Name: open source high content analysis utilizing automated fluorescence lifetime imaging microscopy
Uploaded: 2017-01-18T14:00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy at JoVE.com

The authors gratefully acknowledge funding from the United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC BB/E003621/1, BB/H00713X/1 and BB/M006786/1), the UK Technology Strategy Board (Technology Award CHBT/007/00030, EP/C54269X, in partnership with AstraZeneca, GE Healthcare, GSK, Kentech Instruments Ltd) and the Wellcome Trust (WT 095931/Z/11/Z). FG acknowledges a PhD studentship from the UK Engineering and Physical Sciences Research Council (EPSRC). DA, DK and SW acknowledge PhD studentships from the Institute of Chemical Biology EPSRC-funded Doctoral Training Centre.

1. Preparation of Multiwell Plate Array
<ol>
	<li>Prepare a solution of 75 &#956;M Coumarin 6 in ethanol (&#964; ~ 2.43 ns) for reference measurements to enable determination of t0.</li>
	<li>Seed 150,000 Cos cells into four wells of a 6 well plate and allow to attach overnight in culture medium (see Materials List).</li>
	<li>Transfect one well each with mTq5A, mTq5V, mTq17V and mTq32V using a commercial transfection reagent according to the manufacturer&#39;s instructions and culture for 24 h.</li>
	<li>Detach cells using a commercial trypsin/EDTA solution according to manufacturer&#39;s protocol.</li>
	<li>Pipette 5,000 Cos cells suspended in Hank&#39;s Balanced Salt Solution (HBSS) expressing mTq5A into each of wells A1-G3 of a #1.5 thickness glass-bottomed 96 multiwell plate that has previously been treated with poly-L-lysine. Repeat for mTq5V expressing cells into wells A4-G6, mTq17V into wells A5-G9 and mTq32V into wells A10-G12.</li>
	<li>Leave well H1 empty (to provide measurement of any static background from the multiwell plate).</li>
	<li>Pipette 200 &#181;L of HBSS only into well H2 (to provide measurement of any background signal from the cell culture medium).</li>
	<li>Pipette 5,000 non-transfected cells in HBSS into well H3 (to provide measurement of any background signal from cellular autofluorescence).</li>
	<li>Pipette 200 &#181;L of the 75 &#956;M Coumarin dye solution into well H4 (to provide a check on any variation of t0 during the multiwell plate acquisition, e.g., due to changes in ambient temperature or to fluctuations in laser or timing circuitry.</li>
</ol>
2. openFLIM-HCA Data Acquisition
NOTE: Detailed information can found on the openFLIM-HCA wiki33.
<ol>
	<li>Start &#181;Manager and the OpenFLIM-HCA Plugin</li>
	<li>Select the calibration file for the specific combination of delay generator unit and laser repetition rate under &#34;FLIM control: Delay box calibration file: Calibration Files&#34; .</li>
	<li>Set the folder where the data will be saved under &#34;File: Set Base Folder&#34;.</li>
	<li>Check the excitation beam path to ensure that coupling into the delivery optical fiber is stable and that the coupling efficiency is maximized by measuring power transmitted through delivery fiber using a power meter. Fluctuations lower than 5% are acceptable.</li>
	<li>Check that the GOI triggering is stable by displaying the monitor output signal from the GOI on an oscilloscope triggered by the GOI input trigger signal.</li>
	<li>Check the imaging of the GOI phosphor onto the CCD sensor by covering the photocathode of the GOI, setting its gain to 750 V and focusing one of the relay lenses such that images of sparse single photon events (due to background light leaking though the GOI cover) recorded on the CCD are as sharp as possible.</li>
	<li>Check that the sample field of view is uniformly illuminated by imaging a uniform fluorescent sample, such as a drop of the reference dye solution on a coverslip, using a sufficiently low excitation power (&#60; 1 &#181;W) to ensure that the GOI is not saturated.</li>
	<li>Select the appropriate plate definition file, i.e., select option for 96-well plates (&#34;File: Load Plate properties&#34;).</li>
	<li>Using the &#181;Manager GUI, ensure that there is no dichroic beam splitter cube in the microscope frame by selecting an empty position.</li>
	<li>Manually select a gate width of 4 ns at the GOI for the whole experiment.</li>
	<li>Acquire IRF image data:
	<ol>
		<li>Using the &#181;Manager GUI, ensure that there is no microscope objective in the beam path by selecting an empty position. Manually place a black anodized beam block above the objective turret to prevent any laser light escaping and angle it to minimize any reflection back into the microscope frame.</li>
