We thank Paul O&#39;Brien, Marizel Mina and Isabella Freedman for animal experiments, Jacquie Gonzalez and Danielle Weiner from NIH/NIAID for help with gamma irradiation of rabbit tissues prior to laser capture microdissection and Jansy Sarathy for manuscript thoughts and advice. This work was supported by funding from the Bill and Melinda Gates Foundation (OPP1174780) and NIH shared instrumentation grant 1S10OD018072. We thank Eliseo A. Eugenin for providing access to the Leica LMD 6500 microscope and sharing expertise and advice. The purchase, and ongoing support of, the LMD 6500 was funded by The National Institute of Mental Health grant, MH096625, the National Institute of Neurological Disorders and Stroke, NS105584, PHRI funding (to E.A.E) and GSK contributions (to E.A.E).

All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with approval from the Institutional Animal Care and Use Committee of the NIAID (NIH), Bethesda, MD.
1. Animal Experiments and Tissue Collection
This section of the protocol describes animal procedures and sample collection under Biosafety Level 3 (BSL3) conditions. Detailed protocols of the Mycobacterium tuberculosis aerosol infection procedure and drug administration protocols in rabbits have been described previously11,12.
<ol>
	<li>Infect New Zealand White rabbits (male and female at 4 - 5 months old) with M. tuberculosis HN878 using a nose-only aerosol system, as previously described11.</li>
	<li>Administer the chosen drugs (Ethambutol in the example presented here) via the preferred route and euthanize the animals at 2, 6, and 24 h following administration. First, anesthetize the rabbit by intramuscular injection of Ketamine at 35 mg/kg and Xylazine at 5 mg/kg. Wait for ten minutes and confirm proper anesthetization by pinching the tail and gently touching the eye. If there is no reaction, euthanize by intravenous administration of pentobarbital and phenytoin (see Table of Materials) at 1 mL/4.5 kg in 2 mL sterile saline. 
	NOTE: These timepoints are optimal to cover the pharmacokinetic profile for Ethambutol and may require adjusting/optimizing for other study drugs.</li>
	<li>Using forceps, scissors, and/or scalpel, remove the lungs from the chest cavity, resect lung biopsies containing large necrotic granulomas embedded in surrounding uninvolved lung tissue (as described previously3). Necrotic granulomas appear beige in color and typically protrude slightly from the surrounding red/pink-colored lung. To facilitate easy cryosectioning, ensure that biopsies are no larger than 2 x 1.5 x 1.5 cm.</li>
	<li>Using forceps, place the biopsy onto a pre-labeled cryomold tray with the desired cutting surface in direct contact with the base of the tray. After freezing, this will provide a flat surface from which cryosections will be cut.</li>
	<li>Freeze the biopsy in liquid nitrogen vapor. Fill a styrofoam container to a depth of 2 inches with liquid nitrogen and place a metal wire tube rack. The rack should protrude above the surface of the liquid nitrogen providing a flat surface on which the tissue trays are placed. Place the lid back on the styrofoam container and leave tissues for 10 minutes to fully freeze.</li>
	<li>Remove the tissue trays, quickly wrap in aluminium film and place in individually labeled resealable plastic bags and seal. Transfer to -80 &#176;C freezer for storage. 
	NOTE: Steps 1.1 - 1.6 are performed in BSL3 conditions (including all animal work and handling of infected organs and tissues). Gamma irradiate the lung biopsies at 3 Megarads to enable handling outside of BSL3 containment. Laser capture microdissection on unsterilized tissue may be performed within the BLS3 facility if approved safety protocols are in place. However, the remainder of this protocol describes downstream processing in a BSL-2 facility.</li>
</ol>
2. Tissue Sectioning
<ol>
	<li>Set the cryostat to the desired cutting temperature. Transfer the gamma-irradiated lung biopsy from -80 &#176;C storage to the cryostat and leave for 30 minutes to equilibrate the tissue temperature. Note: -20 to -22 &#176;C is optimal for TB lesion biopsies.</li>
	<li>Using tweezers, fix the biopsy to the cryostat chuck using a small amount of optimal cutting temperature adhesive (OCT) to adhere the base of the tissue to the chuck. Orient the tissue so that the flat surface (that was in contact with the base of the cryomold) is the exposed surface for cutting. Ensure the OCT does not contaminate the tissue surface, as this may interfere with the subsequent mass spectrometry analysis.</li>
