This work was supported by NIH R01AI141439 to J.-X.C and M.S, and R35GM136223 to J.-X.C.

The use of human blood specimens is in accordance with the guidelines of the IRB of Boston University and the National Institutes of Health (NIH). Specifically, the specimens are from a bank and are completely deidentified. These specimens are not considered to be human subjects by institutional review board (IRB)&#160;office at Boston University.
1. Preparation of bacteria and antibiotics stock solution
<ol>
	<li>Prepare the antibiotics (gentamicin sulfate or amoxicillin) stock solution at a concentration of 1 mg/mL dissolved in sterile1x phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO) solvent in 1.5 mL micro tubes. Dissolve gentamicin sulfate in&#160;sterile PBS solution and amoxicillin in sterile DMSO solvent. Thereafter, store the antibiotics solution at 2-8 &#176;C as suggested.</li>
	<li>To make D2O containing cation-adjusted Mueller-Hinton Broth (MHB) media, add 220 mg of MHB broth base to 10 mL of D2O to make 100% D2O containing medium. Sterilize the solution by filtering with filters of 200 nm pore size. 
	NOTE: Use this protocol always for making and sterilizing medium solutions in further steps.</li>
	<li>To prepare bacterial samples for SRS imaging, add 2 mL of normal MHB media, which does not contain deuterium, to a sterile round-bottom culture tube, and then prewarm it at 37 &#176;C.</li>
	<li>Use a sterile loop to select one bacterial (Escherichia coli BW 25113 or Pseudomonas aeruginosa ATCC 47085) colony from the fresh culture on a tryptic soy agar plate. Then suspend it in the prewarmed culture media and gently vortex to prepare the bacteria suspension.</li>
	<li>Incubate bacteria at 37 &#176;C in a shaker at 200 revolutions per minute (rpm) until it reaches the logarithmic phase.</li>
</ol>
2. D2O incorporation treatment in the presence of antibiotics (Figure 2a)
<ol>
	<li>Check the bacterial concentration by measuring the optical density (OD) with a photometer at a wavelength of 600 nm.</li>
	<li>Dilute the bacterial solution using the normal MHB medium, which does not contain deuterium, to reach a final cell concentration of 8 x 105 CFU/mL. Vortex gently to mix the bacterial cells.</li>
	<li>Prepare 300 &#181;L aliquots of the bacterial solution in seven 1.5 mL micro tubes, and 600 &#181;L aliquots of the bacterial solution in one 1.5 mL micro tubes.</li>
	<li>Add 4.8 &#181;L of antibiotic (gentamicin or amoxicillin) stock solution (1 mg/mL) into the micro tube containing 600 &#181;L of the bacterial solution, to make the final antibiotic concentration to 8 &#181;g/mL.</li>
	<li>Take 300 &#181;L of solution out of the 8 &#181;g/mL of antibiotic-containing bacteria solution, and add to another 300 &#181;L of bacterial solution, to make two-fold diluted antibiotic- (4 &#181;g/mL) containing bacteria solution.</li>
	<li>Repeat the two-fold serial dilution of the test antibiotics, gentamicin, or amoxicillin, until the micro tube with the lowest concentration (0.25 &#181;g/mL) is reached, and discard 300 &#181;L from the tube. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 &#181;g/mL - 8 &#181;g/mL.
	<ol>
		<li>Leave one tube with no antibiotics for blank control. This will be the positive control to inspect the bacterial metabolic activity without antibiotics treatment but with D2O treatment.</li>
		<li>Leave one tube with no antibiotics and no&#160;D2O for the negative control.</li>
	</ol>
	</li>
	<li>Incubate the bacterial aliquot with the certain antibiotic (gentamicin or amoxicillin) containing MHB medium for 1 h.</li>
	<li>During incubation, prepare a serial dilution of antibiotics with 100% D2O containing medium with the same concentration gradient of antibiotics prepared in step 2.6. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 &#181;g/mL - 8 &#181;g/mL.</li>
	<li>After 1 h antibiotic treatment, add 700 &#181;L of serially diluted antibiotic and 100% D2O-containing MHB medium to the 300 &#181;L of antibiotic-pretreated bacteria in the same antibiotic concentration (prepared in step&#160;2.6), respectively.
