We thank Daniel Bukala, Natalie Altmeyer and Funda Cay for excellent technical support. We thank Jonathan Cotton, Greg Bowden and Paul Soubiran for editing the manuscript. This work was supported by the Werner Siemens-Foundation and the Medical Faculty of the Eberhard Karls University T&#252;bingen (&#8216;&#8216;Promotionskolleg&#8217;&#8217;) and by the DFG through the CRC 156 (project C3).

All procedures described in this paper, followed the guidelines and international standards of the care and use of laboratory animals and were approved by the local Animal Welfare and Ethics Committee of the Country Commission Tuebingen, Germany. 8 - 12 weeks old BALB/c and C57BL/6 mice were kept on a 12 h:12 h light:dark cycle and were housed in IVCs and standardized environmental conditions at 22 &#177; 1 &#176;C in groups of 2 - 5 with water and food access ad libitum.
1. Material Preparation
<ol>
	<li>Dilute the OI dye for near infrared fluorescence imaging according to the respective data sheet directly before injection. The activatable OI dye (commercial probe with an excitation at 680 nm) to measure MMPs in vivo is ready to use at a concentration of 20 nmol in 1.5 mL 1x PBS. Gently shake or vortex the solution before use. OI dye can be stored at 2 - 8 &#176;C for up to 6 months when protected from light.</li>
	<li>For intravenous (i.v.) injections prepare a venous catheter. Use a 20 U (0.5 mL) insulin syringe filled with 0.9% saline solution (containing 10 injection units of heparin in 50 mL), and a 30-gauge needle attached to a polyethylene catheter. Inject the recommended dose of 2 nmol per mouse of the OI dye.</li>
</ol>
2. Induction of Rheumatoid Arthritis and Chronic Contact Hypersensitivity Reaction
<ol>
	<li>Rheumatoid arthritis:
	<ol>
		<li>Dilute 100 &#181;L of serum (gained from K/BxN mice10) containing antibodies (AB) against glucose-6-phosphate isomerase (GPI) 1:1 with 1x PBS (200 &#181;L) to induce RA. Store the diluted serum at -80 &#176;C. Detailed procedures for induction of RA are described by Monach et al.8.</li>
		<li>To induce RA, lift each mouse gently by its tail and inject 200 &#181;L of the diluted serum intraperitoneal (i.p.) on day 0 of the experiment. After the injection place the mouse directly back into its cage.</li>
		<li>Optionally, measure the ankle diameters of each ankle, before AB injections and define an &#34;arthritic score&#34; (Figure 1A). Continue measurement of ankle swelling daily until day 6 after GPI-serum using a mechanical measuring device (micrometer).</li>
		<li>Inject the activatable OI dye (step 1.1 - 1.2), to measure in vivo MMP activity i.v. into the tail vein of RA mice on day 5 after GPI-serum injection and into the tail vein of control mice. Perform optical imaging experiments 24 h after tail vein injection.</li>
	</ol>
	</li>
	<li>Contact hypersensitivity reaction:
	<ol>
		<li>For sensitization of C57BL/6 mice, prepare a 5% TNCB solution dissolved in a 4:1 mixture of acetone/oil.</li>
		<li>Anaesthetize C57BL/6 mice using 1.5% isoflurane vaporized in 100% oxygen (1.5 L/min). Once the mice are anesthetized, place it in a self-made nose cone (a cut 5 mL polyethylene syringe, connected to the 1.5 vol% isoflurane tube) and maintain anesthesia. Apply vet eye ointment on anesthetized animals to prevent dryness of the eyes while anesthetized.</li>
		<li>Shave the abdomen carefully (2 x 2 cm) using a small animal hair trimmer. Avoid injuring the skin, as this can lead to unspecific fluorescent signals within the animal and can influence the results of the study.</li>
		<li>Apply 80 &#181;L of the 5% TNCB solution slowly at the shaved abdomen area using a 100 &#181;L pipette to sensitize the animal. Place the mouse gently back into its cage taking care to ensure that the mouse&#8217;s eyes do not come in contact with the bedding to avoid corneal injury and avoid low body temperatures during recovery phase.</li>
		<li>Six days after sensitization, prepare a 1% TNCB solution (dissolved in a 9:1 mixture of acetone/oil) and apply 20 &#181;L at the right ear using a pipette to elicit acute and chronic CHSR.1
		<ol>
