Are You FRETting?Are You FRETting?Find Out for Sure With Find Out for Sure With

FLIMFLIM

Frequency Domain FLIM Frequency Domain FLIM for Your Scopefor Your Scope

Intelligent Imaging Innovations

FluorescenceFluorescence

Fluorophores absorb Fluorophores absorb lightlight

Some energy is lost Some energy is lost to heatto heat

Fluorophores emit Fluorophores emit red shifted lightred shifted light

This process takes This process takes time (nanoseconds)time (nanoseconds)

The average time is called the “lifetime” of the probe

Fluorescence LifetimeFluorescence Lifetime

time

coun

ts

What can FLIM do?What can FLIM do?

Probes the molecular environment Probes the molecular environment (FRET, dimerization, pH, mobility, …)(FRET, dimerization, pH, mobility, …)

More specificity (GFP More specificity (GFP ≠ FITC)≠ FITC) Intensity (concentration) independent Intensity (concentration) independent More quantitative and aberration-free More quantitative and aberration-free

measurementsmeasurementsFRET does not require multiple filters FRET does not require multiple filters

(only measure donor)(only measure donor)

FRETFRET

FRET only occurs if…FRET only occurs if…

• • the donor fluorescence emission spectrum the donor fluorescence emission spectrum overlaps with the acceptor absorbanceoverlaps with the acceptor absorbance

• • the donor and acceptor fluorophores are in the donor and acceptor fluorophores are in close proximity close proximity

• • the transition dipole moments of the donor the transition dipole moments of the donor and acceptor fluorophores are not and acceptor fluorophores are not perpendicular.perpendicular.

FRET through FLIMFRET through FLIM

Only the lifetime of the donor needs to be Only the lifetime of the donor needs to be measuredmeasured

If FRET occurs, the lifetime of the donor If FRET occurs, the lifetime of the donor decreasesdecreases

No extensive correction factors are No extensive correction factors are needed as in sensitized emission FRETneeded as in sensitized emission FRET

Efficieny can be easily calculatedEfficieny can be easily calculatedFRET efficiency = (Td- Tda) / TdFRET efficiency = (Td- Tda) / Td

excitation

target

emissionfilter

detector

frequency domain

timedomain

Measuring fluorescence lifetimesMeasuring fluorescence lifetimes

The Instrument

objective

Pockel’s Cell

laser

modulatedintensifier

cam

era

dic

hro

ic

target

phase shifted images

What do we measure?What do we measure?A: Phase lag and demodulationA: Phase lag and demodulation

sin ( ) ( )sin ( )

cos ( ) ( ) cos ( )

tot tot

tot tot

M I M d

M I M d

Only in the case of single lifetimes do these have any obvious meaning.

0

0,5

1

1,5

2

2,5

3

3,5

4

1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 1 1 0 1 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 1 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 1 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 0 1 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 1 5 1 5 2

Radians

Inte

ns

ity Modulated

Emission

ModulatedExcitation

Δφ

M

HomodyneHomodyne

Phase shiftPhase shift

DemodulationDemodulation

M

M

Lifetime VectorsLifetime Vectors

Let the radial distance be Let the radial distance be MM and the angle from and the angle from the x-axis be the x-axis be φφ. .

Call this a Call this a lifetime vectorlifetime vector. .

( ) ( )totr I r d

Polar PlotPolar Plot

0

0,1

0,2

0,3

0,4

0,5

0,6

0 0,2 0,4 0,6 0,8 1 1,2

M

Δφ

Polar PlotPolar Plot

All single exponential lifetimes lie on the All single exponential lifetimes lie on the semicirclesemicircle

Multi-exponetinal Lifetime are a linear Multi-exponetinal Lifetime are a linear combination of their componentscombination of their components

The ratio of the linear combination The ratio of the linear combination determines the fraction of the componentsdetermines the fraction of the components

5ns

R101 and Cy3Polar

0

0.2

0.4

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

has

e ) Cy3R101

Mix

1ns

R101, CY3, and a mixture

CY3

R101

Both

Using the polar plot with imagesUsing the polar plot with images

Two clay

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

ha

se

)

Two clay

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

ha

se

)

Two clay

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

ha

se

)

R101 and Cy3Polar

0

0.2

0.4

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

has

e )

R101 and Cy3Tau Tau

0

1

2

3

4

5

6

0 1 2 3 4 5 6

phase lifetime (ns)

mo

d li

feti

me

(ns)

R101 and Cy3Polar

0

0.2

0.4

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

has

e )

Phase LifetimeMod Lifetime2 Component FitDistance from Point

1ns

5ns

R101 and Cy3Polar

0

0.2

0.4

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

has

e )

