Financial support for this work from the Science Foundation Ireland, grants SFI/12/RC/2276_P2, SFI/17/RC-PhD/3484 and 18/SP/3522, and Breakthrough Cancer Research (Precision Oncology Ireland) is gratefully acknowledged.

All the procedures with animals were performed under authorizations issued by the Health Products Regulatory Authority (HPRA, Ireland) in accordance with the European Communities Council Directive (2010/63/EU) and were approved by the Animal Experimentation Ethics Committee of the University College Cork.
1. Sample preparation
<ol>
	<li>Staining with the probe of live tissue samples ex vivo&#8203;
	<ol>
		<li>For ex vivo applications, use freshly isolated tissue samples from 4 week old female Balb/c mice.</li>
		<li>On the day of the experiment, euthanize a mouse by decapitation, and quickly dissect fragments of the colon (large intestine), approximately 10 mm in size. Wash them immediately with PBS buffer, place in DMEM medium supplemented with 10 mM Hepes buffer (pH 7.2), and incubate at 37 &#176;C36.</li>
		<li>For surface staining of the serosal side of the intestine, transfer the live tissue samples into a mini-dish, apply 2 mL of complete DMEM containing 1 mg/mL NanO2-IR probe to cover the tissue samples, and incubate for 30 min at 37 &#176;C. 
		NOTE: Cells in post-mortem tissue remain live for many hours in culture. NaNO2-IR shows minor cytotoxicity, so all the experiments were completed within 4 h after tissue isolation.</li>
		<li>For deep tissue intraluminal ex vivo staining, transfer the pieces of the intestine to a dry Petri dish, and remove any excess DMEM with filter paper.</li>
		<li>Inject 1 &#181;L of DMEM containing 1 mg/mL NanO2-IR35 into the lumen with a Hamilton syringe, and incubate the samples for 15 min or for up to 4 h. 
		NOTE: NaNO2-IR shows minor long-term cytotoxic effects; therefore, all the experiments should be completed within 4 h after tissue isolation.</li>
	</ol>
	</li>
	<li>Preparation of stained tumor tissue in live animals
	<ol>
		<li>For in vivo applications, pre-stain CT26 cells for 18 h in serum-free medium containing NaNO2-IR probe at 0.05 mg/mL.</li>
		<li>Take a mouse, shave the area of injection in the right flank, and inject with a syringe 200 &#181;L of a mixture of 1 &#215; 105 non-stained cells and 1 &#215; 105 cells pre-stained with NanO2-IR .</li>
		<li>Allow tumors to grow in the mice, monitoring the tumor size with a caliper and the animal weight periodically37. The animals with grafted tumors become ready for imaging on the seventh day of tumor growth. 
		NOTE: The tumor volume was calculated using equation (1): 
		V = (L &#215; W2)/2&#160; &#160; &#160;(1) 
		&#8203;where L is the diameter of the tumor, and W is the diameter perpendicular to the diameter L.</li>
		<li>Sacrifice the animals by cervical dislocation just before the imaging.</li>
	</ol>
	</li>
</ol>
2. PLIM imaging setup
<ol>
	<li>Take the Cricket adaptor, and remove its back side C-mount adapter to gain access to the intensifier housing inside. Insert the MCP-125 image intensifier into this compartment, and put the C-mount adaptor back.</li>
	<li>Remove the Cricket&#39;s front side C-mount adaptor, insert the 760 nm &#177; 50 nm emission filter, and fix it by putting back the C-mount.</li>
	<li>Connect the Tpx3Cam camera module to the back side of the Cricket module via its C-mount adaptor</li>
	<li>Connect the lens to the front side of the Cricket module via its C-mount adaptor.</li>
	<li>Mount the whole camera assembly on top of the optical black box, facing down to the stage on which the samples will be imaged (Figure 1).</li>
	<li>Mount the 624 nm super-bright LED on a post connected to a breadboard inside the black box.</li>
	<li>Connect the LED to a power supply and a pulse generator. Switch on the LED, and focus it to ensure effective and uniform excitation of imaged samples.</li>
	<li>Connect the camera to another pulse generator, and synchronize the pulses sent to the camera and the LED38.</li>