	<li>Cut three tissue sections at 25 &#181;m thickness and mount onto PET membrane slides. Gently touch the membrane to the tissue section and remove. If too much pressure is applied, the thin membrane may tear.
	<ol>
		<li>Avoid excessive handling of the slides prior to mounting as this will result in the PET membrane becoming charged and poor adhesion of the tissue sections. Ensure that the membrane slide is kept at room temperature to enable thaw-mounting and successful adhesion of the tissue to the membrane.</li>
	</ol>
	</li>
	<li>Remove the slide from the cryostat and allow to air-dry for 3 minutes. If LCM-LC/MS/MS will not be performed immediately, seal the slide in a small airtight sealable bag and transfer to -80 &#176;C storage until required for dissection.</li>
	<li>Cut an adjacent section at 10 - 12 &#181;m and thaw-mount on a standard glass slide for Hematoxylin and Eosin (H&#38;E) staining and reference. Additional sections can be cut at this time for other desired histochemistry stains (such as Ziehl-Niellsen for visualizing Mycobacterium tuberculosis (MTB)).</li>
</ol>
3. Microdissection
<ol>
	<li>Remove sealed bag containing the slide from -80 &#176;C storage and allow to reach room temperature for 5 minutes. 
	NOTE: If the cold slide is immediately exposed to the laboratory atmosphere, the tissue will become coated with condensation, and the spatial integrity of the drug may be compromised.</li>
	<li>Turn on the microscope and laser (laser requires 5 - 10 minutes warm up before cutting can commence). Load flat-cap 0.20 mL PCR tubes into the holder.</li>
	<li>Remove slide from the bag and take an optical image of the tissue section on the PET slide using a flatbed scanner.</li>
	<li>Place the slide into the slide holder (tissue side facing down) and assign separate collection tubes to specific granuloma regions of interest using the microscope software. Typically, these will be &#39;uninvolved lung,&#39;&#160;&#39;cellular granuloma,&#39; and &#39;caseum&#39; (necrotic center), but may vary depending upon the specific pathology of the granuloma/biopsy.</li>
	<li>Focus on the tissue using the 5X microscope objective. This magnification should provide a good overview of the tissue containing both cellular and necrotic granuloma areas. In the software select the tube designated &#39;caseum&#39; to move it into position under the tissue.</li>
	<li>Enter the desired dissection parameters. Typical settings for a 25 &#181;m thick lung section are laser power 30, speed 15, and aperture 35 (arbitrary units). However, these will differ depending upon the microscope used and potential declining power due to the age of the laser.</li>
	<li>Select the &#39;free-draw&#39; tool and, using either a mouse or touchscreen pen, outline the desired region for dissection. The surface area of the region will be displayed in the software. Keep selected regions under 500,000 &#181;m2 (0.5 mm2) to facilitate easier dissection. Repeat the dissection until 3 million &#181;m2 (3 mm2) have been collected in total in the tube cap.
	<ol>
		<li>On occasion, the dissected region may remain stuck to the surrounding membrane (for example due to static attraction) and not fall into the collection cap. Remove these regions from the cumulative surface area total by selecting and removing manually within the software.</li>
	</ol>
	</li>
	<li>Select the cap for &#39;cellular lesion&#39; and collect 3 million &#181;m2 of tissue using the same process as outlined in step 3.7.</li>
	<li>Select the cap for &#39;uninvolved lung&#39; and collect 3 million &#181;m2 of tissue using the same process as outlined in step 3.7. Note that uninvolved lung tissue contains many bronchioles and alveolar spaces. Pay careful attention to exclude these from the defined tissue regions for dissection.</li>
	<li>Remove the cap holder and carefully unclip, seal and label each tube. Protect the dissected tissues from surrounding air disturbances (such as from air flow disruption from an opening door). Analyze the dissected tissues immediately, or store at -80 &#176;C and thaw prior to processing and LCMS analysis.</li>
</ol>
4. Extraction and LCMS Analysis
<ol>
	<li>Prepare extraction solution of 1:1 acetonitrile/methanol containing Ethambutol d-10 internal standard. When selecting an internal standard, use a stable labeled form of the analyte drug (such as deuterium-labeled EMB used in this demonstration) with sufficient mass shift to avoid isotope cross talk between the analyte drug and standard (usually a minimum of 4 daltons). 