		<li>Using the &#181;Manager GUI, set the filters in the CSUX (Excitation, Dichroic, Emission) such that the fraction of excitation light scattered from the spinning Nipkow disk can be imaged but ensure the intensity is not sufficient to saturate the system for a 200 ms CCD integration time. This is to avoid exposing the GOI photocathode to too much light, which could damage the photocathode.</li>
		<li>Use the Find maxpoint function over the full range of the delays covered by the programmable fast delay box. Check the output diagram displayed and then adjust the manual course delay box by pushing the forward button so that the full IRF profile is within the scan range of the fast delay box.</li>
		<li>Adjust the acquisition parameters (Neutral density, Exposure time, Frame accumulation) so the CCD is not saturated in the brightest time-gate and the effective integration time is &#62;200 ms.</li>
		<li>Set delay steps of 25 ps over the full delay range (&#34;FLIM control: Fast delay box: Populate delays&#34;).</li>
		<li>Acquire IRF image by pressing &#34;Snap FLIM image&#34;.</li>
		<li>Acquire a background image by blocking the laser (&#34;Light path control: Neutral density: blocked&#34;), reducing the number of delays (&#34;FLIM control: Fast delay box: Current delay setting (ps)&#34;), leave all other properties unaltered and press &#34;Snap FLIM image&#34;.</li>
	</ol>
	</li>
	<li>Acquire reference dye data:
	<ol>
		<li>Select well H4 by using the &#181;Manager GUI (&#34;XYZ control: Go to well: H4&#34;).</li>
		<li>Using the &#181;Manager GUI, select filters (Excitation 434/17 nm, Dichroic, Emission 482/25 nm) for imaging mTqFP.</li>
		<li>Use the Find maxpoint function over the full range of the fast delay box and then adjust the coarse delay box so the full decay profile is within the delay range of the fast delay box.</li>
		<li>Set the delay to the point of maximum intensity by changing &#34;FLIM control: Fast delay box: Current delay setting (ps)&#34;. Adjust the acquisition parameters (Neutral density, Exposure time) so that the CCD is not saturated.</li>
		<li>Set delay steps of 25 ps over the full delay range (&#34;FLIM control: Fast delay box: Populate delays).</li>
		<li>Acquire reference dye data by pressing &#34;Snap FLIM image&#34;.</li>
		<li>Acquire a second background CCD camera image by blocking the excitation laser (see 2.11.7)</li>
	</ol>
	</li>
	<li>Acquire FLIM data of the multiwell plate sample using the &#181;Manager acquisition software:
	<ol>
		<li>Set which wells should be imaged and the number of FOV to be acquired per well (&#34;Setup HCA sequenced acquisition: XYZ positions&#34;).</li>
		<li>Manually select an exemplar FOV containing the sample to be imaged. Use this FOV to determine the optimal neutral density filter, gate width at the GOI and integration time of the camera so as to reach 75% of the dynamic range of the CCD camera.</li>
		<li>Set autofocus (&#34;XYZ control: Autofocus&#34;): Press &#34;Return focus control only&#34;, and then manually focus on the cells or structure of interest. Set &#34;Autofocus search range&#34; to 2000 &#181;m and press &#34;Enter&#34;. Press &#34;AF now&#34; to initiate an autofocus procedure. An offset value will show up in the field &#34;FocusOffset (Autofocus)&#34;.</li>
		<li>Enter the offset value obtained in 2.13.3 into &#34;AF offset&#34; and repeat the autofocus procedure twice (&#34;AF now&#34;) to check that the offset value is now zero, indicating correct functioning of the autofocus system.</li>
		<li>Use the &#34;AutoGate&#34; function in the OpenFLIM-HCA acquisition software to choose a logarithmic sampling of the delay points. Alternatively, these can be selected manually, see reference 36 for more details.</li>
		<li>Set &#34;Accumulate Frames&#34; to a value that yields desired total data acquisition time. Increasing the number of accumulated frames increases signal to noise ratio of the FLIM data at the expense of also increasing total FLIM data acquisition time.</li>
		<li>Run the FLIM acquisition of the multiwell plate by pressing the &#34;Start HCA sequence&#34; button.</li>
	</ol>
	</li>
	<li>Acquire a further background CCD camera image by blocking the excitation laser (see 2.11.7) with identical acquisition settings as used for the FLIM data acquisition.</li>