	<ol>
		<li>For example, add 700 &#181;L of 100% D2O-containing MHB medium (containing 8 &#181;g/mL of antibiotic) to the 300 &#181;L of 8 &#181;g/mL antibiotic-pretreated bacteria. In the same manner, transfer to the corresponding tubes of the next concentration, and homogenize by pipetting up and down several times.</li>
		<li>Add 700 &#181;L of antibiotic-free 100% D2O-containing MHB medium to 300 &#181;L of antibiotic-free bacteria (prepared in step 2.6.1) as a blank control.</li>
		<li>Incubate at 37 &#176;C in an incubation shaker at 200 rpm for an additional 30 min. 
		NOTE: In this step, the final concentration of D2O in the medium for the test is 70%.</li>
	</ol>
	</li>
	<li>First centrifuge the 1 mL of antibiotic and D2O-treated bacterial sample at 6200 x g for 5 min at 4 &#176;C, and then wash twice with purified water. Finally, fix samples in 10% formalin solution and store them at 4 &#176;C.</li>
</ol>
3. Preparation of bacteria in urine environment (Figure 2b)
<ol>
	<li>To prepare E. coli BW 25113 at the logarithmic phase, follow the steps at 1.4 and 1.5.</li>
	<li>Check the bacterial concentration by measuring the OD&#160;with a photometer at a wavelength of 600 nm.</li>
	<li>To mimic the clinical UTI samples14,18,41, spike the E. coli solution into 10 mL of deidentified urine to reach a final cell concentration of 106 CFU/mL.</li>
	<li>Filter the E. coli spiked urine using a 5 &#181;m filter, and then divide the bacterial solution in 300 &#956;L aliquots into seven 1.5 mL micro tubes, and 600 &#181;L aliquots of the bacterial solution in one 1.5 mL micro tubes.</li>
	<li>Perform D2O incorporation treatment in the presence of antibiotics and sample collection&#160;as described in the previous steps from 2.4 to 2.10.</li>
</ol>
4. Preparation of bacteria in blood environment (Figure 2c)
<ol>
	<li>To prepare Pseudomonas aeruginosa ATCC 47085 at the logarithmic phase, follow the steps at 1.4 and 1.5.</li>
	<li>To mimic the clinical bloodstream infections samples42,43, spike P. aeruginosa in 1 mL of deidentified human blood to reach a&#160;concentration of 107 CFU/mL.</li>
	<li>Add 9 mL of sterile purified water to lyse the blood.</li>
	<li>Filter the P. aeruginosa spiked blood using a 5 &#181;m filter. Then harvest bacteria to 1 mL volume by centrifugation at 6200 x g for 5 min at 4 &#176;C.&#160;Add 9 mL of pre-warmed normal MHB into the bacteria solution and gently vortex. The final concentration of bacteria is 106 CFU/mL</li>
	<li>Divide the P. aeruginosa spiked blood solution in 300 &#956;L aliquots into seven 1.5 mL micro tubes, and 600 &#181;L aliquots of the bacterial solution in one 1.5 mL micro tubes.</li>
	<li>Perform D2O incorporation treatment in the presence of antibiotics and sample collection as described in the previous steps from 2.4 to 2.10.</li>
</ol>
5. SRS imaging of D2O metabolic incorporation in a single bacterium
<ol>
	<li>Wash 1 mL of fixed bacteria solution with purified water and then centrifuge at 6200 x g for 5 min at 4 &#176;C. Remove the supernatant. Enrich the bacterial solution to about 20 &#181;L.</li>
	<li>Deposit the bacterial solution on a poly-L-lysine coated coverglass. Sandwich and seal the sample for SRS imaging.</li>
	<li>Image bacteria at the C-D vibrational frequency at 2168 cm-1 using an SRS microscope.