	NOTE: Creating standards in homogenate of each respective tissue type is difficult because there is very limited control tissue with which to create homogenate standards. As an alternative to making standards from a spiked homogenate sample, a standard can be created by adding blank tissue and test compound together and extracting. A volume of control tissue in a homogenate that matches the target volume of the study sample tissue sections is combined directly with an amount of the test compound that would be present at a given concentration.</li>
	<li>Calculate the targeted tissue volume based on the surface area and thickness of the tissue section and determine the necessary dilution factor for the homogenate using the volume of homogenate that will be added to the standard and QC samples. Calculations are illustrated below for a 3 million &#181;m2 (3 mm2) target dissected area with a 25 &#181;m thickness and 2 &#181;L volume of homogenate. 
	<img alt="Equation 1" src="/files/ftp_upload/57402/57402eq1.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/57402/57402eq2.jpg" /> 
	<img alt="Equation 3" src="/files/ftp_upload/57402/57402eq3.jpg" /> 
	<img alt="Equation 4" src="/files/ftp_upload/57402/57402eq4.jpg" /></li>
	<li>Assuming a tissue density of 1 g/mL, prepare the homogenate stock by weighing 50 mg of control tissue and adding PBS buffer to dilute (using the 26.67 homogenate dilution factor calculated in step 4.2, the diluent is 1.283 mL). Homogenize by bead beating lung tissue and PBS buffer for 5 minutes at 1750 rpm on a bead homogenizer. 
	<img alt="Equation 5" src="/files/ftp_upload/57402/57402eq5.jpg" /> 
	<img alt="Equation 6" src="/files/ftp_upload/57402/57402eq6.jpg" /></li>
	<li>Dilute 1 mg/mL drug stocks concentration in 1:1 acetonitrile/water to create standard curve spiking solutions. Determine spiking standard concentrations based on the spike volume and the target tissue volume. The illustrated example is for a 100 ng/mL standard using a 10 &#181;L spike volume. 
	<img alt="Equation 7" src="/files/ftp_upload/57402/57402eq7.jpg" /> 
	<img alt="Equation 8" src="/files/ftp_upload/57402/57402eq8.jpg" /></li>
	<li>Remove the tubes containing the microdissected tissues from -80 &#176;C storage and allow to reach room temperature for 5 minutes.</li>
	<li>Add 10 &#181;L of 1:1 acetonitrile/water solution and 2 &#181;L of PBS buffer to the tubes containing microdissected tissue.</li>
	<li>For standard curve and quality control tubes, add 10 &#181;L of spiking solution to 2 &#181;L of control lung homogenate.</li>
	<li>Add 50 &#181;L of extraction solution to each tube.</li>
	<li>Vortex each tube for 5 minutes, sonicate for 5 minutes and centrifuge at 5000 RPM for 5 minutes to form a pellet of film and tissue in each tube.</li>
	<li>Transfer 50 &#181;L of supernatant to a 96-well deep-well plate and dilute with an additional 50 &#181;L of deionized water in each well.</li>
	<li>Perform LC/MS/MS analysis using optimized instrument parameters for Ethambutol and Ethambutol-d10 internal standard (as previously described in detail12).</li>
	<li>Use a dilution factor to correct for the exact amount of tissue dissected for each sample. 
	<img alt="Equation 9" src="/files/ftp_upload/57402/57402eq9.jpg" /></li>
</ol>
5. Method Validation
<ol>
	<li>Create a homogenate in control lung tissue by combining 1 part lung, 2 parts PBS, and 3 - 4 steel beads. Beat lung tissue and PBS buffer for 5 minutes at 1750 rpm using a bead homogenizer.</li>
	<li>Spike the homogenate by adding 10 &#181;L of 1 mg/mL Ethambutol DMSO stock into 990 &#181;L homogenate to create a final concentration of 10,000 ng/mL (10 mg/mL) and vortex for 1 minute.</li>
	<li>Create a frozen homogenate block by pouring the homogenate into a cryomold and rapidly freezing on dry ice for 5 minutes.</li>
	<li>Prepare 25 &#181;m thick sections from the homogenate block as described in steps 2.1 - 2.5.</li>
	<li>Dissect the target tissue area as specified in steps 3.2 - 3.10.</li>
	<li>Add 10 &#181;L 1:1 acetonitrile/water and 2 &#181;L of PBS buffer to the tubes containing microdissected tissue.</li>
	<li>Add 50 &#181;L of extraction solution to each tube. Follow steps 4.9 - 4.12 to create a standard curve and determine the drug concentration in the tissue homogenate block.</li>
	<li>Calculate extraction efficiency using the formula below: 
	<img alt="Equation 10" src="/files/ftp_upload/57402/57402eq10.jpg" /></li>
</ol>