	<li>Using the special cable and socket on the Cricket unit, connect the intensifier to a standard power supply, and set the gain to 2.7 V.</li>
	<li>Using the focusing capabilities of the lens and Cricket adaptor, focus the camera optics on the sample stage to generate clear images of samples with good contrast and brightness.</li>
	<li>For imager use with Pt-porphyrin dyes, replace the 625 nm LED with a 390 nm LED for excitation, and replace the 760 nm &#177; 50 nm filter with a 650 nm &#177; 50 nm filter in the Cricket module.</li>
</ol>
3. Image acquisition
<ol>
	<li>Place the sample in front of the camera lens. 
	NOTE: Use an x-y-z adjustable stage as the sample holder in order to adjust the sample position for good focus.</li>
	<li>Turn off all the lights in the room.</li>
	<li>Switch on the Sophy software for tuning the operational parameters, such as the focusing and sample alignment. 
	NOTE: Sophy software is provided together with the camera to set up the imaging parameters and record the data. Make sure that the software is connected to the camera by checking the camera code. However, we used a different program for data acquisition.</li>
	<li>In Modules, select infinite frames, and set the pixel operation mode to time over threshold.</li>
	<li>In Modules, go to Preview, and select Active module. This opens the Medpix/Timpix Frames window.</li>
	<li>In this window, change the color scale, and rotate the image to the desired orientation.</li>
	<li>Switch on the intensifier, and start the recording. 
	NOTE: Use the recording screen of the Sophy software to visually confirm the alignment and focus of the sample and to optimize the LED excitation parameters for recording.</li>
	<li>Stop the recording, and close the Sophy software.</li>
	<li>Go to the terminal, and use the custom-designed software to acquire the raw data in the binary format and post-process it (https://github.com/svihra/TimePix3).
	<ol>
		<li>In the terminal, run the following commands to record the data: 
		Cd Document/SPIDR/trunk/Release/ 
		&#160;ls 
		./Tpx3daq - i 1 - b 50 - m - s {Name of the file} - t {acquisition time} 
		NOTE: Upon typing &#34;ls,&#34; check the list of files in the current directory; confirm that Tpx3daq is seen.</li>
		<li>Wait until all the frames are recorded.</li>
		<li>To process the data, run the following commands in the terminal: 
		cd Documents/DataProcessing/Timepix3/Timepix3/ 
		root 
		.L dataprocess.cpp++ 
		&#8203;.x DRGUI.C</li>
		<li>Wait for an RRGui window to open. Select all the variables on the left and All Data, Single File, and Centroid on the right.</li>
		<li>Select the file to process and run the data reducer. 
		&#8203;NOTE: All the processed files will appear in the same folder as the raw file.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Analyze the post-processed data with a dedicated program written in C-language that will write the data into an .ics image file (https://github.com/lmhirvonen/timepix3cam).</li>
	<li>Open the .ics image files using the freely available Time Resolved Imaging software (see Table of Materials). Use two-exponential functions to fit the phosphorescence decays.</li>
	<li>Open the fitted .ics image files with the available image analysis software (see Table of Materials).</li>
	<li>Using Lookup Tables, generate phosphorescence lifetime images, and encode them in pseudocolor scale (e.g., blue color for short lifetimes and red for long lifetimes). Use the Measure function to calculate the average lifetime values for the entire image or specific regions of interest (ROIs).</li>
	<li>Convert the lifetime values into the oxygen concentration by using the equation obtained from the fitting of the O2 calibration of the probe36. 
	NOTE: Equation (2) was used for this work: 
	O2 [&#181;M] = &#8722;86.16 + 770.35 &#215; e&#8722;0.049 &#215; LT&#160; &#160; &#160;(2)</li>
</ol>

