Name: fluorescence lifetime macro imager for biomedical applications
Uploaded: 2023-04-07T12:30:00
Description: Watch this Scientific Journal Video about Fluorescence Lifetime Macro Imager for Biomedical Applications at JoVE.com

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 t...

<ol>
	<li>Papkovsky, D. B., Dmitriev, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+oxygen+and+hypoxia+in+cell+and+tissue+samples.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+the+partial+oxygen+pressure+of+human+tissues+a+crucial+parameter?+Small+molecules+and+hypoxia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+imaging+of+living+cells+and+tissues+using+luminescent+molecular+probes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescence+based+oxygen+sensors+essential+tools+for+cell+biology+and+life+science+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+brain+metabolism+activity+using+a+phosphorescent+oxygen-sensitive+probe.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescent+oxygen-sensitive+materials+for+biological+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+probes+and+techniques+for+O+2+measurement+in+live+cells+and+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescence+based+oxygen+sensors+and+probes+for+biomedical+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+phosphorescence:+A+novel+method+for+measuring+oxygen+distribution+in+perfused+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescence+quenching+method+for+measurement+of+intracellular+PO+2+in+isolated+skeletal+muscle+fibers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+imaging+of+oxygen+by+phosphorescence+lifetime.">Tomographic imaging of oxygen by phosphorescence lifetime.</a> Applied Optics. 45 (33), 8547-8559 (2006).</li><li>Becker, W., Shcheslavskiy, V., R&#252;ck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+phosphorescence+and+fluorescence+lifetime+imaging+by+multi-dimensional+TCSPC+and+multi-pulse+excitation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescent+sensing+and+imaging+of+oxygen:+Fierce+competition+to+the+Clark+electrode.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+neurosphere+oxygenation+with+phosphorescent+probes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+fluorescence+and+phosphorescence+lifetime+imaging.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+phosphorescence+imaging+of+pO2+using+planar+oxygen+sensors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcutaneous+pO2+imaging+during+tourniquet-induced+forearm+ischemia+using+planar+optical+oxygen+sensors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PO2+measurements+in+the+microcirculation+using+phosphorescence+quenching+microscopy+at+high+magnification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+oxygen+gradients+in+giant+umbrella+cells:+An+ex+vivo+PLIM+study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+-+Techniques+and+applications.">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., Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+and+tissue+O+2+by+TCSPC-PLIM.">Imaging cell and tissue O 2 by TCSPC-PLIM.</a> Advanced Time-Correlated Single Photon Counting Applications. , 225-247 (2015).</li><li>Becker, W., K&#246;nig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+TCSPC-FLIM+techniques.">Advanced TCSPC-FLIM techniques.</a> Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine. , 23-52 (2018).</li><li>Wei, L., Yan, W., Ho, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+fluorescence+lifetime+analytical+microsystems:+Contact+optics+and+CMOS+time-resolved+electronics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+TCSPC:+Methods+and+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+time-correlated+single-photon+counting+(TCSPC)+lifetime+microscopy+with+microsecond+time+resolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+new+gated+optical+image+intensifiers+for+fluorescence+lifetime+imaging.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progress in Photon Science: Emerging New Directions. 115, (2017).</li><li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+macro-imager+based+on+Tpx3Cam+optical+camera+for+PLIM+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TimepixCam:+A+fast+optical+imager+with+time-stamping.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+time+stamping+of+photons+with+nanosecond+resolution+in+Timepix+based+optical+cameras.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timepix3:+A+65K+channel+hybrid+pixel+readout+chip+with+simultaneous+ToA/ToT+and+sparse+readout.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+TimepixCam,+a+fast+imager+for+the+time-stamping+of+optical+photons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+counting+phosphorescence+lifetime+imaging+with+TimepixCam.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDR:+A+read-out+system+for+Medipix3+&amp;+Timepix3.">SPIDR: A read-out system for Medipix3 &amp; Timepix3.</a> Journal of Instrumentation. 10, 12028 (2015).</li><li>Tsytsarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+brain+metabolism+activity+using+a+phosphorescent+oxygen-sensitive+probe.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+O2+concentration+in+ex-vivo+tissue+samples+on+a+fast+PLIM+macro-imager.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+tumor+volume+measurement+in+the+awake,+manually+restrained+mouse+using+MRI.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+luminescence+lifetime+macro-imager+based+on+a+Tpx3Cam+optical+camera.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescent+polymer+films+for+optical+oxygen+sensors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+planar+phosphorescence+based+oxygen+sensors+on+a+TCSPC-PLIM+macro-imager.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging.">Fluorescence lifetime imaging.</a> Analytical Biochemistry. 202 (2), 316-330 (1992).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#177; 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 &#177; 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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Mylar plastic film, 75 micron&#160;</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 &#39;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.&#39; or provided by our lab.&#160;</td>
		</tr>
		<tr>
			<td>NMV-50M11&#8221; 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 ...