<ol>
	<li>Dartois, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+of+anti-tuberculosis+drugs:+From+blood+to+lesions+to+mycobacterial+cells.">The path of anti-tuberculosis drugs: From blood to lesions to mycobacterial cells.</a> Nat Rev Microbiol. 12 (3), 159-167 (2014).</li><li>Prideaux, 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=The+association+between+sterilizing+activity+and+drug+distribution+into+tuberculosis+lesions.">The association between sterilizing activity and drug distribution into tuberculosis lesions.</a> Nat Med. 21 (10), 1223-1227 (2015).</li><li>Prideaux, 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=High-sensitivity+MALDI-MRM-MS+imaging+of+moxifloxacin+distribution+in+tuberculosis-infected+rabbit+lungs+and+granulomatous+lesions.">High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.</a> Anal Chem. 83 (6), 2112-2118 (2011).</li><li>Roscioli, K. 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=Desorption+electrospray+ionization+(DESI)+with+atmospheric+pressure+ion+mobility+spectrometry+for+drug+detection.">Desorption electrospray ionization (DESI) with atmospheric pressure ion mobility spectrometry for drug detection.</a> Analyst. 139 (7), 1740-1750 (2014).</li><li>Prideaux, 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=Mass+spectrometry+imaging+of+levofloxacin+distribution+in+TB-infected+pulmonary+lesions+by+MALDI-MSI+and+continuous+liquid+microjunction+surface+sampling.">Mass spectrometry imaging of levofloxacin distribution in TB-infected pulmonary lesions by MALDI-MSI and continuous liquid microjunction surface sampling.</a> Int J Mass Spectrom. 377, 699-708 (2015).</li><li>Griffiths, R. L., Randall, E. C., Race, A. M., Bunch, J., Cooper, H. 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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=Cavitary+penetration+of+levofloxacin+among+patients+with+multidrug-resistant+tuberculosis.">Cavitary penetration of levofloxacin among patients with multidrug-resistant tuberculosis.</a> Antimicrob Agents Chemother. 59 (6), 3149-3155 (2015).</li><li>Zhao, Y., 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=Unraveling+drug+penetration+of+echinocandin+antifungals+at+the+site+of+infection+in+an+intra-abdominal+abscess+model.">Unraveling drug penetration of echinocandin antifungals at the site of infection in an intra-abdominal abscess model.</a> Antimicrob Agents Chemother. , (2017).</li><li>Pascal, 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=Mechanistic+modeling+identifies+drug-uptake+history+as+predictor+of+tumor+drug+resistance+and+nano-carrier-mediated+response.">Mechanistic modeling identifies drug-uptake history as predictor of tumor drug resistance and nano-carrier-mediated response.</a> ACS Nano. 7 (12), 11174-11182 (2013).</li></ol>