<ol><li>Papkovsky, D. B., Dmitriev, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+oxygen+and+hypoxia+in+cell+and+tissue+samples%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Imaging of oxygen and hypoxia in cell and tissue samples.</a> Cellular and Molecular Life Sciences. 75 (16), 2963-2980 (2018).</li>
<li>Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C., Kieda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+is+the+partial+oxygen+pressure+of+human+tissues+a+crucial+parameter%3F+Small+molecules+and+hypoxia%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia.</a> Journal of Cellular and Molecular Medicine. 15 (6), 1239-1253 (2011).</li>
<li>Yoshihara, T., Hirakawa, Y., Hosaka, M., Nangaku, M., Tobita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+imaging+of+living+cells+and+tissues+using+luminescent+molecular+probes%5BTitle%5D)+AND+%22Journal+of+Photochemistry+and+Photobiology+C%3A+Photochemistry+Reviews%22%5BJournal%5D)">Oxygen imaging of living cells and tissues using luminescent molecular probes.</a> Journal of Photochemistry and Photobiology C: Photochemistry Reviews. 30, 71-95 (2017).</li>
<li>Papkovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorescence+based+oxygen+sensors+essential+tools+for+cell+biology+and+life+science+research%5BTitle%5D)+AND+%2217th+International+Meeting+on+Chemical+Sensors+-+IMCS%22%5BJournal%5D)">Phosphorescence based oxygen sensors essential tools for cell biology and life science research.</a> 17th International Meeting on Chemical Sensors - IMCS. , 71-72 (2018).</li>
<li>Tsytsarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+brain+metabolism+activity+using+a+phosphorescent+oxygen-sensitive+probe%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe.</a> Journal of Neuroscience Methods. 216 (2), 146-151 (2013).</li>
<li>O'Donovan, C., Hynes, J., Yashunski, D., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorescent+oxygen-sensitive+materials+for+biological+applications%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry%22%5BJournal%5D)">Phosphorescent oxygen-sensitive materials for biological applications.</a> Journal of Materials Chemistry. 15, 2946-2951 (2005).</li>
<li>Dmitriev, R. I., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+probes+and+techniques+for+O+2+measurement+in+live+cells+and+tissue%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Optical probes and techniques for O 2 measurement in live cells and tissue.</a> Cellular and Molecular Life Sciences. 69 (12), 2025-2039 (2012).</li>
<li>Papkovsky, D. B., Zhdanov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorescence+based+oxygen+sensors+and+probes+for+biomedical+research%5BTitle%5D)+AND+%22Advanced+Environmental%2C+Chemical%2C+and+Biological+Sensing+Technologies+XIV%22%5BJournal%5D)">Phosphorescence based oxygen sensors and probes for biomedical research.</a> Advanced Environmental, Chemical, and Biological Sensing Technologies XIV. 10215, 102150(2017).</li>
<li>Rumsey, W. L., Vanderkooi, J. M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+phosphorescence%3A+A+novel+method+for+measuring+oxygen+distribution+in+perfused+tissue%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Imaging of phosphorescence: A novel method for measuring oxygen distribution in perfused tissue.</a> Science. 241 (4873), 1649-1651 (1988).</li>
<li>Hogan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorescence+quenching+method+for+measurement+of+intracellular+PO+2+in+isolated+skeletal+muscle+fibers%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">Phosphorescence quenching method for measurement of intracellular PO 2 in isolated skeletal muscle fibers.</a> Journal of Applied Physiology. 86 (2), 720-724 (1999).</li>
<li>Apreleva, S. V., Wilson, D. F., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tomographic+imaging+of+oxygen+by+phosphorescence+lifetime%5BTitle%5D)+AND+%22Applied+Optics%22%5BJournal%5D)">Tomographic imaging of oxygen by phosphorescence lifetime.</a> Applied Optics. 45 (33), 8547-8559 (2006).</li>
<li>Becker, W., Shcheslavskiy, V., Rück, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+phosphorescence+and+fluorescence+lifetime+imaging+by+multi-dimensional+TCSPC+and+multi-pulse+excitation%5BTitle%5D)+AND+%22Advances+in+Experimental+Medicine+and+Biology%22%5BJournal%5D)">Simultaneous phosphorescence and fluorescence lifetime imaging by multi-dimensional TCSPC and multi-pulse excitation.</a> Advances in Experimental Medicine and Biology. 1035, 19-30 (2017).</li>
<li>Wolfbeis, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Luminescent+sensing+and+imaging+of+oxygen%3A+Fierce+competition+to+the+Clark+electrode%5BTitle%5D)+AND+%22BioEssays%22%5BJournal%5D)">Luminescent sensing and imaging of oxygen: Fierce competition to the Clark electrode.</a> BioEssays. 37 (8), 921-928 (2015).</li>
