Presentation on theme: "Development and application of an automated FLIM multiwell plate reader for high content analysis Douglas Kelly1,2,3, Anca Margineanu2, Sean Warren1,2,"— Presentation transcript:
1 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. French21Institute 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
2 Outline Introduction Data acquisition Applications Fluorescence Lifetime ImagingFörster Resonance Energy TransferData acquisitionSchematic of instrumentOverview of softwareApplicationsTest experiments: dyes, FRET constructsFOXM1-SUMOylation in response to doxorubicinRac1 biosensor response to CdGAP overexpressionRassf-family protein interaction screen
3 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.intensitytt
4 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.
5 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, 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. Talbot, C. B. et al. J. Biophoton. 1, 514–521 (2008).
6 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.
7 Förster Resonance Energy Transfer Intramolecular FRET:Single chain biosensorsIn 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.Intermolecular FRET:Separate labelled proteins
9 FLIM plate reader: control software Software written in-house for flexibility and expandabilityUser can construct FLIM experiments with sequences of arbitrarily-ordered functions, including:Movement between FOVsZ-stacksTime course imagingPhase contrast imagingChange of filters for FLIM in different spectral windowsSoftware implements hardware autofocus to ensure high image quality across multiwell platesSimple liquid handling is supportedCell pre-find reduces experiment time and sample exposure...
10 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.
11 FLIM plate reader: dye calibration experiment Simplest possible experimentTwo spectrally indistinguishable dyes mixed in different ratiosFit globally to return lifetime values of pure dyes and ratio in each wellResults match TCPSC-measured valuesAcquisition in 700s1.000.950.250.750.050.500.900.100.600.000.40Mix ratio R6G:RBContribution of long lifetimeDyeGlobally fitted lifetime (ns)TCSPC measured lifetime (ns)Rhodamine B1.71.7±0.05Rhodamine 6G3.93.9±0.1
12 Typical experiment pipeline: FRET plate 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 labCells are sufficiently sparsely seeded that cell prefind is required, c.f. typical samples. We seek 8 “acceptable” fields of view per well.1.000.950.900.750.600.500.400.250.100.050.00Ratio mCerulean to mCerulean-5-mVenusby plasmid weight Koushik, S. V. et al., Biophysical Journal 91, L99–L101 (2006).
13 Typical experiment pipeline: FRET plate Large areas of the plate are screened with 10x magnification objectiveLow magnification fields are interrogated for suitable cellsAn 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.Prefind FOVEventual FLIM FOVThis is most necessary in cases where cell seeding is sparse or transfection efficiency is low.We scan the plate at low magnification to identify suitable fluorescent cells. We store a user defined number of fields of view centred on these cells, that will be non-overlapping when imaged at high magnification in our FLIM experiment.Depending on acquisition parameters and cell density, this step can take ten minutes to half an hour.Entire plate prefind imageSingle wellFinal, centred FLIM FOV
14 Typical experiment pipeline: FRET plate Following prefind, an acquisition sequence is set up to reflect a typical experimentWe acquire FLIM images at donor-wavelength, a single acceptor fluorescence image and a phase contrast image for each saved field of view.FLIM sequenceDonor integrated intensityAcceptor intensityPhase contrast
15 Typical experiment pipeline: analysis and results Acceptor intensity imaging shows that expression of FRETting construct follows expected patternApply global double exponential fit with lifetimes fixed to values obtained from each construct alone.Fitted results show mean lifetime decreasing with increasing FRETFRETting contribution (biexponential fit) follows expected trendMean fitted lifetime, ps3000 ps1500 ps
16 Typical experiment pipeline: FRET plate 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.Sample preparationCell prefindImaging:FLIMAcceptor channelPhase contrastControls:BackgroundIRF/reference dyeAnalysis:SegmentationFittingInterpretation?25 min120 min>1 min>1 minUnsupervised~150 min
17 FOXM1-SUMOylation: doxorubicin response 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.(a)(b)FOXM1mutFOXM1WTSS(a) Cartoon representation of fluorescent fusion proteins FOXM1WT-eGFP, SUMO1-tRFP and negative control, non-SUMOylatable mutant FOXM1mut-eGFP. (b) Plate layout.
18 FOXM1-SUMOylation: doxorubicin response 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.(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).
19 Rac1 activation by cdGAP 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 – upon activation, Rac1 can bind PAK, causing a conformational change and an increase in FRET.GFPmRFPPAKRac1GDPGTPGEFGAP Itoh, R. E. et al. Molecular and cellular biology 22, 6582–6591 (2002).
20 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.
21 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.(a)(b)mChSARAHMST1mCherry-MST1 (full length)GFPRSARAHeGFP-Classical RASSFmChSARAHmCherry-SARAHRMST1mCherry-MST1ΔGFPReGFP-N-terminal RASSF(a) Cartoon showing FRET donor proteins. (b) Cartoon showing acceptor proteins: full length mCh-MST1, non-interacting mCh-MST1Δ and truncated mCh-SARAH.
22 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.
23 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:Ras/Raf interaction in response to gefitinib treatementFOXM1 SUMOylation in response to DNA damageAutofluorescence as readout for cytotoxicityGag protein aggregation and response to NMT inhibitorAutofluorescence of structural proteins in osteoarthritisRho activation at cell-cell junctionsScreening for PPIs during mitosisRac activation at cell-cell junctionsRASSF-family interaction screen
24 SummaryOur 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.