R101 and Cy3Polar

0

0.2

0.4

0.6

0 0.2 0.4 0.6 0.8 1

x = mod * cos( phase )

y =

mo

d *

sin

( p

has

e )

R101 and Cy3Tau Tau

0

1

2

3

4

5

6

0 1 2 3 4 5 6

phase lifetime (ns)

mo

d li

feti

me

(ns)

R101 and Cy3Tau Tau

0

1

2

3

4

5

6

0 1 2 3 4 5 6

phase lifetime (ns)

mo

d li

feti

me

(ns)

R101 and Cy3Tau Tau

0

1

2

3

4

5

6

0 1 2 3 4 5 6

phase lifetime (ns)

mo

d li

feti

me

(ns)

Cy3

Cy3

R101R101

Mix

Mix

x

R101 distortR101 distort

C5A and C5V FRET StandardsC5A and C5V FRET Standards(Steve Vogel Lab)(Steve Vogel Lab)

Intensity single tau

Where there is no difference in intensity or color, the lifetimes can be different

C5A and C5V FRET StandardsC5A and C5V FRET Standards(Steve Vogel Lab)(Steve Vogel Lab)

Donor OnlyDonor Only

Donor/AcceptorDonor/Acceptor

Donor+ Donor/AcceptorDonor+ Donor/Acceptor

With good data you can determine the % FRET and the fraction of donors that are FRETting

How to use SlideBookHow to use SlideBook

Start SlideBookStart SlideBook Select focus windowSelect focus window Focus on your sampleFocus on your sample Set Modulation FrequencySet Modulation Frequency Set Pockel Cell BiasSet Pockel Cell Bias Set number of PhasesSet number of Phases

4 minimum4 minimum

Calibrate to known LifetimeCalibrate to known Lifetime Take an image of FluoresceinTake an image of Fluorescein

or Rhodamine dye and imageor Rhodamine dye and image

using the aforementioned using the aforementioned

protocolprotocol

• After image is collected, select a After image is collected, select a

Region and click on calibrate lifetimeRegion and click on calibrate lifetime

Capture Lifetime ImagesCapture Lifetime Images Select FLIM filter, capture FLIM image of the donor onlySelect FLIM filter, capture FLIM image of the donor only

Capture FLIM image of the donor/ acceptorCapture FLIM image of the donor/ acceptor

Make sure to have a good S/N ratio for the FLIM imagesMake sure to have a good S/N ratio for the FLIM images

S/N vs # of photons

0

20

40

60

80

100

120

0 2000 4000 6000 8000 10000

# of photons

S/N

Intensity Lifetime

10% precision: Intensity needs 100 photons,Lifetime needs 1000 photons

Lifetime Vector AnalysisLifetime Vector Analysis

The microscope captures a series of The microscope captures a series of images at different phasesimages at different phases

From these, 3 images are created:From these, 3 images are created:

1.1. Lifetime XLifetime X

2.2. Lifetime YLifetime Y

3.3. Fluorescence IntensityFluorescence Intensity Real physical measurements (such as Real physical measurements (such as

FRET) are calculated from these imagesFRET) are calculated from these images

Lifetime Vector AnalysisLifetime Vector Analysis

• Mark a region on the Image Mark a region on the Image • Click on get Lifetime Histogram Click on get Lifetime Histogram • Double click on the point on the polar plotDouble click on the point on the polar plot

nnss

Lifetime Vector AnalysisLifetime Vector Analysis

• Mark a region on the Lifetime Histogram • Create Mask (Region of Interest) from selection • Highlighted regions within the cell of the lifetime are given

FRET Efficiency MeasurementFRET Efficiency Measurement

• Enter the Donor Lifetime

• Click on Apply and purple

portion shows up on unit circle

Showing range of Fret

Efficiencies for the donor to

Acceptor

• Double click on the point

on the unit circle to show green

Line and Efficiency appears

The Lifetime Vector PlotThe Lifetime Vector Plot

HomodyneHomodyne

Photons NeededPhotons NeededS/N vs # of photons

0

20

40

60

80

100

120

0 2000 4000 6000 8000 10000

# of photons

S/N

Intensity Lifetime

10% precision: Intensity needs 100 photons, lifetime needs 1000

FRET ExampleFRET Example

Lifetime of the Donor with no Lifetime of the Donor with no Acceptor = Acceptor = TTdd

Lifetime of the Donor with Lifetime of the Donor with Acceptor = TdaAcceptor = Tda

FRET efficiency = (Td- Tda) / TdFRET efficiency = (Td- Tda) / Td