			<li>Start with the 1st challenge on both sides of the right ear with 20 &#181;L of 1% TNCB solution using a 100 &#181;L pipette and repeat the challenge every second day up to five times (day 15 after sensitization) to elicit CHR (Figure 1B). Measure ear thickness daily, using a micrometer measuring device.</li>
		</ol>
		</li>
		<li>Inject the activatable OI dye (a commercial probe with excitation at 680 nm) (step 1.1-1.2) to measure MMP activity in vivo in CHR mice 12 h after the challenge. Perform optical imaging experiments 24 h post tail vein injection (i.v.).</li>
	</ol>
	</li>
</ol>
3. Animal Preparation for Optical Imaging
<ol>
	<li>Switch mouse chow (at least 3 days before imaging) to low or non-fluorescence chow (e.g., manganese free) to avoid the interference of auto-fluorescence (around 700 nm) with the fluorescence signal of the used OI agents.</li>
	<li>Place the animal into an anesthesia box and anesthetize using 1.5 vol% isoflurane vaporized with oxygen (1.5 L/min) (oxygen can be replaced by air but needs to be standardized within one experiment).</li>
	<li>When the mouse is visibly anesthetized, place it in a self-made nose cone (build by a cut 5 mL polyethylene syringe, connected to the 1.5 vol% isoflurane tube) and maintain anesthesia.</li>
	<li>Shave the animal carefully on the target site (2 cm x 2 cm) using a small animal hair trimmer. Avoid injuring the skin, as this can lead to unspecific fluorescent signals within the animal and can influence the results of the study. 
	NOTE: Hairs can absorb the fluorescence signal during OI (dependent on the mouse strain, more or less absorption is observed) depending on the region of interest (ROI).</li>
	<li>For i.v. injection, place the tail of the mouse in warmed water, to induce vasodilation, gently cleanse the skin at the injection site with alcohol, and start by placing the catheter at the distal site of the tail. Place the &#34;cut&#34; edge of the needle in, at an angle of 20&#176; into the tail vein and test the correct placing of the catheter by re-suspending the syringe.</li>
	<li>If the catheter is placed correctly, replace the syringe and inject the OI probe (2 nmol). After injection, replace the syringe and inject 25 &#181;L of 0.9% saline solution to fully clear the dead volume of the polyethylene tube. 
	NOTE: The tissue half-life time of the used OI dye (a commercial probe with excitation at 680 nm) for measuring MMP activity in vivo is 72 h. To ensure a complete clearance, a re-injection of the dye is not recommended earlier than 7 days after a prior injection.</li>
</ol>
4. Optical Imaging
<ol>
	<li>Place a black plastic or paper sheet in the black box of the OI-scanner in the center of the field of view (FOV).</li>
	<li>Set up a measuring protocol and choose the right wavelength (excitation: 680 &#177; 10 nm and emission: 700 &#177; 10 nm) and imaging parameters. 
	NOTE: In some OI systems, the setup for several imaging dyes is pre-defined.</li>
	<li>To choose the correct protocol for fluorescence imaging, open the imaging software (provided by the manufacturer) and initialize the system. Most CCD cameras need to cool down to their working temperature, and this can take 10 minutes. For reliable results, wait until the system is ready.</li>
	<li>Observe the in vivo imaging system acquisition control panel pop up and each filter pair selected will represent one image in the sequence. In this case, acquire one image with the filter pairs for the commercial dye with and an excitation and emission wavelength of 680 &#177; 10 nm and 700 &#177; 10 nm, respectively, and start the measurement (Press &#34;Acquire sequence&#34;). For more detailed instructions, please see the manual of the manufacturer.</li>
	<li>Label the images appropriately and save the information in the &#34;Edit Image Labels&#34; window, which will pop up following the &#34;Acquire sequence&#34;.</li>
	<li>Take a baseline scan of each animal before the injection of the OI dye, or use na&#239;ve control animals to differentiate the background signal.</li>
	<li>h after the tail vein injection of the OI dye, place the animals in the center of the FOV, in a position to measure the highest signal in the OI system, and start the measurements. 