<li>Dmitriev, R. I., Zhdanov, A. V., Nolan, Y. M., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+neurosphere+oxygenation+with+phosphorescent+probes%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Imaging of neurosphere oxygenation with phosphorescent probes.</a> Biomaterials. 34 (37), 9307-9317 (2013).</li>
<li>Shcheslavskiy, V. I., Neubauer, A., Bukowiecki, R., Dinter, F., Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+fluorescence+and+phosphorescence+lifetime+imaging%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">Combined fluorescence and phosphorescence lifetime imaging.</a> Applied Physics Letters. 108, 091111(2016).</li>
<li>Babilas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+phosphorescence+imaging+of+pO2+using+planar+oxygen+sensors%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">In vivo phosphorescence imaging of pO2 using planar oxygen sensors.</a> Microcirculation. 12 (6), 477-487 (2005).</li>
<li>Babilas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcutaneous+pO2+imaging+during+tourniquet-induced+forearm+ischemia+using+planar+optical+oxygen+sensors%5BTitle%5D)+AND+%22Skin+Research+and+Technology%22%5BJournal%5D)">Transcutaneous pO2 imaging during tourniquet-induced forearm ischemia using planar optical oxygen sensors.</a> Skin Research and Technology. 14 (3), 304-311 (2008).</li>
<li>Golub, A. S., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PO2+measurements+in+the+microcirculation+using+phosphorescence+quenching+microscopy+at+high+magnification%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">PO2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification.</a> American Journal of Physiology-Heart and Circulatory Physiology. 294 (6), 2905-2916 (2008).</li>
<li>Zhdanov, A. V., Golubeva, A. V., Okkelman, I. A., Cryan, J. F., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+oxygen+gradients+in+giant+umbrella+cells%3A+An+ex+vivo+PLIM+study%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Cell+Physiology%22%5BJournal%5D)">Imaging of oxygen gradients in giant umbrella cells: An ex vivo PLIM study.</a> American Journal of Physiology - Cell Physiology. 309 (7), 501-509 (2015).</li>
<li>Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+lifetime+imaging+-+Techniques+and+applications%5BTitle%5D)+AND+%22Journal+of+Microscopy%22%5BJournal%5D)">Fluorescence lifetime imaging - Techniques and applications.</a> Journal of Microscopy. 247 (2), 119-136 (2012).</li>
<li>Jenkins, J., Dmitriev, R. I., Papkovsky, D. B. Imaging cell and tissue O 2 by TCSPC-PLIM. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJenkins%2C+J+%22Advanced+Time-Correlated+Single+Photon+Counting+Applications%22">Advanced Time-Correlated Single Photon Counting Applications</a>. Becker, W. , Springer. Cham, Switzerland. 225-247 (2015).</li>
<li>Becker, W. Advanced TCSPC-FLIM techniques. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBecker%2C+W+%22Multiphoton+Microscopy+and+Fluorescence+Lifetime+Imaging%3A+Applications+in+Biology+and+Medicine%22">Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine</a>. König, K. , De Gruyter. Berlin, Germany. 23-52 (2018).</li>
<li>Wei, L., Yan, W., Ho, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+fluorescence+lifetime+analytical+microsystems%3A+Contact+optics+and+CMOS+time-resolved+electronics%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">Recent advances in fluorescence lifetime analytical microsystems: Contact optics and CMOS time-resolved electronics.</a> Sensors. 17 (12), 2800(2017).</li>
<li>Hirvonen, L. M., Suhling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wide-field+TCSPC%3A+Methods+and+applications%5BTitle%5D)+AND+%22Measurement+Science+and+Technology%22%5BJournal%5D)">Wide-field TCSPC: Methods and applications.</a> Measurement Science and Technology. 28, 012003(2017).</li>
<li>Hirvonen, L. M., Festy, F., Suhling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wide-field+time-correlated+single-photon+counting+%28TCSPC%29+lifetime+microscopy+with+microsecond+time+resolution%5BTitle%5D)+AND+%22Optics+Letters%22%5BJournal%5D)">Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution.</a> Optics Letters. 39 (19), 5602(2014).</li>
<li>Sparks, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterisation+of+new+gated+optical+image+intensifiers+for+fluorescence+lifetime+imaging%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging.</a> Review of Scientific Instruments. 88 (1), 013707(2017).</li>
<li>Chelushkin, P. S., Tunik, S. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChelushkin%2C+PS+%22Progress+in+Photon+Science%3A+Emerging+New+Directions%22">Progress in Photon Science: Emerging New Directions</a>. 115, Springer. Cham, Switzerland. (2017).</li>