The authors have nothing to disclose.

Spatially-resolved quantification of drugs within pulmonary TB lesions is required to determine whether drug exposure reaches sterilizing concentrations to bacterial populations residing within the different lesion compartments. The LCM-LC/MS method described here enables absolute quantification of anti-TB drugs within all lesion compartments, including the bacteria-rich caseum, using only 1 - 3 tissue sections in total. Traditional tissue homogenization and LC/MS approaches for drug quantification in tissue often lack t...

The ability to spatially resolve and quantify drug levels is a crucial requirement for determining whether anti-tuberculosis drugs reach bacterial subpopulations within pulmonary lesions at sterilizing concentrations1. Of particular importance is determining drug penetration into the necrotic core of the lesion (called caseum), which typically contains the highest number of bacilli and may be poorly accessible to drugs due to the absence of vascularization.
Traditional methods to assess lesion penetration, which involve homogenization of excised pulmonary lesions followed by solvent extraction and liquid chromatography mass spectrometry (LC/MS) analysis, are highly sensitive and selective for the drugs of interest. However, these methods offer poor spatial information, limited to the size of the original homogenized tissue. Mass spectrometry-based imaging approaches, such as matrix-assisted laser desorption ionization (MALDI)2,3, desorption electrospray ionization (DESI)4 or liquid-enhanced surface extraction5,6 offer highly spatially-resolved imaging capabilities, but direct quantification can be extremely challenging or impossible due to heterogeneous ion suppression effects and differing extraction efficiencies of analyte from the various cell or tissue types7. Additionally, most direct tissue MS imaging approaches are inherently less sensitive than LC/MS due to the lack of chromatographic separation of endogenous species competing for ionization and the lower solvent extraction efficiency of the drug from tissue.
Laser capture microdissection (LCM) combined with LC/MS analysis has been routinely applied to isolate and characterize distinct tissue regions for proteomic studies8,9 and recently utilized for drug quantification in dosed animal tissue10. Here we present an optimized protocol applying LCM combined with LC/MS (LCM-LC/MS) analysis to quantify anti-TB drugs within distinct granuloma compartments. In the laser capture microdissection process, a UV laser is focused through the microscope objective onto the tissue section, which cuts and isolates the desired tissue area by following a path defined by the user. For gravity-assisted LCM (the technique used for this research), the tissue section is mounted onto a thin polymer membrane slide (PET or PEN) and the tissue is captured in a collection tube cap sited below the slide. The drugs are extracted from the excised tissue and quantified using standard LC/MS approaches. The amount of tissue required to be collected is ultimately determined from the expected concentration of the drug present in the tissue and the sensitivity of the LC/MS method. For most analyses of drugs dosed at therapeutic levels and analyzed using a routine triple quadrupole mass spectrometer, 3 million &#181;m2 (3 mm2) of tissue surface area is sufficient.
This protocol describes the powerful combination of spatial profiling and full quantification offered by LCM-LC/MS, providing absolute drug concentrations within all compartments of TB granulomas. The technique may also be applied to determining drug concentrations in many different diseased tissues providing vital drug discovery and development information.

