bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2023,
Номер
unknown
Опубликована: Ноя. 10, 2023
Microstructures
arrayed
over
a
substrate
have
shown
increasing
interest
due
to
their
ability
provide
advanced
3D
cellular
models,
which
open
new
possibilities
for
cell
culture,
proliferation,
and
differentiation.
Still,
the
mechanisms
by
physical
cues
impact
phenotype
are
not
fully
understood,
hence
necessity
interrogate
behavior
at
highest
resolution.
However,
high-resolution
optical
imaging
on
such
microstructured
substrates
remains
challenging
complexity,
as
well
axial
calibration
issues.
In
this
work,
we
address
issue
leveraging
self-referenced
characteristics
of
fractal-like
structures,
simultaneously
modulate
growth
serve
tools.
To
end,
use
multiscale
SiO
2
consisting
spatially
octahedral
features
few
micrometers
hundreds
nanometers.
Through
optimizations
both
structures
conditions,
demonstrate
potential
these
tools
super-resolution
microscopy.
We
intrinsic
nature
perform
lateral
calibrations
in
single-molecule
localization
microscopy
(SMLM)
assess
resolutions.
then
utilize
platform
bioimaging.
As
proof
concept,
cultivate
human
mesenchymal
stem
cells
substrates,
revealing
very
different
patterns
compared
flat
glass.
Specifically,
spatial
distribution
cytoskeleton
proteins
is
vastly
modified,
with
SMLM
assessment.
Nano Letters,
Год журнала:
2022,
Номер
22(15), С. 6454 - 6461
Опубликована: Июль 6, 2022
A
recent
addition
to
the
toolbox
of
super-resolution
microscopy
methods
is
fluorescence-lifetime
single-molecule
localization
(FL-SMLM).
The
synergy
SMLM
and
imaging
(FLIM)
combines
superior
image
resolution
with
lifetime
information
can
be
realized
using
two
complementary
experimental
approaches:
confocal-laser
scanning
(CLSM)
or
wide-field
microscopy.
Here,
we
systematically
comprehensively
compare
these
novel
FL-SMLM
approaches
in
different
spectral
regions.
For
FL-SMLM,
use
a
commercial
camera,
for
CLSM-based
employ
home-built
system
equipped
rapid
scan
unit
single-photon
detector.
We
characterize
performances
systems
localizing
single
emitters
3D
by
combining
metal-induced
energy
transfer
(MIET)
along
third
dimension
lifetime-based
multiplexed
bioimaging
DNA-PAINT.
Finally,
discuss
advantages
disadvantages
confocal
provide
practical
advice
on
rational
experiment
design.
Annual Review of Physical Chemistry,
Год журнала:
2024,
Номер
75(1), С. 163 - 183
Опубликована: Фев. 16, 2024
By
superlocalizing
the
positions
of
millions
single
molecules
over
many
camera
frames,
a
class
super-resolution
fluorescence
microscopy
methods
known
as
single-molecule
localization
(SMLM)
has
revolutionized
how
we
understand
subcellular
structures
past
decade.
In
this
review,
highlight
emerging
studies
that
transcend
outstanding
structural
(shape)
information
offered
by
SMLM
to
extract
and
map
physicochemical
parameters
in
living
mammalian
cells
at
levels.
encoding/decoding
high-dimensional
information-such
emission
excitation
spectra,
motion,
polarization,
lifetime,
beyond-for
every
molecule,
mass
accumulating
these
measurements
for
molecules,
such
multidimensional
multifunctional
approaches
open
new
windows
into
intracellular
architectures
dynamics,
well
their
underlying
biophysical
rules,
far
beyond
diffraction
limit.
Light Science & Applications,
Год журнала:
2023,
Номер
12(1)
Опубликована: Март 10, 2023
3D
super-resolution
microscopy
with
nanometric
resolution
is
a
key
to
fully
complement
ultrastructural
techniques
fluorescence
imaging.
Here,
we
achieve
by
combining
the
2D
localization
of
pMINFLUX
axial
information
graphene
energy
transfer
(GET)
and
single-molecule
switching
DNA-PAINT.
We
demonstrate
<2
nm
precision
in
all
3
dimension
reaching
below
0.3
nm.
In
DNA-PAINT
measurements,
structural
features,
i.e.,
individual
docking
strands
at
distances
nm,
are
directly
resolved
on
DNA
origami
structures.
GET
represent
particular
synergetic
combination
for
imaging
near
surface
such
as
cell
adhesion
membrane
complexes
each
photon
used
both
information.
Furthermore,
introduce
local
PAINT
(L-PAINT),
which
imager
equipped
an
additional
binding
sequence
upconcentration
improving
signal-to-background
ratio
speed
clusters.
L-PAINT
demonstrated
triangular
structure
6
side
lengths
within
seconds.
Angewandte Chemie International Edition,
Год журнала:
2024,
Номер
63(10)
Опубликована: Янв. 2, 2024
Water
molecules
play
an
important
role
in
the
structure,
function,
and
dynamics
of
(bio-)
materials.