	NOTE: Important: Hypothermia can significantly influence the distribution of imaging agents. Make sure that the stage is heated up to 37 &#176;C to avoid hypothermia of the animal. Imaging can be performed simultaneously with animals in groups of 1 to 5 mice. Choose the size of the FOV depending on the number of animals to measure at the same time. You can take a bright field image to check whether each mouse is visible.</li>
</ol>
5. Data Analysis
NOTE: Perform data analysis using the image software following manufacturer&#39;s protocol.
<ol>
	<li>For measurements of RA, use the calibrated unit of the photon emission as shown in Figure 2, whereas chronic CHSR are pictured as signal intensity in percent (efficiency).</li>
	<li>To draw ROIs manually, use the tool plate. For the analysis of the RA images use a standardized circle, placed around the highest signal in all ankles and paws of each animal. To analyze the CHSR, place the ROIs around the whole right and left ear according to the bright field image.</li>
	<li>To measure photon emission or signal intensity values in the specific drawn ROI, press &#34;measure&#34;. The system will provide values in the drawn ROI for descriptive statistical analysis.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+mechanisms+in+the+pathogenesis+of+rheumatoid+arthritis.">Immunologic mechanisms in the pathogenesis of rheumatoid arthritis.</a> J Clin Rheumatol. 11, S39-S44 (2005).</li><li>Pap, 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=Differential+expression+pattern+of+membrane-type+matrix+metalloproteinases+in+rheumatoid+arthritis.">Differential expression pattern of membrane-type matrix metalloproteinases in rheumatoid arthritis.</a> Arthritis Rheum. 43 (6), 1226-1232 (2000).</li><li>Firestein, G. S., Paine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromelysin+and+tissue+inhibitor+of+metalloproteinases+gene+expression+in+rheumatoid+arthritis+synovium.">Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium.</a> Am J Pathol. 140 (6), 1309-1314 (1992).</li><li>Schwenck, 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=In+vivo+optical+imaging+of+matrix+metalloproteinase+activity+detects+acute+and+chronic+contact+hypersensitivity+reactions+and+enables+monitoring+of+the+antiinflammatory+effects+of+N-acetylcysteine.">In vivo optical imaging of matrix metalloproteinase activity detects acute and chronic contact hypersensitivity reactions and enables monitoring of the antiinflammatory effects of N-acetylcysteine.</a> Mol Imaging. 13, (2014).</li><li>Monach, P. A., Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+K/BxN+arthritis+model.">The K/BxN arthritis model.</a> Curr Protoc Immunol. 15, 22 (2008).</li><li>Zelmer, A., Ward, T. 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A., Maricevich, M., Mahmood, U. <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+optical+molecular+imaging+of+matrix+metalloproteinase+activity+in+abdominal+aortic+aneurysms+correlates+with+treatment+effects+on+growth+rate.">In vivo optical molecular imaging of matrix metalloproteinase activity in abdominal aortic aneurysms correlates with treatment effects on growth rate.</a> Atherosclerosis. 212 (1), 181-187 (2010).</li><li>Chen, 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=Near-infrared+fluorescent+imaging+of+matrix+metalloproteinase+activity+after+myocardial+infarction.">Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.</a> Circulation. 111 (14), 1800-1805 (2005).</li><li>Wallis de Vries, B. M., 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=Images+in+cardiovascular+medicine.+Multispectral+near-infrared+fluorescence+molecular+imaging+of+matrix+metalloproteinases+in+a+human+carotid+plaque+using+a+matrix-degrading+metalloproteinase-sensitive+activatable+fluorescent+probe.">Images in cardiovascular medicine. Multispectral near-infrared fluorescence molecular imaging of matrix metalloproteinases in a human carotid plaque using a matrix-degrading metalloproteinase-sensitive activatable fluorescent probe.</a> Circulation. 119 (20), e534-e536 (2009).</li><li>Weissleder, R., Tung, C. H., Mahmood, U., Bogdanov, A. <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+tumors+with+protease-activated+near-infrared+fluorescent+probes.">In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.</a> Nat Biotechnol. 17 (4), 375-378 (1999).</li><li>Cortez-Retamozo, 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=Real-time+assessment+of+inflammation+and+treatment+response+in+a+mouse+model+of+allergic+airway+inflammation.">Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.</a> J Clin Invest. 118 (12), 4058-4066 (2008).