<li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+macro-imager+based+on+Tpx3Cam+optical+camera+for+PLIM+applications%5BTitle%5D)+AND+%22Proceedings+of+SPIE%22%5BJournal%5D)">A new macro-imager based on Tpx3Cam optical camera for PLIM applications.</a> Proceedings of SPIE. , 113591(2020).</li>
<li>Fisher-Levine, M., Nomerotski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TimepixCam%3A+A+fast+optical+imager+with+time-stamping%5BTitle%5D)+AND+%22Journal+of+Instrumentation%22%5BJournal%5D)">TimepixCam: A fast optical imager with time-stamping.</a> Journal of Instrumentation. 11, (2016).</li>
<li>Nomerotski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+and+time+stamping+of+photons+with+nanosecond+resolution+in+Timepix+based+optical+cameras%5BTitle%5D)+AND+%22Nuclear+Instruments+and+Methods+in+Physics+Research%2C+Section+A%3A+Accelerators%2C+Spectrometers%2C+Detectors+and+Associated+Equipment.+937%22%5BJournal%5D)">Imaging and time stamping of photons with nanosecond resolution in Timepix based optical cameras.</a> Nuclear Instruments and Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 937. 937, 26-30 (2019).</li>
<li>Poikela, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timepix3%3A+A+65K+channel+hybrid+pixel+readout+chip+with+simultaneous+ToA%2FToT+and+sparse+readout%5BTitle%5D)+AND+%22Journal+of+Instrumentation%22%5BJournal%5D)">Timepix3: A 65K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout.</a> Journal of Instrumentation. 9, 05013(2014).</li>
<li>Nomerotski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+TimepixCam%2C+a+fast+imager+for+the+time-stamping+of+optical+photons%5BTitle%5D)+AND+%22Journal+of+Instrumentation%22%5BJournal%5D)">Characterization of TimepixCam, a fast imager for the time-stamping of optical photons.</a> Journal of Instrumentation. 12, 01017(2017).</li>
<li>Hirvonen, L. M., Fisher-Levine, M., Suhling, K., Nomerotski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photon+counting+phosphorescence+lifetime+imaging+with+TimepixCam%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Photon counting phosphorescence lifetime imaging with TimepixCam.</a> Review of Scientific Instruments. 88, 013104(2017).</li>
<li>Visser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SPIDR%3A+A+read-out+system+for+Medipix3+%26+Timepix3%5BTitle%5D)+AND+%22Journal+of+Instrumentation%22%5BJournal%5D)">SPIDR: A read-out system for Medipix3 & Timepix3.</a> Journal of Instrumentation. 10, 12028(2015).</li>
<li>Tsytsarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+brain+metabolism+activity+using+a+phosphorescent+oxygen-sensitive+probe%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe.</a> Journal of Neuroscience Methods. 216 (2), 146-151 (2013).</li>
<li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+O2+concentration+in+ex-vivo+tissue+samples+on+a+fast+PLIM+macro-imager%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Mapping O2 concentration in ex-vivo tissue samples on a fast PLIM macro-imager.</a> Scientific Reports. 10, 19006(2020).</li>
<li>Kersemans, V., Cornelissen, B., Allen, P. D., Beech, J. S., Smart, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcutaneous+tumor+volume+measurement+in+the+awake%2C+manually+restrained+mouse+using+MRI%5BTitle%5D)+AND+%22Journal+of+Magnetic+Resonance+Imaging%22%5BJournal%5D)">Subcutaneous tumor volume measurement in the awake, manually restrained mouse using MRI.</a> Journal of Magnetic Resonance Imaging. 37 (6), 1499-1504 (2013).</li>
<li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+luminescence+lifetime+macro-imager+based+on+a+Tpx3Cam+optical+camera%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">New luminescence lifetime macro-imager based on a Tpx3Cam optical camera.</a> Biomedical Optics Express. 11 (1), 77-88 (2020).</li>
<li>Papkovsky, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorescent+polymer+films+for+optical+oxygen+sensors%5BTitle%5D)+AND+%22Biosensors+and+Bioelectronics%22%5BJournal%5D)">Phosphorescent polymer films for optical oxygen sensors.</a> Biosensors and Bioelectronics. 7 (3), 199-206 (1992).</li>
<li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+planar+phosphorescence+based+oxygen+sensors+on+a+TCSPC-PLIM+macro-imager%5BTitle%5D)+AND+%22Sensors+and+Actuators%2C+B%3A+Chemical%22%5BJournal%5D)">Characterization of planar phosphorescence based oxygen sensors on a TCSPC-PLIM macro-imager.</a> Sensors and Actuators, B: Chemical. 321, 128459(2020).</li>
<li>Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., Berndt, K. W., Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+lifetime+imaging%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Fluorescence lifetime imaging.</a> Analytical Biochemistry. 202 (2), 316-330 (1992).</li>
</ol>

