensitivity
these
variants,
determine
spatial
structures.
A
comparative
analysis
allowed
optimization
GCaMP6s-BrUS
construct,
resulting
an
combining
behavior
time
domain
enhanced
brightness.
Our
data
may
serve
starting
point
further
engineering
efforts
towards
improved
GECI
variants
fine-tuned
lifetimes.
Fluorescence
microscopy
is
nowadays
one
of
the
most
diffused
techniques
to
study
photophysical
processes
and
molecular
interactions
in
both
biomedical
material
science
fields.
Since
each
fluorophore
characterized
by
a
specific
emission
spectrum
lifetime,
it
essential
not
only
spatially
localize
its
position
but
also
measure
spectral
temporal
properties
with
multispectral
fluorescence
lifetime
imaging
microscope
(λFLIM).
Moreover,
rapidly
evolving
samples,
measurement
system
capable
fast
acquisitions
needed.
λFLIM
systems
are
currently
limited
on
acquisition
speed
cannot
reach
high
throughput.
In
this
work,
we
propose
novel
wide-field
based
16-channel
silicon
photomultiplier
(SiPM)
array.
Our
system,
thanks
SiPM
technology
along
single
pixel
camera
compress
sensing
approaches
able
acquire
multidimensional
measurements
(space,
spectrum,
time)
at
frame
rate.
We
validate
moving
fluorescent
samples
capturing
snapshots
rate
up
13
fps.
The
developed
could
enable
enhanced
specificity
real-time
biological
biosensing
applications.
Proceedings of the National Academy of Sciences,
Год журнала:
2024,
Номер
121(40)
Опубликована: Сен. 25, 2024
Fluorescence
lifetime
imaging
microscopy
(FLIM)
is
a
powerful
technique
that
enables
the
visualization
of
biological
samples
at
molecular
level
by
measuring
fluorescence
decay
rate
fluorescent
probes.
This
provides
critical
information
about
interactions,
environmental
changes,
and
localization
within
systems.
However,
creating
high-resolution
maps
using
conventional
FLIM
systems
can
be
challenging,
as
it
often
requires
extensive
scanning
significantly
lengthen
acquisition
times.
issue
further
compounded
in
three-dimensional
(3D)
because
demands
additional
along
depth
axis.
To
tackle
this
challenge,
we
developed
computational
called
light-field
tomographic
(LIFT-FLIM).
Our
approach
allows
for
volumetric
images
highly
data-efficient
manner,
reducing
number
steps
required
compared
to
point-scanning
or
line-scanning
imagers.
Moreover,
LIFT-FLIM
measurement
high-dimensional
data
low-dimensional
detectors,
which
are
typically
low
cost
feature
higher
temporal
bandwidth.
We
demonstrated
linear
single-photon
avalanche
diode
array
on
various
systems,
showcasing
unparalleled
detection
sensitivity.
Additionally,
expanded
functionality
our
method
spectral
its
application
high-content
multiplexed
lung
organoids.
has
potential
open
up
broad
avenues
both
basic
translational
biomedical
research.
Journal of Materials Chemistry C,
Год журнала:
2024,
Номер
12(48), С. 19371 - 19385
Опубликована: Янв. 1, 2024
The
mechanism
of
new
mechanofluorochromic
is
resolved
using
established
and
unexplored
techniques,
including
electron
diffraction
fluorescence
lifetime
mapping.
Driving
factors
posited
include
conformational
flexibility
partial
amorphisation.
Biophysica,
Год журнала:
2024,
Номер
4(2), С. 207 - 226
Опубликована: Апрель 25, 2024
Drug
delivery
systems
play
a
pivotal
role
in
targeted
pharmaceutical
transport
and
controlled
release
at
specific
sites.
Liposomes,
commonly
used
as
drug
carriers,
constitute
fundamental
part
of
these
systems.
Moreover,
the
drug–liposome
model
serves
robust
platform
for
investigating
interaction
processes
both
cellular
molecular
levels.
To
advance
our
understanding
carrier
uptake
mechanisms,
we
employed
fluorescence
lifetime
imaging
microscopy
(FLIM)
correlation
spectroscopy
(FCS),
leveraging
unique
benefits
two-photon
(2P)
excitation.
Our
approach
utilized
giant
unilamellar
vesicles
(GUVs)
simplified
system
cell
membranes,
labelled
with
amphiphilic
fluorescent
dye
3,3′-dioctadecyloxa-carbocyanine
(DiOC18(3)).
Additionally,
large
(LUVs)
functioned
system,
incorporating
spectrally
distinct
sulforhodamine
101
(SRh101)
surrogate
drug.
The
investigation
emphasized
diverse
interactions
between
GUVs
LUVs
based
on
charged
lipids
employed.
We
examined
exchange
kinetics
structural
alterations
liposome
carriers
during
process.
study
underscores
significance
employing
2P
excitation
conjunction
FLIM
FCS.
This
powerful
combination
offers
valuable
methodological
studying
interactions,
positioning
them
an
exceptionally
versatile
technical
advantage.
International Journal of Molecular Sciences,
Год журнала:
2024,
Номер
25(23), С. 12493 - 12493
Опубликована: Ноя. 21, 2024
The
calcium
cation
is
a
crucial
signaling
molecule
involved
in
numerous
cellular
pathways.
Beyond
its
role
as
messenger
or
modulator
intracellular
cascades,
calcium's
function
excitable
cells,
including
nerve
impulse
transmission,
remarkable.
central
of
nervous
activity
has
driven
the
rapid
development
fluorescent
techniques
for
monitoring
this
living
cells.
Specifically,
genetically
encoded
indicators
(GECIs)
are
most
in-demand
molecular
tools
their
class.
In
work,
we
address
two
issues
imaging
by
designing
based
on
successful
GCaMP6
backbone
and
protein
BrUSLEE.
first
indicator
variant
(GCaMP6s-BrUS),
with
reduced,
calcium-insensitive
fluorescence
lifetime,
potential
dynamics
high
temporal
resolution
combination
advanced
microscopy
techniques,
such
light
beads
microscopy,
where
lifetime
limits
acquisition
speed.
Conversely,
second
(GCaMP6s-BrUS-145),
flexible,
calcium-sensitive
relevant
static
measurements,
particularly
determining
absolute
concentration
values
using
(FLIM).
To
identify
structural
determinants
sensitivity
these
variants,
determine
spatial
structures.
A
comparative
analysis
allowed
optimization
GCaMP6s-BrUS
construct,
resulting
an
combining
behavior
time
domain
enhanced
brightness.
Our
data
may
serve
starting
point
further
engineering
efforts
towards
improved
GECI
variants
fine-tuned
lifetimes.