</li><li>McIntyre, J. O., 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=Development+of+a+novel+fluorogenic+proteolytic+beacon+for+in+vivo+detection+and+imaging+of+tumour-associated+matrix+metalloproteinase-7+activity.">Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.</a> Biochem J. 377, 617-628 (2004).</li><li>Scherer, R. L., VanSaun, M. N., McIntyre, J. O., Matrisian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+matrix+metalloproteinase-7+activity+in+vivo+using+a+proteolytic+nanobeacon).">Optical imaging of matrix metalloproteinase-7 activity in vivo using a proteolytic nanobeacon).</a> Mol Imaging. 7 (3), 118-131 (2008).</li><li>Olson, E. S., 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+characterization+of+activatable+cell+penetrating+peptides+for+targeting+protease+activity+in+cancer.">In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer.</a> Integr Biol (Camb. 1 (5-6), 382-393 (2009).</li><li>Duijnhoven, S. M., Robillard, M. S., Nicolay, K., Grull, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+targeting+of+MMP-2/9+activatable+cell-penetrating+imaging+probes+is+caused+by+tumor-independent+activation).">Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation).</a> J Nucl Med. 52 (2), 279-286 (2011).</li><li>Schafers, M., Schober, O., Hermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-metalloproteinases+as+imaging+targets+for+inflammatory+activity+in+atherosclerotic+plaques.">Matrix-metalloproteinases as imaging targets for inflammatory activity in atherosclerotic plaques.</a> J Nucl Med. 51 (5), 663-666 (2010).</li><li>Wagner, S., 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+18F-labelled+derivative+of+the+MMP+inhibitor+CGS+27023A+for+PET:+radiosynthesis+and+initial+small-animal+PET+studies.">A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal PET studies.</a> Appl Radiat Isot. 67 (4), 606-610 (2009).</li><li>Waschkau, B., Faust, A., Schafers, M., Bremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+new+fluorescence-labeled+MMP+inhibitor+to+image+tumor+MMP+activity+in+vivo+in+comparison+to+an+MMP-activatable+probe.">Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.</a> Contrast Media Mol Imaging. 8 (1), 1-11 (2013).</li><li>Vogl, 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=Alarmin+S100A8/S100A9+as+a+biomarker+for+molecular+imaging+of+local+inflammatory+activity.">Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.</a> Nat Commun. 5, 4593 (2014).</li></ol>

The authors have nothing to disclose.

OI is a very useful, fast and inexpensive tool for non-invasive in vivo molecular imaging in preclinical research. A particular strength of OI is the capability to monitor highly dynamic processes like inflammatory responses. Moreover, OI allows one to follow the course of a disease for an extended period of time, ranging from days to weeks.
OI has several advantages over other in vivo imaging modalities such as positron-emission tomography (PET) or magnetic resonance imaging (MRI), as it is very time-, and cost-efficient. High-throughput analysis with up to five animals per acquisition is possible. Furthermore, a large number of probes, targeting many different biological processes, are available.12 To provide further anatomical information, OI can be combined with other imaging techniques such as MRI or x-ray. Another limitation is its low tissue penetration depth compared e.g. to functional PET imaging. Furthermore a large amount of probes aiming at many different biological targets are available.12
Effects of anesthesia should not be underestimated in in vivo imaging. While specific effects of anesthesia on OI results are not well characterized, our group has shown in several studies the influence of anesthesia on PET imaging results.13,14,15 For example, Fuchs et al. analyzed blood parameters, such as pCO2, pH and lactate before and after PET scans using different breathing and anesthesia protocols. It was shown that, significant changes in pCO2 and lactate levels in anesthetized compared to conscious oxygen or air breathing mice led to significant changes in PET results.14 They conclude that this effect is mainly caused by oxygen breathing and subsequent to respiratory acidosis in animals, leading to the different results in the PET imaging.
Standardization of anesthesia parameters, like O2-concentrations and doses of anesthetic drugs, as well as prevention of hypothermia help to avoid systematic failures. Of course, OI is limited by physical properties e.g. light scattering, tissue auto-fluorescence and light attenuation9. Several groups are addressing these problems by combining the macroscopic results of OI with other techniques like in vivo fluorescence microscopy or flow cytometry.