The authors have no conflicts of interest to declare.

The above protocols give a detailed description of the assembly of the new imager and its operation in the microsecond FLIM/PLIM mode. The TCSPC-based new generation Tpx3Cam camera, coupled by means of the opto-mechanical adaptor Cricket with the image intensifier, emission filter, and macro-lens, produces a stable, compact, and flexible optical module that is easy to operate. The imager was shown to perform well with a range of different samples and analytical tasks, which included the characterization of phosphorescent...

O2 is one of the key environmental parameters for living systems, and knowledge of the distribution of O2 and its dynamics is important for many biological studies1,2,3. The assessment of tissue oxygenation by means of phosphorescent probes4,5,6,7,8 and PLIM9,10,11,12,13 are gaining popularity in biological and medical research3,9,14,15,16,17,18,19. This is because PLIM, unlike fluorescence or phosphorescence intensity measurements, is not affected by external factors such as probe concentration, photobleaching, excitation intensity, optical alignment, scattering, and autofluorescence.
However, current O2 PLIM platforms are limited by their sensitivity, image acquisition speed, accuracy, and general usability. Time-correlated single photon counting (TCSPC), combined with a raster scanning procedure, is frequently used in PLIM and fluorescence lifetime imaging microscopy (FLIM) devices20,21,22. However, since PLIM requires a long pixel dwell time (in the millisecond range), the time of image acquisition is much longer than what is required for FLIM applications20,22,23. Other techniques, such as gated CCD/CMOS cameras, lack single photon sensitivity and have low frame rates20,24,25,26. Moreover, the existing PLIM systems are mostly used in the microscopic format, while macroscopic systems are less common27.
The TCSPC-based PLIM macro imager28 was set up to overcome many of these limitations. The design of the imager was greatly facilitated by the use of a new opto-mechanical adapter, Cricket, which has the following: i) two C-mount adapters, which provide easy coupling of the camera module on the back side and objective lens on the front side; ii) an internal housing for an image intensifier and a power socket for the latter on the outer side of the Cricket; iii) an internal space behind the front-side C-mount adapter where a standard 25 mm emission filter can be housed in front of the intensifier; and iv) a built-in light collimating optics with ring regulators, which allow optical alignment/focusing between the lens and the camera to produce crisp images on the camera chip.
In the assembled imager, the camera module is coupled to the back side of the Cricket adapter, which also houses an image intensifier consisting of a photocathode followed by a microchannel plate (MCP), an amplifier, and a fast scintillator, P47 phosphor. A 760 nm &#177; 50 nm emission filter is fitted inside the Cricket, and an objective lens, NMV-50M11&#39;&#39;, is attached to the front side C-mount adapter. Finally, the lens and the camera are aligned optically with ring regulators.
The role of the intensifier is to detect incoming photons and convert them into fast bursts of light on the camera chip, which are registered and used to generate emission decays and lifetime images. The camera module comprises an advanced TCSPC-based optical sensor array (256 pixels x 256 pixels) and a new generation readout chip29,30,31,32,33, which allow the simultaneous recording of the time of arrival (TOA) and the time over threshold (TOT) of photon bursts at each pixel of the imaging chip with a time resolution of 1.6 ns and an 80 Mpixel/s readout rate.
In this configuration, the camera with the intensifier has single-photon sensitivity. It is data-driven and based on the speedy pixel detector readout (SPIDR) system34. The spatial resolution of the imager was previously characterized with planar phosphorescent O2 sensors and a resolution plate mask. The instrument response function (IRF) was measured by the imaging of a planar fluorescent sensor under the same settings as used for all the other measurements. The lifetime of the dye of around 2.6 ns was short enough for it to be used for the IRF measurement in PLIM mode. The imager can image objects of up to 18 mm x 18 mm in size with spatial and temporal resolutions of 39.4 &#181;m and 30.6 ns (full width at half-maximum), respectively28.
The following protocols describe the assembly of the macro imager and its subsequent use for mapping the O2 concentration in biological samples stained with the previously characterized near-infrared O2 probe, NanO2-IR35. The probe is a bright, photostable, cell-permeable O2-sensing probe based on platinum (II) benzoporphyrin (PtBP) dye. It is excitable at 625 nm, emits at 760 nm, and provides a robust optical response to O2 in the physiological range (0%-21% or 0-210 &#181;M of O2). The imager is also demonstrated to characterize different sensor materials based on Pt(II)-porphyrin dyes. Overall, the imager is compact and flexible, similar to a common photographic camera. In the current setup, the imager is appropriate for different widefield PLIM applications. Substituting the LED with a fast laser source will further improve the performance of the imager and could potentially enable nanosecond FLIM applications.