<table><tbody><tr><td>New Zealand White rabbits</td><td>Covance</td><td>N/A</td><td></td></tr><tr><td>HN878 Mycobacterium tuberculosis</td><td>BEI Resources</td><td>NR-13647</td><td></td></tr><tr><td>Ketathesia (Ketamine) 100 mg/mL C3N</td><td>Henry Schein Animal Health</td><td>56344</td><td></td></tr><tr><td>Anased (Xylazine) 100 mg/mL</td><td>Henry Schein Animal Health</td><td>33198</td><td></td></tr><tr><td>Euthasol (pentobarbital sodium and phenytoin sodium) Solution</td><td>Virbac</td><td>710101</td><td></td></tr><tr><td>Acetonitrile (LC-MS grade)</td><td>Fisher</td><td>A955-212</td><td></td></tr><tr><td>Methanol (LC-MS grade)</td><td>Fisher</td><td>A456-212</td><td></td></tr><tr><td>Formic Acid (LC-MS grade)</td><td>Fisher</td><td>A117-50</td><td></td></tr><tr><td>Water (LC-MS grade)</td><td>Fisher</td><td>W6212</td><td></td></tr><tr><td>0.2 mL flat-cap PCR tubes</td><td>Corning</td><td>07-200-392</td><td></td></tr><tr><td>Steel frames, PET-membrane</td><td>Leica</td><td>11505151</td><td></td></tr><tr><td>Premium Frosted Microscope Slides</td><td>Fisher</td><td>12-544-2</td><td></td></tr><tr><td>96 Deep well plate 2.0ML PP RB</td><td>Fisher</td><td>NC0363259</td><td></td></tr><tr><td>Zorbax SB-C8 column (4.6 by 50 mm; particle size, 3.5 &amp;mu;m)</td><td>Agilent</td><td>820631-001D</td><td></td></tr><tr><td>&quot;Zipper&amp;rdquo; Seal Sample Bags</td><td>Fisher</td><td>01-816-1B</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CM1850 cryostat</td><td>Leica</td><td>Discontinued</td><td>Leica CM1860 is the current model</td></tr><tr><td>Laser Microdissection System 6500</td><td>Leica</td><td>Discontinued</td><td>Leica LMD 6 is the current model</td></tr><tr><td>Agilent 1260 Infinity II HPLC</td><td>Agilent</td><td></td><td></td></tr><tr><td>API 4000 QTRAP Mass Spectrometer</td><td>Sciex</td><td></td><td></td></tr></tbody></table>

spatial quantification of drugs in pulmonary tuberculosis lesions by laser capture microdissection liquid chromatography mass spectrometry (lcm-lc/ms)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

An overview of the LCM-LC/MS approach is shown in Figure 1. After sterilizing the tissue by gamma-irradiation, all subsequent steps (from tissue sectioning onwards) take place outside of BSL3 conditions. Figure 2 shows the lesion biopsy sections before and after tissue isolation by LCM. Necrotic and cellular areas of TB lesions can be easily identified and isolated by visual inspection of optical images alone (without the require...

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Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

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9.0K Views.  Rutgers, The State University of New Jersey. Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Video: Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

Engineering

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Biochemistry

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

Immunology and Infection

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Biology

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Chemical Cartography Approaches to Study Trypanosomatid Infection

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. Human-infective trypanosomatid parasites include Trypanosoma cruzi, the causative agent of Chagas disease; Trypanosoma brucei, the&#160;causative agent of sleeping sickness; and Leishmania species, causative agents of leishmaniasis. Jointly, they affect 20 million people across the globe. These parasites show specific tropism: heart, esophagus, colon for T. cruzi, adipose tissue, pancreas, skin, circulatory system and central nervous system for T. brucei, skin for dermotropic Leishmania strains, and liver, spleen, and bone marrow for viscerotropic Leishmania strains. A spatial perspective is therefore essential to understand trypanosomatid disease pathogenesis. Chemical cartography generates 3D visualizations of small molecule abundance generated via liquid chromatography-mass spectrometry, in comparison to microbiological and immunological parameters. This protocol demonstrates how chemical cartography can be applied to study pathogenic processes during trypanosomatid infection, beginning from systematic tissue sampling and metabolite extraction, followed by liquid chromatography-tandem mass spectrometry data acquisition, and concluding with the generation of 3D maps of metabolite distribution. This method can be used for multiple research questions, such as nutrient requirements for tissue colonization by T. cruzi, T. brucei, or Leishmania, immunometabolism at sites of infection, and the relationship between local tissue metabolic perturbation and clinical disease symptoms, leading to comprehensive insight into trypanosomatid disease pathogenesis.

This protocol describes the steps to generate a 3D model of metabolite distribution during trypanosomatid infection, including sample collection, metabolite extraction, an overview of liquid chromatography-tandem mass spectrometry data acquisition, 3D model generation, and finally, data visualization.

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

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Chapters in this video

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Analysis of ¹⁸FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Chemical Cartography Approaches to Study Trypanosomatid Infection