In vivo imaging of MMP activity by MMP-specific activatable fluorescent probes has been used in several experimental models of inflammation like CHS,7 but also in diseases like cerebral ischemia,16 aortic aneurysms,17 myocardial infarction18 and atherosclerotic plaques19 as well as various tumor models.20
For example, Cortez-Retamozo et al. investigated the in vivo activity of MMPs in a model of allergic airway inflammation by injecting an MMP activatable probe, 24 h after intranasal application of ovalbumin in previously sensitized mice.21 To identify the location of MMP activity further this group combined tomographic OI, NIR fiber optic bronchoscopy and intravital microscopy in order to monitor the course of disease and treatment response in greater detail.21
Other approaches to measure in vivo MMP activity mostly use inhibitors of MMP that bind to the active site of MMPs. McIntyre et al. described an in vivo tumor associated-MMP-7 detection using a &#34;polymer-based fluorogenic substrate&#34;, which acts as a &#34;proteolytic beacon&#34; for MMPs in a mouse xenograft model.22 They show that the activity of MMP-7 could be detected selectively by optical imaging.23 Further, Olson et al. investigated &#34;activatable cell penetrating peptides&#34; (ACPPs) which are able to target many xenograft tumor models, but cannot adsorb into the cell until the linker is proteolyzed. They investigated ACPPs which are selective for MMP-2 and MMP-9.24,25
Labeling of MMP inhibitors with radioactive isotopes for single-photon emission computed tomography (SPECT) or PET imaging was used to study MMP expression in atherosclerosis26 and cancer27. In a recent study a fluorescence-labeled MMP inhibitor was compared to an MMP-activatable probe.28 The fluorescently-labeled MMP inhibitor showed a lower signal to noise ratio compared to an activatable probe but required a shorter uptake time.
New biomarkers to assess key features of pathophysiology can be utilized and validated by OI. For example, the response to danger associated molecular patterns (DAMPs) is an early event during an inflammatory reaction. Vogl et al. used a Cy5.5 labeled antibody against alarmin S100A9 in models of acute ear skin dermatitis, collagen-induced arthritis and infection with Leishmania major to develop sensitive biomarker for early signs of inflammation.29
In conclusion, OI represents a fast and efficient method for preclinical research, which is capable of uncovering new mechanisms of diseases in vivo, characterizing novel molecular targets, as well as monitoring therapeutic effects. Nevertheless, a subsequent validation process with more quantitative techniques like PET might be required.

Autoimmune diseases such as rheumatoid arthritis (RA) or psoriasis vulgaris are graded as delayed-type hypersensitivity reactions (DTHRs).1 RA is a common autoimmune disease characterized by erosive synovitis and joint destruction.2 Inflamed arthritic joints demonstrate infiltration and proliferation of inflammatory cells, an increased expression of pro-inflammatory cells leading to pannus formation, cartilage and bone destructions.3,4 The cleavage of extracellular matrix molecules, such as collagen by matrix metalloproteinases (MMPs), is essential for tissue conversion and angiogenesis and causes tissue destructions.5,6 Contact hypersensitivity reactions (CHR) are characterized by aggregation of neutrophils leading to an oxidative burst.7 Similar to RA, MMPs in CHR are involved in tissue conversion, cell migration and angiogenesis in order to establish chronic inflammation.
To investigate RA, the glucose-6-phosphate isomerase (GPI)-serum injection mouse model was used.8 Serum from transgenic K/BxN mice containing antibodies against GPI, was injected into na&#239;ve BALB/c mice after which rheumatic inflammation began to develop within 24 h with a maximum of ankle swelling on day 6 after GPI-serum injection (see 1.1). To analyze chronic CHR, C57BL/6 mice were sensitized with trinitrochlorobenzene (TNCB) on the abdomen. The right ear was challenged up to 5 times starting 1 week after sensitization (see also 1.1 and 1.2).
Noninvasive small animal OI is a technique based on the in vivo investigation of fluorescent-, chemiluminescent- and bioluminescent-signals, which are mainly used in preclinical research. The acquired semi-quantitative data gives insights into the molecular mechanisms in the organs and tissues of healthy as well as diseased experimental animal models, and enables longitudinal follow up measurements (e.g. to assess therapeutic response profiles in vivo). A big advantage of longitudinal studies is the reduction of animal numbers, as the same animals can be measured in follow up studies at several time points instead of using different mice per time point. The resolution of OI allows detailed functional imaging of organs and even smaller tissue structures in experimental animals.
