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Printable Handouts
Navigable Slide Index
- Introduction
- Talk structure (1)
- Time-resolved imaging in fluorescence microscopy of the brain
- Example: measuring intracellular [Ca2+] using ratiometric dyes
- Ratiometric indicators in brain tissue (1)
- Ratiometric indicators in brain tissue (2)
- Talk structure – Part 1
- Fluorescence lifetime imaging (FLIM): a time-domain measure
- FLIM: Oregon Green 488 BAPTA-1 (OGB-1)
- FLIM: Calibration of OGB-1 Ca2+ sensitivity
- Typical imaging system setting for FLIM measurements in live brain tissue
- FLIM: monitoring neuronal and astrocytic Ca2+ in acute brain slices
- Monitoring Ca2+ landscapes in CA1 pyramidal cells (1)
- Monitoring Ca2+ landscapes in CA1 pyramidal cells (2)
- Monitoring Ca2+ landscapes in CA1 pyramidal cells (3)
- Fast FLIM (dendritic spike recording)
- Direct comparison of basal Ca2+ in neurons and astroglia with 3D FLIM (quiescent tissue)
- Ca2+ landscapes in gap-junction-connected (unperturbed) astrocytes
- Interim conclusion
- Talk structure – Part 2
- Rationale
- Time-resolved fluorescence anisotropy imaging (TR-FAIM): first principles
- TR-FAIM: first principles
- Question (1)
- Measuring translational D in brain slices
- Comparing rotational and translation diffusion measurements for AF350
- TR-FAIM map of extracellular diffusivity in the slice
- Question (2)
- TR-FAIM map of intracellular diffusivity in CA3 pyramids
- Exploring the structure individual MF – CA3 synapses
- Exploring the structure individual MF – CA3 synapses (2)
- Co-ordinated intra- extracellular TR-FAIM nano-diffusivity maps (1)
- Co-ordinated intra- extracellular TR-FAIM nano-diffusivity maps (2)
- Take home message
- Acknowledgements
Topics Covered
- Measuring intracellular [Ca2+]
- Ratiometric indicators in brain tissue
- Fluorescence lifetime imaging (FLIM)
- Monitoring Ca2+ landscapes
- Fast FLIM
- Time-resolved fluorescence anisotropy imaging (TR-FAIM)
Talk Citation
Rusakov, D. (2024, June 30). Time-resolved fluorescence microscopy in high-resolution brain imaging [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved October 13, 2024, from https://doi.org/10.69645/TUKR5222.Export Citation (RIS)
Publication History
Financial Disclosures
- Prof. Dmitri Rusakov has not informed HSTalks of any commercial/financial relationship that it is appropriate to disclose.
A selection of talks on Methods
Transcript
Please wait while the transcript is being prepared...
0:00
Today, we'll talk about
time-resolved
fluorescence microscopy
in high-resolution
brain imaging.
0:10
This lecture will
consist of two parts;
the first one will be about
fluorescence lifetime
imaging, or FLIM,
exemplified by the monitoring
of intracellular nanomolar
calcium concentrations,
and the second
part will be about
time-resolved fluorescence
anisotropy imaging which
you'll try to explain by
measuring molecular
mobility on the nanoscale.
0:41
First, we have to
understand why and when we
actually need
time-resolved imaging
inflorescence microscopy
of the brain.
Well, you probably know that
in the majority of cases,
fluorescent indicators increase
their emission intensity
upon binding to the signal
molecule of interest.
However, the emission
intensity signal
might depend directly on
the local concentration of
the indicator as well as
on the optical properties
of the tissue.
This actually makes it pretty
difficult to assess
quantitatively how
much of the signal and molecule
is reported by the
fluorescence indicator,
or historically,
concentration measurements
of signaling molecules
were, therefore, achieved using
so-called ratiometric
indicators.
The ratiometric
indicators changed
either the absorption or
emission spectrum
upon binding to
the signaling molecule so that
the ratio between
different parts
of the emission spectrum
reports the signaling
molecule concentration which,
therefore, does not depend on
the indicator concentration.
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