fluorescence lifetime imaging microscopy (flim · jan 27th / 2017 nick weilinger eli york macvicar...
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Fluorescence Lifetime Imaging Microscopy (FLIM)
DATA BINGE
Jan 27th / 2017
Nick Weilinger
Eli York
MacVicar Lab
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)
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
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
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
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
Bastiaens & Squire, TICB (2009)
acquiring a FLIM signal
TCSPC:
Time
Correlated
Single
Photon
Counting
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
Becker & Hickl TCSPC Handbook (6th ed)
photon counting
Becker & Hickl TCSPC Handbook (6th ed)
photon counting
Becker & Hickl TCSPC Handbook (6th ed)
photon counting
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
applications
Imaging Ca2+ with FLIM:
• OGB-1 is a Ca2+ sensitive dye that is appropriate for FLIM
Zheng et al., Neuron (2015)
applications
Imaging Ca2+ with FLIM:
• OGB-1 is a Ca2+ sensitive dye that is appropriate for FLIM
Zheng et al., Neuron (2015)
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
+ =
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
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
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)
applications
Dore et al., PNAS (2015)
applications
Dore et al., PNAS (2015)
• 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
• 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
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
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
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
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