The use of specific excitation and emission filters with a narrow transmission spectrum, a protection against scattered light by a lightproof &#34;dark box&#34; and a sensitive charged-coupled device (CCD) camera, which is cooled in many devices down to -70 &#176;C, allows highly specific and sensitive measurements of fluorescence signals.
By using fluorescent agents with excitation- and emission-spectra in the near-infrared fluorescence window (650 - 950 nm), signal-to-noise ratios can be improved significantly. The near-infrared fluorescence window is characterized by a relatively low absorption of the signal by hemoglobin and water as well as a low background auto-fluorescence.9 This allows a penetration depth of up to 2 cm in the tissue of small animals. OI-probes can address a target directly (e.g. by a fluorescence labeled antibody) or can be activated in the target tissue (e.g. by proteases). Activatable OI probes are optically silent in their inactivated form due to the F&#246;rster resonance energy transfer (FRET) to a quenching moiety, which transfers the excitation energy within the molecule to another domain. If the dye is cleaved (by a protease for example) the energy is no longer transferred within the molecule and a fluorescent signal can be detected by OI. This allows the design of OI probes with high specificity for distinct biological processes and excellent signal-to-noise-ratios.
The following protocol explains in detail the preparation of the animals, the OI measurements using an Activatable OI probe to image MMP-2, -3, -9 and -13 activity in vivo and two experimental models of inflammation (RA, CHR).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
		</tr>
		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
		</tr>
		<tr>
			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
		</tr>
		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
		</tr>
		<tr>
			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
		</tr>
		<tr>
			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
		</tr>
		<tr>
			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
		</tr>
		<tr>
			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
		</tr>
		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
		</tr>
		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
		</tr>
		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
		</tr>
	</tbody>
</table>

non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

To induce rheumatoid arthritis (RA) in na&#239;ve BALB/c mice, animals were injected i.p. with auto-antibodies (1:1 dilution with 1x PBS) against GPI on day 0. The maximum inflammation (ankle swelling) in this GPI-serum induced RA model is on day 6 post injection11. Therefore, 2 nmol of the activatable OI dye was prepared and injected i.v. in the tail vein of arthritic mice and healthy control animals on day 5. 24 h after injection (day 6), mice were anesthetized and imaged as described in in section 3 (Figure 1A).
To induce CHR, C57BL/6 mice were sensitized with 5% TNCB on day 0 at the shaved abdomen. On day 7 post sensitization the 1st challenge on the right ear using 1% TNCB started. Up to five challenges (day 15) were performed to induce chronic inflammation.7 12 h after the last challenge, 20 nmol of the OI dye was injected i.v. On day 15, 24 h post injection, OI was performed as described above (Figure 1B).
Figure 2 displays a clearly enhanced fluorescence signal in the hind paws, ankles and front paws of the RA mouse on day 6 after disease induction, compared to control ankles (Figure 2A). Biodistribution analysis of the MMP activity in organs of RA mice and healthy mice exhibited a strongly enhanced signal in the hind paws, ankles and fore paws exclusively in the joints of RA mice. Interestingly, we determined an enhanced MMP signal in both kidneys of all measured RA mice, when compared to the kidneys of healthy control mice (Figure 2A; right). Moreover, we measured an enhanced MMP signal in the liver and intestine regardless of whether mice suffer from RA. No differences between RA and healthy mice were found in the pancreas, lung, spleen and heart.
During chronic CHR (five TNCB challenges at the right ear), we measured highly increased MMP activity on the right inflamed ear compared to the healthy left control ear (Figure 2B).
Thus, our data is indicating a major role of MMP activity during disease progression in both inflammatory diseases models.
Toolsets for Bioluminescence and Fluorescence allow the quantification of, for example pmol of fluorochrome or the number of cells which express fluorescence. Data can be displayed in 1. Counts: displays an uncalibrated measurement of the photon incident on the camera, 2. Radiance (photons): indicates a calibrated measurement of the photon emission in &#34;photons/second/cm2/steradian&#34;, which is recommended for luminescence imaging or 3. Radiant Efficiency (fluorescence): shows fluorescence emission radiance per incident excitation power, recommended for fluorescence measurements.