</ol>
3. openFLIM-HCA Data Analysis Using FLIMfit
<ol>
	<li>Check that necessary ancillary files for FLIM data analysis are present (i.e., IRF and reference dye measurement).</li>
	<li>Load the data from the empty well (H1), the well containing culture medium (H2) and the well containing unlabeled cells in culture medium (H3) into FLIMfit (&#34;File: Load FLIM data&#34;) to check whether there are any background contributions larger than 1% of the signal from the cells expressing &#34;donor-only&#34;, i.e., in wells A1-G3.</li>
	<li>Prepare a time-varying background (TVB) file by loading the well with culture medium into FLIMfit (&#34;File: Load FLIM data&#34;). Load the camera background data acquired in section 2.14 (&#34;Background: Load Background&#34;) and use the FLIMfit option &#34;Tools: Create TVB Intensity Map&#34; to generate the TVB. See reference 31 for more detail on the TVB.</li>
	<li>Prepare the spatially varying IRF Shift Map by loading the IRF data acquired in section 2.11 and subtract the CCD camera background acquired in section 2.11.7. Use the FLIMfit option &#34;Tools: Create IRF Shift Map&#34; to calculate the spatially varying IRF Shift Map. See reference [31] for more detail on the IRF Shift Map.</li>
	<li>Fit a monoexponential decay to the reference dye data acquired in section 2.12. Load reference dye and the spatially varying IRF Map calculated in section 3.4, set fit parameters (&#34;Lifetime: No. Exp:1&#34;) with t0 set as a fitted parameter (&#34;Lifetime: FIT IRF Shift: Fitted&#34;). The value of t0 obtained is then reported in the table shown in the &#34;Parameters&#34; tab.</li>
	<li>Analyze the FLIM data by loading the experimental FLIM data acquired in section 2.13, the spatially varying IRF Map calculated in section 3.4, the camera background acquired in section 2.14, the TVB file prepared in section 3.3 and type in the negative value of t0 obtained in section 3.5 (&#34;IRF: IRF shift: t0&#34;). Then fit the experimental data to the desired decay model using FLIMfit36 according to the requirements of the experiment.</li>
</ol>

The authors have nothing to disclose. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We have described an automated multiwell plate microscope designed for HCA using time-gated FLIM. This instrument is controlled using open source software written in &#181;Manager with the option of saving the data to an OMERO server and the FLIM data analysis being undertaken using FLIMfit36, an open source program written in MatLab and available as an OMERO client32 The &#181;Manager instrument control software, openFLIM-HCA, is available online33 with a list of the system components48 to enable academic researchers to construct their own FLIM HCA instruments.
In order to acquire robust FLIM data, it is necessary to optimize the GOI and CCD acquisition settings and to take account of any variations in t0. As described in 3.8.2, the GOI gain setting and CCD camera integration time should be set to reach ~75% of the CCD dynamic range. Using higher GOI voltages and multiple CCD frame accumulations at each time gate delay increases the signal to noise ratio49 but this increases the total FLIM acquisition time (since fewer fluorescence photons are acquired per frame before the CCD camera saturates and so the number of frame accumulations should be increased) and so this trade-off should be decided for each experiment. The calibration of the delay generator depends on the laser repetition rate and it is therefore necessary to ensure that the correct calibration file for the repetition rate used is loaded into the acquisition software. The procedure for calibrating the delay box can be found on the openFLIM-HCA wiki33.
If the cellular autofluorescence is low compared to the fluorescence signal, we use the signal from a well containing just culture medium to provide the time-varying background (TVB). To minimize background fluorescence from the cell culture medium, we avoid media containing phenol red. If the cellular autofluorescence is significant, then the TVB may be derived from measurements of the unlabeled cells. However, in this case care must be taken, as this assumes that the autofluorescence background is the same for every pixel in the image. This assumption will not be valid if the autofluorescence varies significantly across a cell or if the excitation beam or detection sensitivity is not uniform over the field of view.
