fluorescence lifetime imaging microscopy (flim · jan 27th / 2017 nick weilinger eli york macvicar...

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