Our protocol provides detailed 2D mapping of phosphorescence lifetime and oxygen concentration in macroscopic objects, particularly live animal tissue and whole animals, by means of dedicated probes and sensing materials. This information is important for many areas of research. Our protocol uses an integrated and compact imaging module that operates in TCSPC mode and provides easy and accurate visualization of lifetime and oxygen distribution in complex biological samples, such as life tissue.Take the Cricket adapter and inspect it from different sides. Remove the front side C-mount adapter to show the Photonis PP0360EF intensifier housed in the Cricket, and then put the C-mount adapter back. Remove the Cricket's front side C-mount adapter and insert the 650 plus/minus 50 nanometer emission filter.Fix it by putting back the C-mount. Then, connect the TPX three-cam camera module to the backside of the Cricket module via their ring adapters. Connect the lens to the front side of the Cricket module via the C-mount adapter.Connect the LED to a power supply and a pulse generator. Now, mount the 390-nanometer super bright LED on a post attached to a breadboard inside the black box. Turn on the LED and focus it to ensure effective and uniform excitation of the sample being imaged.Mount the camera assembly on top of the optical black box facing down towards the stage where the samples will be imaged. Connect the camera and LED to two-pulse generators and a four-channel oscilloscope. Using settings on the oscilloscope and pulse generators, synchronize the pulses sent to the camera and the LED.Utilize the special cable and socket on the Cricket unit to connect the intensifier to a standard power supply and set the gain to 2.7 volts. Position the sample before the camera lens. Then, turn on the camera and all the electrical modules, except the intensifier.Activate the SOFI software to tune the operational parameters, like focusing and sample alignment. In the modules, set frame exposure to 0.01 seconds. Select Infinite frames and set the pixel operation mode to time over threshold.Then, go to Preview and select Active module to open the Medipix/Timepix frames window. In the next step, after starting the recording, adjust the color scale and rotate the image to the desired orientation. After that, switch off the lights in the room and switch on the intensifier.Focus the camera optics on the sample stage with the focusing capabilities of the lens and Cricket adapter to generate clear images of samples with good contrast and brightness. Once the focus is complete, close the SOFI software. Then, go to the terminal, execute the commands of the custom-design software to acquire the raw data in the binary format and close the terminal.Open a new terminal to process the acquired data. Load the data file and run the data reducer. Analyze the post-process data with a dedicated program written in C language that will write the data into a ICS image file.Subsequently, open the ICS image files using the freely-available Time Resolved Imaging software, and use two exponential functions to fit the phosphorescence decays. Lastly, open the Fitted. ics image files with the available image analysis software.Generate phosphorescence lifetime images using lookup tables and code them in pseudo color scale. Utilize the Measure function to calculate the average lifetime values for the entire image or regions of interest. Phosphorescence intensity and phosphorescence lifetime imaging microscopy images of the platinum octaethylporphyrin dye sensor spot in the oxygenated and deoxygenated states are shown.Make sure that the right emission filter is mounted inside the Cricket and the right LED, cable connections, and pulse settings are used. Currently, this protocol is limited to animal models, but potentially can be applied to diagnose and treat pathological states associated with tissue hypoxia, such as cancer, stroke, and inflammation.

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