<table><tbody><tr><td>627 nm LED</td><td>Parts Express</td><td></td><td>Can be replaced with different LED based on the excitation wavelength of the sensor. Used 390 nm LED for Pt-porphyrin dyes.</td></tr><tr><td>760 &amp;plusmn; 50 nm emission filter</td><td>Edmund Optics</td><td>84-788</td><td>Can be replaced with different filter based on the emission wavelength of the sensor. Used 650 &amp;plusmn; 50 nm bandpass filter for Pt-porphyrin dyes.</td></tr><tr><td>Balb/c mice</td><td>Envigo, UK</td><td>Balb/c</td><td></td></tr><tr><td>Black box</td><td>Thorlabs</td><td>XE25C9/M</td><td></td></tr><tr><td>Cricket Adapter</td><td>Photonis</td><td>Cricket-2</td><td></td></tr><tr><td>CT26 cells&amp;nbsp;</td><td>ATCC</td><td>CT26.WT</td><td>https://www.atcc.org/products/crl-2638</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D0697</td><td>Other media can also be used</td></tr><tr><td>ImageJ Software</td><td>ImageJ</td><td></td><td>Free Image analysis software. Can be downloaded from: https://imagej.nih.gov/ij/index.html</td></tr><tr><td>MCP-125 image intensifier with P47 phosphor screen</td><td>Photonis</td><td>PP0360EF</td><td></td></tr><tr><td>Mini dishes</td><td>Sarstedt</td><td>83.3900.300</td><td>35 mm diameter&amp;nbsp;</td></tr><tr><td>Mylar plastic film, 75 micron&amp;nbsp;</td><td>RS Ireland</td><td>785-0795</td><td>Othe plastic substrates can also be used</td></tr><tr><td>NanO2-IR</td><td>home-made</td><td>n/a</td><td>The probe can be synthesised according to the published method &#039;Tsytsarev V, Arakawa H, Borisov S, Pumbo E, Erzurumlu RS, Papkovsky DB. In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe. J Neurosci Methods. 2013 Jun 15;216(2):146-51. doi: 10.1016/j.jneumeth.2013.04.005. Epub 2013 Apr 25. PMID: 23624034; PMCID: PMC3719178.&#039; or provided by our lab.&amp;nbsp;</td></tr><tr><td>NMV-50M11&amp;rdquo; 50 mm lens</td><td>Navitar</td><td></td><td>Other lenses compatibel with C-mount adators can be used</td></tr><tr><td>Optical breadboard</td><td>Thorlabs</td><td>MB1836</td><td></td></tr><tr><td>Petri Dishes</td><td>Sarstedt</td><td>82.1472.001</td><td>92 mm diameter</td></tr><tr><td>Power Supply</td><td>Tenma</td><td>72-10495</td><td></td></tr><tr><td>Pulse Generator</td><td>Tenma</td><td>TGP110</td><td></td></tr><tr><td>Sophy</td><td>Amsterdam Scientific Instruments</td><td>n/z</td><td>Provided by ASI together with the Tpx3Cam</td></tr><tr><td>Tpx3Cam</td><td>Amsterdam Scientific Instruments</td><td>TPXCAM</td><td></td></tr><tr><td>Tri2 Software</td><td>University of Oxford</td><td>n/a</td><td>Free Time Resolved Imaging software, can be downloaded from: https://users.ox.ac.uk/~atdgroup/index.shtml</td></tr><tr><td>XYZ Translation Stage</td><td>Thorlabs</td><td>LT3</td><td></td></tr></tbody></table>

