Development and application of an automated FLIM multiwell plate reader for high content analysis

Douglas Kelly1,2,3, Anca Margineanu2, Sean Warren1,2, Mesayamas Kongsema3, Jia Chan4, Eric W.-F. Lam3, Matilda Katan4, Chris Dunsby2, Paul M. W. French2

1Institute of Chemical Biology, Imperial College London, UK2Photonics Group, Imperial College London, UK3Department of Surgery and Cancer, Imperial College London, UK4Department of Structural and Molecular Biology, University College London

Outline

• Introduction– Fluorescence Lifetime Imaging

– Förster Resonance Energy Transfer

• Data acquisition– Schematic of instrument

– Overview of software

• Applications– Test experiments: dyes, FRET constructs

– FOXM1-SUMOylation in response to doxorubicin

– Rac1 biosensor response to CdGAP overexpression

– Rassf-family protein interaction screen

Fluorescence Lifetime Imaging Microscopy• Fluorescence lifetime imaging may be considered to be the construction of images

with contrast originating from differences in the characteristic decay time of fluorescence following excitation.

• This characteristic lifetime depends on radiative and non-radiative decay rates kr and knr as the fluorophore returns to the ground state.

rnr kk

1

inte

nsity

t

inte

nsity

t

Fluorescence Lifetime Imaging Microscopy

• FLIM is most commonly performed using time correlated single photon counting on a laser scanning confocal microscope.

• In TCSPC, the relative delay between photon arrival and excitation pulses is used to build up a histogram of photon arrival times, which can be fitted to a decay model to yield fluorescence lifetimes.

• This method gives best S/N per photon, but is slow. This is linked to a number of factors: point scan speed, detector dead time, limits on excitation power related to sample photodamage, and the inherent limit that the probability of detecting a photon following an excitation pulse must be << 1 to avoid miscounting.

Fluorescence Lifetime Imaging Microscopy• Increasing throughput of FLIM microscopy mitigates for biological noise and permits

screening/dose response experiments to be carried out easily.

• Employing a gated optical intensifier (GOI) to implement wide field time gated FLIM, all pixels are interrogated in parallel. This gives a higher S/N per unit acquisition time[1], and makes increased throughput viable.

• The GOI amplifies signal when in the ‘on’ state, occluding signal in the ‘off’ state.

• Acting like an “ultrafast shutter”, each gate provides a snapshot of the decay at a given delay after excitation, allowing reconstruction of the decay and fitting of fluorescence lifetimes.

[1] Talbot, C. B. et al. J. Biophoton. 1, 514–521 (2008).

Förster Resonance Energy Transfer

• FRET is a phenomenon whereby energy is transferred non-radiatively between donor and acceptor fluorophores when certain spectral, orientation and separation criteria are met.

• FRET typically occurs over nanometres, making it a useful readout for interactions on biologically relevant scales.

• Since FRETting donors have an additional non-radiative pathway accessible from the excited state, the fluorescence lifetime is shorter than for non-FRETting counterparts: FRET can be read out by FLIM.

D

DAE

1

nm~ ,

1

106

0

RRR

E

Förster Resonance Energy Transfer

• In biological contexts, FLIM-FRET can read out signalling events or environment changes reported by genetically encoded FRET biosensors. The presence of a signalling molecule prompts a conformational change, and hence a change in FRET.

• Alternatively, two different proteins of interest can be modified to be expressed fused to fluorescent proteins. Here, an increase in FRET reflects an interaction between the proteins of interest.

• In either case, it can be informative to fit a biexponential model, which allows both FRET efficiency and FRETting donor to be extracted from data.

Intramolecular FRET:Single chain biosensors

Intermolecular FRET:Separate labelled proteins

FRETtingFRETting

FRETtingnonFRETtingnon

tI

tItI

exp...

exp)(

0

0

FLIM plate reader: instrument schematic

Hamamatsu ORCA ER-II CCD

Fianium SC400-6supercontinuum

source

ND/Spectral filter wheels

Olympus IX-81 chassis: automated nosepiece, AF, filter selection and

stage

KentechHRI GOI

Spinning diffuser

Sample

FLIM plate reader: control software

• Software written in-house for flexibility and expandability

• User can construct FLIM experiments with sequences of arbitrarily-ordered functions, including:

• Movement between FOVs

• Z-stacks

• Time course imaging

• Phase contrast imaging

• Change of filters for FLIM in different spectral windows

• Software implements hardware autofocus to ensure high image quality across multiwell plates

• Simple liquid handling is supported

• Cell pre-find reduces experiment time and sample exposure...

FLIM plate reader: control software• Cell pre-find eliminates time wasted imaging non-fluorescent cells when fluorescent probes are introduced by transient transfection, or

empty regions when stably transfected cell lines are seeded sparsely.