The acquisition of an IRF image using scattered excitation light pulses provides the temporal IRF profile that is convolved with the data model in the fitting process and also provides a map of the spatial variation of the IRF across the field of view. The IRF can be measured by imaging a scattering sample such as a colloidal suspension although, in our instrument, using excitation light scattered from the Nipkow disk provides a cleaner measurement of the IRF. Accurate determination of the timing of the IRF is important because errors in the time delay between the excitation and time-gating can significantly impact the fitting process, particularly when fitting to complex exponential decay models. The IRF acquired using scattered excitation light is not exactly what is needed for the fitting process because it is recorded at the excitation wavelength rather than at the fluorescence emission wavelength, which can affect its timing, although the spatial variation of the IRF should be the same at both wavelengths. In addition, the scattered IRF may be globally shifted in time relative to the true IRF due to a differing optical path length from the scattering object, as is the case for an IRF acquired from the Nipkow disk in optically sectioned FLIM. This correction, t0, which is the required global temporal shift that should be applied to the acquired scattered excitation light IRF, is calculated from the monoexponential reference dye data as indicated in Protocol step 4.4. The following dyes can be used for different excitation wavelengths: a 75 &#956;M Coumarin 153 solution in methanol (&#964; ~ 4.3 ns50) can be used for excitation in the range 295-442 nm; a 75 &#956;M Coumarin 6 solution in ethanol (&#964; ~ 2.43 ns37) can be used for excitation in the range 430-500 nm; and a 75 &#956;M Rhodamine B solution in water (&#964; ~ 1.7 ns37) can be used for excitation in the range 488-575 nm. We note that, although it is possible in FLIMfit to use a different IRF for each pixel of the system, usually it is reasonable to make the assumption that the spatially varying IRF has the same temporal profile for every pixel in the image but presents a range of spatially varying temporal offsets relative to the global t0. We describe this as the IRF shift map, which is determined from the scattered light IRF. Together, the IRF profile, t0 and the IRF shift map are used to ensure that each pixel in the FOV is fitted with an appropriate IRF. Importantly, t0 can vary slowly over time so it should be measured before any FLIM data acquisition.
The user should decide how to sample the fluorescence decay profiles and is required to set the width of the time-gates and their relative delays after excitation. In general, it is desirable to use broad gate widths to maximize the detected signal and typically we use gate widths set to 4 ns for FLIM of cells labeled with fluorescence proteins. The number of time-gate delays should be sufficient to sample the fluorescent decay profile (including one gate set to measure the signal just before the excitation pulse) given the complexity of fitting model that will be used in the analysis. The optimum value can depend on the lifetimes and relative amplitudes of the decay components. In general, 4 gates are sufficient for fitting monoexponential decays while 7 or more gates may be used for more complex decay models.
We note that FLIM can be implemented using a range of time-domain or frequency domain techniques and automated FLIM HCA of multiwell plate arrays was first implemented in a (non-sectioning) wide-field microscope using frequency domain lifetime readouts of FRET51. The first automated optical sectioning FLIM multiwell plate reader for HCA utilizing wide-field time-gated imaging28 was then reported. Subsequently, wide-field (non-sectioning) frequency domain FLIM HCA was applied to screen for post translational modifications (tyrosine phosphorylation) across a gene library using FRET52 and time-gated FLIM HCA has been applied to FRET assays of HIV-1 Gag oligomerization53 and of SUMOylation of FOXM154 and of Raichu RhoA and Rac1 biosensors33. It is also possible to use TCSPC for multiwell plate FLIM26 but to date it has proved challenging to match the throughput of techniques exploiting wide-field detection. One emerging approach that could address this issue is the use of arrays of single photon counting detectors such as SPAD arrays55.
We note that any readouts of FRET using fluorescent proteins should be treated with caution and that the absolute values of parameters obtained from FLIMfit or any other FLIM analysis software will depend on the assumptions associated with the fitting model. For example, where the FRET donor has a significant second decay component, the use of a biexponential model to fit the FRET FLIM data can result in the contribution of the faster decay component, typically associated with the FRETing population appearing higher than it should. It is therefore desirable to use donors with a mono-exponential lifetime such as mTq2FP. Even then, recent studies have shown that FRET analysis can still be affected by dark states of acceptor fluorescence proteins or by heterogeneity of the orientation factor &#954;2 due to a distribution of fluorophore conformations with a variety of chromophore angles and distances56. Nevertheless, we and others have shown that fluorescence lifetime readouts of FRET do produce useful results that correlate with biochemical measurements and can be used to screen for protein interactions or to follow dynamics of biosensors. Thus this openFLIM HCA platform can be applied to a wide range of fluorescence lifetime-based assays utilizing FRET or cellular autofluorescence8, as well as providing quantitative readouts of dye-based probes25.