A big advantage of working with radiance units while analyzing OI images is, that camera settings can be changed during one experiment, and this has no impact on the images itself or the measured ROI data, which further allows comparing images from different IVIS systems.
The region of interest (ROI) indicates the specific area of interest from the user in an optical image. The toolbar enables to draw 3 different ROIs: measurement (measures the signal intensity in the ROI of the image), average background (measures the average signal intensity in the user defined area for the background), or subject ROI (identifies a subject animal in an image).
If the subject displays a high unspecific background signal itself, obtain a background-corrected ROI measurement by subtracting the background signal (ROI) from the target ROI. Set the image minimum close to zero to determine the auto fluorescence or naturally occurring background luminescence in your image. Link the image to a self-defined (user specific) background image (ROI) which were measured primarily to the measured specific image.
Semi-quantitative analyses of the images were performed by drawing standardized regions of interest (ROIs) in both arthritic and control ankles, as well as in inflamed and healthy ears using Living Image software. Only descriptive statistical analyses were performed on in vivo data in this manuscript, due to insufficient number of animals. Analysis of the signal intensity of arthritic and healthy ankles or paws showed a 7-fold enhanced MMP signal intensity in arthritic, compared to control ankles. A 3-fold higher MMP signal intensity was detected in the front paws of arthritic mice compared to that of the healthy animals (Figure 3A).
Analysis of ears with acute and chronic CHR compared to the left unchallenged control ear showed a significantly increased MMP signal even after the 1st challenge, which further increased after the 3rd and remained at the same level until the 5th challenge (Figure 3B). As a consequence of contamination with the contact allergen on the left ear due to scratching of the mice, we determined a slightly increased MMP signal in the left (not TNCB challenged) control ear. The results indicate an important role of MMPs, evinced by a highly increased fluorescence signal during GPI-arthritis and chronic inflammation, as measured by OI.
<img alt="Figure 1" src="/files/ftp_upload/55180/55180fig1.jpg" /> 
Figure 1: RA and CHR Model, Time Course of Disease Induction and Time Points for OI. (A) Time course of RA induction in BALB/c mice (B) and chronic CHR induction in C57BL/6 mice. Overview of the respective in vivo imaging time point and associated injection time points of the OI dye for measuring MMP activity in both models. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55180/55180fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55180/55180fig2.jpg" /> 
Figure 2: MMP OI in RA, CHR and Control Animals. (A) Representative results of the measured MMP activity in an arthritic mouse 6 days after GPI-arthritis induction and a healthy control mouse (left side). Significantly higher signal (Radiance (p/s/cm2/sr)) intensity was observed in the arthritic compared to the healthy mouse. The image on the right displays the MMP activity in the hind paws and includes the ankles, fore paws, pancreas, lung, spleen, heart, kidneys, liver and intestine of a sacrificed GPI-arthritis mouse after OI dye injection. OI uncovered a high signal in the fore paws, ankles and hind paws of RA mice and the liver and intestine regardless of whether the mice were injected with GPI serum (RA mice) or control serum (healthy control mice). (B) MMP fluorescence signal in ears with chronic CHR (left) and control animals (right). The inflamed right ear (challenged 5 times) displays a highly enhanced signal compared to control ears. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55180/55180fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55180/55180fig3.jpg" /> 
Figure 3: Semi Quantitative Analysis of Inflamed and Healthy Ankles and Ear Tissue Using OI. (A) Semi-quantitative analysis of the fluorescence signal in RA and healthy animals in the front paws and ankle joints. Arthritic front paws and ankle joints display a significantly (**p &#60; 0.05) (n = 3) higher MMP signal compared to healthy ankle joints and front paws. The data is presented as the mean &#177; SD. (B) Semi-quantitative analysis of the left and right (challenged) ears after the 1st, 3rd and 5th challenge. MMP signal is increased significantly from the first to the 5th challenge and already showed a maximum signal intensity in the right challenged ear after the 3rd challenge (**p &#60; 0.05) (n = 3) (Control ears are partly contaminated with TNCB due to the mice scratching. This produced an enhanced signal which was not significant). The data is presented as the mean &#177; SEM. The 2-tailed Student&#39;s t-test was used to analyze differences between inflammation (GPI-arthritic ankles, right treated ears) and control (control ankles, left untreated ears) for both models. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55180/55180fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Eberhard Karls University of Tübingen. This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Video: Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...