• Low magnification fluorescence or phase contrast images are used to find objects of interest using internal functions or by calling CellProfiler.

• Positions are mapped such that cells are located in the centre of eventual high magnification FLIM fields of view.

(a)(b)

(c)

(a) Set up for low magnification phase contrast prefind showing raw image, variance image, and eventual cell identification following variance, area and intensity thresholding.

(b) Search pattern showing low magnification search FOVs in relation to the well.

(c) High magnification FLIM camera FOV resulting from prefind.

FLIM plate reader: dye calibration experimentC

ontribution of long lifetime

Dye Globally fitted lifetime (ns)

TCSPC measured lifetime (ns)

Rhodamine B 1.7 1.7±0.05

Rhodamine 6G 3.9 3.9±0.1

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

0.95 0.25 0.75 0.05 0.50 0.95 0.25 0.75 0.05 0.50

0.90 0.10 0.60 0.00 0.40 0.90 0.10 0.60 0.00 0.40

0.75 0.05 0.50 0.95 0.25 0.75 0.05 0.50 0.95 0.25

0.60 0.00 0.40 0.90 0.10 0.60 0.00 0.40 0.90 0.10

0.50 0.95 0.25 0.75 0.05 0.50 0.95 0.25 0.75 0.05

0.40 0.90 0.10 0.60 0.00 0.40 0.90 0.10 0.60 0.00

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Mix ratio R

6G:R

B

• Simplest possible experiment

• Two spectrally indistinguishable dyes mixed in different ratios

• Fit globally to return lifetime values of pure dyes and ratio in each well

• Results match TCPSC-measured values

• Acquisition in 700s

RBRB

GRGR

tItItI expexp)( 06

60

Typical experiment pipeline: FRET plate

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

1.00 0.95 0.90 0.75 0.60 0.50 0.40 0.25 0.10 0.05 0.00

Ratio mCerulean to mCerulean-5-mVenusby plasmid weight

• To illustrate the experiment pipeline and the performance of the plate reader with more typical samples, we prepare a plate to simulate intermolecular FRET using a donor fluorophore (mCerulean) and a donor-acceptor construct (mCerulean-5-mVenus) developed in the Vogel lab[2]

• Cells are sufficiently sparsely seeded that cell prefind is required, c.f. typical samples. We seek 8 “acceptable” fields of view per well.

[2] Koushik, S. V. et al., Biophysical Journal 91, L99–L101 (2006).

Typical experiment pipeline: FRET plate

Entire plate prefind image

Prefind FOVEventual FLIM FOV

• Large areas of the plate are screened with 10x magnification objective

• Low magnification fields are interrogated for suitable cells

• An image of the entire plate is accumulated during prefind – particularly useful as an overview if multiple transfection conditions are being trialled in a single plate.

Single well Final, centred FLIM FOV

Typical experiment pipeline: FRET plate

• Following prefind, an acquisition sequence is set up to reflect a typical experiment

• We acquire FLIM images at donor-wavelength, a single acceptor fluorescence image and a phase contrast image for each saved field of view.

Donor integrated intensity

Acceptor intensity Phase contrast

FLIM sequence

Typical experiment pipeline: analysis and results

Mean fitted lifetim

e, ps

3000 ps

1500 ps

• Acceptor intensity imaging shows that expression of FRETting construct follows expected pattern

• Apply global double exponential fit with lifetimes fixed to values obtained from each construct alone.

• Fitted results show mean lifetime decreasing with increasing FRET

• FRETting contribution (biexponential fit) follows expected trend

Typical experiment pipeline: FRET plate

? 25 min

120 min

>1 min

>1 min

~150 min

Unsupervised

• Excluding sample preparation time which varies depending on application, users can expect to have quantitative FRET results within three hours of starting the experiment.

• In this case, 8 FOV per well = 56 FOV per condition > 56 cells per condition.

• FOXM1 is a transcription factor, aberrant expression of which is linked to tumorigenesis, angiogensis and metastasis across a range of malignancies.

• Biochemical experiments undertaken in parallel to our FLIM-FRET studies show that small ubiquitin-like modifier (SUMO1) is covalently attached to FOXM1 in response to DNA damage in breast cancer cell lines.

• Doxorubicin is an anthracycline commonly used to treat breast cancer. Its mode of action is not completely understood; however it has been shown to intercalate into DNA and promote double strand breaks (DSB), interrupting cell cycle progression and eventually contributing to cell death.

• We seek to probe interaction of FOXM1 and SUMO1 in response to doxorubicin treatment of MCF7 cells using fluorescent fusion proteins, reading out the noisy FRET data on the FLIM plate reader.

FOXM1-SUMOylation: doxorubicin response

(a) Cartoon representation of fluorescent fusion proteins FOXM1WT-eGFP, SUMO1-tRFP and negative control, non-SUMOylatable mutant FOXM1mut-eGFP. (b) Plate layout.