fluorescence lifetime macro imager for biomedical applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.
The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

For ex vivo imaging applications, fragments of intestinal tissues were stained by the topical application of the NanO2-IR probe on the serosal side of the tissue. For deeper staining, 1 &#181;L of the probe was injected into the lumen. In the latter case, the 0.2-0.25 mm thick intestinal wall shielded the probe from the camera. The two staining processes are demonstrated in Figure 2A.
The resulting intensity and PLIM images are presented in <strong class=...

Watch this Scientific Journal Video about Fluorescence Lifetime Macro Imager for Biomedical Applications at JoVE.com

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

fluorescence-lifetime-macro-imager-for-biomedical-applications

Research

JoVE Journal

Biology

688 Views.  University College Cork. This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

Video: Fluorescence Lifetime Macro Imager for Biomedical Applications

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Bioengineering

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Biochemistry

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials.

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise ...

Engineering

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting endogenous fluorescence. Viscosity is measured using a fluorescent molecular rotor.

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and ...

Cancer Research

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs O2 sensitive luminescent sensor foils (planar optodes) in combination with a specialized camera system for imaging luminescence lifetime in the frequency-domain. Planar optodes are prepared by dissolving the O2-sensitive indicator dye in a polymer and spreading the mixture on a solid support in a defined thickness via knife coating. After evaporation of the solvent, the planar optode is placed in close contact with the sample of interest - here demonstrated with the roots of the aquatic plant Littorella uniflora. The O2 concentration-dependent change in the luminescence lifetime of the indicator dye within the planar optode is imaged via the backside of the transparent carrier foil and aquarium wall using a special camera. This camera measures the luminescence lifetime (&#181;s) via a shift in phase angle between a modulated excitation signal and emission signal. This method is superior to luminescence intensity imaging methods, as the signal is independent of the dye concentration or intensity of the excitation source, and solely relies on the luminescence decay time, which is an intrinsically referenced parameter. Consequently, an additional reference dye or other means of referencing are not needed. We demonstrate the use of the system for macroscopic O2 imaging of plant rhizospheres, but the camera system can also easily be coupled to a microscope.

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs ...

Chemistry

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Immunology and Infection

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Neuroscience

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

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.

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 ...