A
direct
access
to
number
water
nanoscopic
volumes
can
thus
give
new
molecular
insights
into
materials
allow
for
fine-tuning
their
properties
sophisticated
applications.
The
determination
local
content
has
become
possible
by
finding
that
H
Nature Communications,
Год журнала:
2024,
Номер
15(1)
Опубликована: Фев. 27, 2024
Abstract
Out-of-plane
fluctuations,
also
known
as
stochastic
displacements,
of
biological
membranes
play
a
crucial
role
in
regulating
many
essential
life
processes
within
cells
and
organelles.
Despite
the
availability
various
methods
for
quantifying
membrane
dynamics,
accurately
complex
systems
with
rapid
tiny
such
mitochondria,
remains
challenge.
In
this
work,
we
present
methodology
that
combines
metal/graphene-induced
energy
transfer
(MIET/GIET)
fluorescence
correlation
spectroscopy
(FCS)
to
quantify
out-of-plane
fluctuations
simultaneous
spatiotemporal
resolution
approximately
one
nanometer
microsecond.
To
validate
technique
resolution,
measure
bending
undulations
model
membranes.
Furthermore,
demonstrate
versatility
applicability
MIET/GIET-FCS
studying
diverse
systems,
including
widely
studied
fluctuating
system
human
red
blood
cells,
well
two
unexplored
pore-spanning
membrane,
mitochondrial
inner/outer
Annual Review of Physical Chemistry,
Год журнала:
2023,
Номер
74(1), С. 391 - 414
Опубликована: Фев. 8, 2023
Super-resolution
fluorescence
microscopy
techniques
are
powerful
tools
to
investigate
polymer
systems.
In
this
review,
we
address
how
these
have
been
applied
hydrogel
nano-
and
microparticles,
so-called
or
microgels.
We
outline
which
research
questions
on
microgels
could
be
addressed
what
new
insights
achieved.
Studies
of
the
morphology,
shape,
deformation
microgels;
their
internal
compartmentalization;
cross-linker
distribution
polarity
inside
them;
dynamics
diffusion
summarized.
particular,
abilities
super-resolve
structures
in
three
dimensions
boosted
field
also
allowed
researchers
obtain
impressive
3D
images
deformed
Accessing
information
beyond
localization,
such
as
spectral
lifetime
properties
correlative
imaging
combination
data
with
other
methods,
shines
light
onto
systems
helps
us
understand
complexity
detail.
Such
future
trends
developments
addressed.
Journal of the American Chemical Society,
Год журнала:
2024,
Номер
146(29), С. 19756 - 19767
Опубликована: Июль 11, 2024
In
recent
work
to
develop
cyanine
dyes
with
especially
large
Stokes
shifts,
we
encountered
a
"blueing"
reaction,
in
which
the
heptamethine
dye
Cy7
(IUPAC:
1,3,3-trimethyl-2-((1E,3E,5E)-7-((E)-1,3,3-trimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3H-indol-1-ium)
undergoes
shortening
two-carbon
steps
form
pentamethine
(Cy5)
and
trimethine
(Cy3)
analogs.
Each
step
blue-shifts
resulting
absorbance
wavelength
by
ca.
100
nm.
Though
photochemical
oxidative
chain-shortening
reactions
had
been
noted
previously,
it
is
simple
heating
alone
or
amine
bases
that
effects
this
unexpected
net
C2H2
excision.
Explicit
acetylene
loss
would
be
too
endothermic
merit
consideration.
Our
mechanistic
studies
using
2H
labeling,
mass
spectrometric
NMR
spectroscopic
analyses,
quantum
chemical
modeling
point
instead
electrocyclic
closure
aromatization
of
chain
forming
Fischer's
base
FB
(1,3,3-trimethyl-2-methyleneindoline),
reactive
carbon
nucleophile
initiates
attack
on
their
polymethine
backbones.
The
byproduct
cationic
indolium
species
TMP
1,3,3
trimethyl-2-phenyl
indolium).
ACS Nano,
Год журнала:
2023,
Номер
17(9), С. 8242 - 8251
Опубликована: Март 30, 2023
Metal-induced
energy
transfer
(MIET)
imaging
is
an
easy-to-implement
super-resolution
modality
that
achieves
nanometer
resolution
along
the
optical
axis
of
a
microscope.
Although
its
capability
in
numerous
biological
and
biophysical
studies
has
been
demonstrated,
implementation
for
live-cell
with
fluorescent
proteins
still
lacking.
Here,
we
present
applicability
capabilities
diverse
cell
types
(adult
human
stem
cells,
osteo-sarcoma
Dictyostelium
discoideum
cells),
various
(GFP,
mScarlet,
RFP,
YPet).
We
show
MIET
axial
mapping
living
cellular
subcellular
components
across
multiple
time
scales,
from
few
milliseconds
to
hours,
negligible
phototoxic
effects.