fluorescence lifetime imaging microscopy (flim · jan 27th / 2017 nick weilinger eli york macvicar...
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![Page 1: Fluorescence Lifetime Imaging Microscopy (FLIM · Jan 27th / 2017 Nick Weilinger Eli York MacVicar Lab. intensity vs lifetime imaging ... (2015) applications Imaging Ca2+ with FLIM:](https://reader036.vdocuments.us/reader036/viewer/2022081612/5f7507c3032ca8775b7e29e1/html5/thumbnails/1.jpg)
Fluorescence Lifetime Imaging Microscopy (FLIM)
DATA BINGE
Jan 27th / 2017
Nick Weilinger
Eli York
MacVicar Lab
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intensity vs lifetime imaging
Zeiss Microscopy Campus
Fluorescence (a refresher)
Recall:
• Absorption of photon leads to
excitation of electron in
fluorophore to excited singlet
state
• Electrons exist in excited
vibrational energy states for ps
to ns before red-shifted radiative
relaxation (emission)
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intensity vs lifetime imaging
Becker & Hickl TCSPC Handbook (6th ed)
Intensity-based fluorescent imaging:
• Can be used to monitor relative changes in fluorophore signal, which
changes proportionately to the amount of input excitation and
concentration
• Cannot be used to directly measure concentration of a fluorophore
binding partner
• e.g. Fluo-4/5/etc, GECIs
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intensity vs lifetime imaging
Mainen et al., Nature (1999)
Becker & Hickl TCSPC Handbook (6th ed)
Intensity-based fluorescent imaging:
• Can be used to monitor relative changes in fluorophore signal, which
changes proportionately to the amount of input excitation and
concentration
• Cannot be used to directly measure concentration of a fluorophore
binding partner
• e.g. Fluo-4/5/etc, GECIs
![Page 5: Fluorescence Lifetime Imaging Microscopy (FLIM · Jan 27th / 2017 Nick Weilinger Eli York MacVicar Lab. intensity vs lifetime imaging ... (2015) applications Imaging Ca2+ with FLIM:](https://reader036.vdocuments.us/reader036/viewer/2022081612/5f7507c3032ca8775b7e29e1/html5/thumbnails/5.jpg)
intensity vs lifetime imaging
Leica Science Lab
Intensity-based fluorescent imaging:
• Ratiometric dyes can be used to directly measure
[cofactor] (e.g. Ca2+)
• Requires differential sensitivity to Ca2+ at either
two excitation OR emission wavelengths
• e.g. fura-2, Indo-1
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intensity vs lifetime imaging
Becker & Hickl TCSPC Handbook (6th ed)
Intensity-based fluorescent imaging:
• Can be used to monitor relative changes in fluorophore signal, which
changes proportionately to the amount of input excitation and
concentration
• Cannot be used to directly measure concentration of a fluorophore
binding partner
• e.g. Fluo-4/5/etc, GECIs
![Page 7: Fluorescence Lifetime Imaging Microscopy (FLIM · Jan 27th / 2017 Nick Weilinger Eli York MacVicar Lab. intensity vs lifetime imaging ... (2015) applications Imaging Ca2+ with FLIM:](https://reader036.vdocuments.us/reader036/viewer/2022081612/5f7507c3032ca8775b7e29e1/html5/thumbnails/7.jpg)
Bastiaens & Squire, TICB (2009)
acquiring a FLIM signal
TCSPC:
Time
Correlated
Single
Photon
Counting
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Becker & Hickl TCSPC Handbook (6th ed)
acquiring a FLIM signal
FLIM requires (in addition to a
microscope):
• Hardware suitable for photon-counting
(coming up)
• Pulsed laser (e.g. Ti:Sa 2P)
• Sensitive detectors (e.g. GaAsp
hybrid detectors)
• Module hub (e.g. Simple-Tau,
Becker & Hickl)
• Computer
• Software
• Coordinate laser scan signals from
laser & scan head
• Acquisition and analysis software
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Becker & Hickl TCSPC Handbook (6th ed)
photon counting
![Page 10: Fluorescence Lifetime Imaging Microscopy (FLIM · Jan 27th / 2017 Nick Weilinger Eli York MacVicar Lab. intensity vs lifetime imaging ... (2015) applications Imaging Ca2+ with FLIM:](https://reader036.vdocuments.us/reader036/viewer/2022081612/5f7507c3032ca8775b7e29e1/html5/thumbnails/10.jpg)
Becker & Hickl TCSPC Handbook (6th ed)
photon counting
![Page 11: Fluorescence Lifetime Imaging Microscopy (FLIM · Jan 27th / 2017 Nick Weilinger Eli York MacVicar Lab. intensity vs lifetime imaging ... (2015) applications Imaging Ca2+ with FLIM:](https://reader036.vdocuments.us/reader036/viewer/2022081612/5f7507c3032ca8775b7e29e1/html5/thumbnails/11.jpg)
Becker & Hickl TCSPC Handbook (6th ed)
photon counting
![Page 12: Fluorescence Lifetime Imaging Microscopy (FLIM · Jan 27th / 2017 Nick Weilinger Eli York MacVicar Lab. intensity vs lifetime imaging ... (2015) applications Imaging Ca2+ with FLIM:](https://reader036.vdocuments.us/reader036/viewer/2022081612/5f7507c3032ca8775b7e29e1/html5/thumbnails/12.jpg)
the good & bad of FLIM
PRO:
• FLIM value can be used to
directly measure levels of your
[favourite ion]
• Not sensitive to [dye] or intensity
or scattering or photobleaching
(if used correctly)
CON:
• Must be calibrated correctly
(not trivial)!