<table border="0" cellpadding="0" cellspacing="0" width="549">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">microscope frame&#160;</td>
			<td style="width:64px;">Olympus</td>
			<td style="width:64px;">IX81</td>
			<td style="width:101px;">&#160;http://www.olympusmicro.com/brochures/pdfs/ix71.pdf&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">optical autofocus</td>
			<td style="width:64px;">Olympus</td>
			<td style="width:64px;">ZDC</td>
			<td>&#160;http://www.olympusmicro.com/brochures/pdfs/ix71.pdf&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">motorized (x-y-z) microscope stage</td>
			<td style="width:64px;">Marzhauser</td>
			<td style="width:64px;">00-24-565-0000</td>
			<td>Stage with Corvus controlle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">excitation laser source</td>
			<td style="width:64px;">Fianium</td>
			<td style="width:64px;">SC400-4</td>
			<td>Supercontinuum laser http://www.nktphotonics.com/product/sc400-4-compact-supercontinuum-laser/</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:320px;">gated optical intensifier&#160;</td>
			<td style="width:64px;">Kentech</td>
			<td style="width:64px;">High Rate Imager (HRI)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">delay generators</td>
			<td style="width:64px;">Kentech</td>
			<td style="width:64px;">---</td>
			<td>See agile delay generator and precision delay generator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">camera</td>
			<td style="width:64px;">Hamamatsu</td>
			<td style="width:64px;">ORCA-ER-II</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Nipkow disc scanner unit&#160;</td>
			<td style="width:64px;">Yokogawa</td>
			<td style="width:64px;">CSU-X1&#160;</td>
			<td>http://www.yokogawa.com/solutions/products-platforms/life-science/confocal-scanner-unit-csu/csu-x1/</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Top hat diffuser</td>
			<td style="width:64px;">Thorlabs</td>
			<td style="width:64px;">ED1-S20-MD</td>
			<td>Engineered diffusers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"></td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">mTq5V&#160;</td>
			<td style="width:64px;">Oxford Genetics</td>
			<td style="width:64px;">P3850</td>
			<td>mTurquoise Venus construct with 5 amino acid linker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">mTq17V</td>
			<td style="width:64px;">Oxford Genetics</td>
			<td style="width:64px;">P3847</td>
			<td>mTurquoise Venus construct with 17 amino acid linker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">mTq32V</td>
			<td style="width:64px;">Oxford Genetics</td>
			<td style="width:64px;">P3848</td>
			<td>mTurquoise Venus construct with 32 amino acid linker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">mTq5A</td>
			<td style="width:64px;">Oxford Genetics</td>
			<td style="width:64px;">P3849</td>
			<td>mTurquoise Amber construct</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">HEK293T</td>
			<td style="width:64px;">---</td>
			<td style="width:64px;">---</td>
			<td>cell type used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"></td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">phenol red-free HBSS</td>
			<td style="width:64px;">Sigma Aldrich</td>
			<td>55021C-1000ML</td>
			<td>imaging media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">culture media</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td>DMEM + 10%FBS + 0.5% Antibiotics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">DMEM</td>
			<td style="width:64px;">Sigma Aldrich</td>
			<td>D6046-500ML</td>
			<td>for culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">FBS</td>
			<td style="width:64px;">Sigma Aldrich</td>
			<td>12003C-500ML</td>
			<td>for culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">xTremeGene9</td>
			<td style="width:64px;">Sigma Aldrich</td>
			<td>6.37E+09</td>
			<td>for transfection, follow online protocol from La Roche</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">trypsin/EDTA Solution</td>
			<td style="width:64px;">Thermo Fischer</td>
			<td style="width:64px;">R001100</td>
			<td>for detaching cells, follow online protocol from Thermo Fischer</td>
		</tr>
	</tbody>
</table>

open source high content analysis utilizing automated fluorescence lifetime imaging microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

Watch this Scientific Journal Video about Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy at JoVE.com

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

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