(a) (b)

SFOXM1WT

S

FOXM1mut

• Of all conditions, only FOXM1-eGFP + SUMO1-tRFP shows a response to doxorubicin treatment. FOXM1-mut is a mutated form of FOXM1 lacking SUMOylation sites; the basal decrease in lifetime exhibited by this mutant and the offset in lifetime between conditions at zero treatment time suggests an additional interaction mechanism contributing to the FRET signal – we hypothesise that this may be linked to the presence of SUMO-interacting motifs (SIMs) on FOXM1.

FOXM1-SUMOylation: doxorubicin response

(a) Map showing plating strategy. (b) Gallery view illustrating heterogeneity in cell response. (c) Map showing single fitted lifetime. (d) Plot showing fitted lifetime response of FOXM1-eGFP + SUMO1-tRFP cells to 0.1 µM doxorubicin treatment time course (Dunnett’s test).

Rac1 activation by cdGAP

GFPmRF

PPAK Rac1

GDP

GFP

mRFP

PAK

Rac1

GTP

GEF

GAP

• In keratinocytes, the dysregulation of cell-cell junctions has implications in the study of tumour metastasis.

• Activity of small GTPases such as Rac1 is modulated by GTPase activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs).

• Our collaborators are interested in spatio-temporal activation of Rac1 in response to overexpression of cdGAP, with a view to examining the role played by cdGAP in maintenance of adherens junctions.

• Rac1 activation is probed using a single chain biosensor based on those developed in the Matsuda lab[3] – upon activation, Rac1 can bind PAK, causing a conformational change and an increase in FRET.

[3] Itoh, R. E. et al. Molecular and cellular biology 22, 6582–6591 (2002).

Rac1 activation by cdGAP

• Data acquired on the FLIM plate reader show a decrease in Rac1 activation upon overexpression of cdGAP, supporting findings of bulk biochemical experiments.

• The ability to image multiple spectral channels allows confirmation that cdGAP is overexpressed: immunofluorescence techniques are used such that the presence of the near-IR dye Cy5 indicates expression of exogeneous cdGAP. .

• Furthermore, the imaging capabilities of FLIM allows segmentation to distinguish between membrane-associated activation and bulk-cell activation.

(a) Exemplar fields in the absence and presence of exogeneous cdGAP. (b) Lifetime results at the cell membrane. (c) Lifetime results for whole cells.

Rassf-family interaction screen

• RASSF family proteins are implicated in regulation of apoptosis, microtubule stabilisation and DNA damage response: they have been shown to play a role in tumour suppression.

• Classical RASSF proteins (1-6) contain a SARAH domain; N-terminal RASSF proteins lack this domain.

• MST1 protein has also been shown to play a role in cancer signalling, and has been shown to interact with RASSF proteins via heterodimerisation of the SARAH domain.

• We coexpress eGFP-RASSF family proteins MST1-mCherry, SARAH-deficient MST1Δ-mCherry and a truncated MST1 consisting of the SARAH-domain only in order to determine which RASSF proteins interact with MST1.

GFP RSARA

H

eGFP-Classical RASSF

GFP R

eGFP-N-terminal RASSF

mCh

SARAHMST1

mCherry-MST1 (full length)

mCh

SARAH

mCherry-SARAH

mCh RMST1

mCherry-MST1Δ

(a) (b)

(a) Cartoon showing FRET donor proteins. (b) Cartoon showing acceptor proteins: full length mCh-MST1, non-interacting mCh-MST1Δ and truncated mCh-SARAH.

Rassf-family interaction screen

• As expected, classical RASSF proteins show shortened lifetime (increased FRET) when coexpressed with both mCherry-SARAH and mCherry-MST1 compared to donor-only wells. N-terminal RASSFs, lacking the SARAH domain, do not show any evidence of binding.

FLIM plate reader: flexibility

• The ability to acquire large amounts of data quickly on the FLIM plate reader makes it a useful tool for assay characterisation and optimisation.

• The FLIM plate reader has been applied to many FRET-based experiments as well as UV-wavelength autofluorescence studies:

Summary

• Our multiwell plate reading instrument allows high content FLIM experiments to be performed in a practical timescale – as fast as 1 plate per 15 minutes.

• The instrument and acquisition software are flexible, allowing a large range of different FLIM experiments to be automated.

• FLIM-FRET can provide robust, information-rich high-content data.

• We have illustrated application of FLIM-FRET in drug response studies and interaction screening.

Thanks

Imperial College Photonics:

• Paul French

• Chris Dunsby

• Anca Margineanu

• Sean Warren

• Dominic Alibhai

• Sunil Kumar

Collaborators:

• Eric Lam

• Mesayamas Kongsema

• Matilda Katan

• Jia Chan

• Jess McCormack

• Vania Braga

Funding/commercial partners:

Kentech Instruments Ltd