• Can be sensitive to
temperature, pH, environmental
medium (e.g. intracellular vs
extracellular viscosity)
• Computationally demanding &
can require long acquisition
time
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applications
Imaging Ca2+ with FLIM:
• OGB-1 is a Ca2+ sensitive dye that is appropriate for FLIM
Zheng et al., Neuron (2015)
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applications
Imaging Ca2+ with FLIM:
• OGB-1 is a Ca2+ sensitive dye that is appropriate for FLIM
Zheng et al., Neuron (2015)
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applications
Imaging Cl- with FLIM:
• MQAE is a Cl- sensitive dye that is appropriate for FLIM
• FLIM is advantageous in the context because the dye photobleaches
easily and becomes dimmer as Cl- increases
+ =
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applications
Imaging FRET with FLIM:
• Perhaps the most advantageous
application for FLIM is paired with
Förster’s (Fluorescence) Resonance
Energy Transfer (FRET)
• Depends on non-radiative energy
transfer between fluorescent proteins
• Requires close physical proximity
between proteins and spectral overlap
• FRET can be used ratiometrically to
calculate [favourite ion] with careful
calibration
Leica Science Lab
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applications
Imaging FRET with FLIM:
• Perhaps the most advantageous
application for FLIM is paired with
Förster’s (Fluorescence) Resonance
Energy Transfer (FRET)
BUT you have to deal with:
• Inconsistent protein expression
• Spectral crosstalk (cannot directly
access pure acceptor emission)
• Photobleaching
• Distance between donor/acceptor
muddling your signal AND fraction of
interacting donor molecules
Leica Science Lab
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applications
Imaging FRET with FLIM:
• Single exponential FLIM-FRET
exploits the non-radiative energy
loss of donor proteins as
measured by a loss of lifetime
signal (measures donor only)
• Double exponential FLIM-FRET
solves the issue of unknown
fraction of donor molecules due
to the two different lifetimes
(quenched or unquenched)
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applications
Dore et al., PNAS (2015)
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applications
Dore et al., PNAS (2015)
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• NADH is autofluorescent
– (ex/em: 750/460 nm with 2p)
– Free NADH is created in
glycolysis and the TCA
• Lifetime of ~400 ps
– NADH is bound by complex 1 in
the ETC
• Lifetime of ~2000 ps
• Can use the amount of free
and bound protein to determine
the metabolic state of
cells/tissue
glucose
pyruvate
gly
coly
sis
TCA
Cycle
ETC
NADH
Complex
1
NADH
FLIM to measure metabolism
NADH
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• Since free and bound NADH have different lifetimes, the FLIM signal is multiexponential
• SPCImage calculates the best decay curve to fit both components
• Goodness of fit = χ2 value
NADH FLIM
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Intensity Colour-coded
Chi-
squaredMeasured decay trace (dotted)
Fit curve (red line)
Pix
el N
um
be
r
Avg
lifetime
components
lifetimes
Import from SPCM acquisition software to SPCImage:
NADH in SPCImage
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Binning: Number of surrounding pixels which are summed into each decay trace. Useful for low intensity images, but lose spatial resolution.
Mask: To define region of interest in image. Only pixels inside the mask are used to create the histogram.Tedious to do in SPCImage… Matlab to the rescue!
Colour-coded value: To change the data in the colour image, and define output parameter of interest.
To the real SPCImage
3
2
1
0
SPCImage parameters
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1. Save .lsm file (green and red channels)
GFP (microglia) autofluor
5. Plot average
values across
time or
treatments.
NADH data analysis2. Export files from SPCImage
3. In Matlab: mask GFP image and erode
(to account for binning).
Mask red channel and dilate.
microglia mask autofluor mask
4: Overlay microglia and neuropil masks onto FLIM data.
microglia Tm neuropil Tm
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30 min anoxia (0% oxygen), and reperfusion:Example data
Change in Average NADH Lifetime in Anoxia
0 10 20 30 40-300
-200
-100
0
100
200
Microglia
Neuropil
***
***
****** *** ***
*****
** = p<0.001, *** = p<0.0001
Anoxia Reperfusion
increasingglycolysis/decreasingOXPHOS
decreasingglycolysis/increasingOXPHOS
Time (min)
Ch
an
ge i
n M
ean
Lif
eti
me f
rom
Co
ntr
ol
(ps)
Change in Average NADH Lifetime in Anoxia
Ch
an
ge i
n M
ea
n L
ifeti
me
(p
s)
GFP (microglia) Control
NADH Τm
30 min anoxia
NADH Τm