	<ol>
		<li>Input and tune the pump wavelength to 852 nm using the control software on a computer.</li>
		<li>Measure the laser power using a power meter. Set the power of pump laser at the sample to ~8 mW and the power of Stokes laser at the sample to ~40 mW by adjusting the half-wave plate in front of the laser output. 
		NOTE: In the SRS microscope, a tunable femtosecond laser with an 80-MHz repetition rate provides the pump (680 to 1300 nm) and Stokes (1045 nm) excitation lasers.</li>
	</ol>
	</li>
	<li>By adjusting the screws of the reflection mirrors, spatially align the pump and Stokes beams and direct the two beams into an upright microscope equipped with 2D galvo mirror system for laser scanning.
	<ol>
		<li>Use a 60x water immersion objective to focus the pump and Stokes lasers on the sample.</li>
		<li>Use an oil condenser to collect the signals from the sample in the forward direction.</li>
		<li>Use a bandpass filter to filter out the Stokes laser before directing it into a photodiode.</li>
		<li>Extract the stimulated Raman signal by a lock-in amplifier and detect the signals by the photodiode.</li>
	</ol>
	</li>
	<li>Set each SRS image to contain 200 x 200 pixels and the pixel dwell time for 30 &#181;s in the software&#39;s control panel. The total acquisition time for one image is ~1.2 s. Set the Step size to 150 nm, so the image size is about 30 x 30 &#181;m2. Image at least three field of views for each sample.</li>
</ol>
6. Image processing and data analysis (&#160;Figure 3)
<ol>
	<li>To obtain the average C-D signal intensity, open and process SRS images with ImageJ software.</li>
	<li>First, convert SRS images into 8-bit type images with inverted color by clicking Image | Type | 8-bit, and then Edit | Invert buttons in the ImageJ software.</li>
	<li>Then, filter the images with Gaussian blur by clicking Process | Filters | Gaussian blur buttons and set the Sigma (Radius) to 1.</li>
	<li>Use image threshold adjustment to select the bacterial area. Click Image | Adjust | Threshold to ensure the selected bacterial sizes match those in the original SRS images. Eliminate small particles by adjusting the size threshold to determine the particles. Click Apply.</li>
	<li>Apply Analyze | Particles Analysis buttons to label and determine the area of bacteria.</li>
	<li>By clicking the Show All button in the ROI manager to the original unprocessed SRS image, label the same area of bacteria, determine the average intensity of each data point by clicking the Measure button in the ROI manager.</li>
	<li>Circle the background area in the original SRS image and measure the average intensity of the background. The average C-D intensities of each bacterium is obtained by deducting the background signal intensity.</li>
</ol>
7. Quantitation of antimicrobial susceptibility via SC-MIC
NOTE: The cut-off value at 0.60 to determine the SC-MIC is established according to the statistical analysis of the SRS C-D intensities of the metabolism-active and metabolism-inhibited conditions for bacteria upon various concentrations of drug exposure40. The C-D intensities for the antibiotic-susceptible and antibiotic-resistant groups were fitted with normal distribution.
<ol>
	<li>Plot the receiver operating characteristic (ROC) curve and evaluate the cut-off threshold at 0.60. Based on this cut-off value, the SC-MIC as an indicator of the efficacy of antibiotics can be defined to determine the metabolically inactive and metabolically active group.</li>
	<li>To quantitatively analyze the SRS imaging data, plot the histograms of C-D signal intensities for each bacteria group treated with the serially diluted antibiotic concentration. The colored data points stand for&#160;different individual bacterium.</li>
	<li>Normalize the C-D intensities of antibiotic-treated group to the mean intensity of the control group without antibiotic treatment. Determine the SC-MIC results of different bacteria and antibiotic combinations by quantifying the SRS signal intensities at C-D region versus various concentration of antibiotics using the cut-off value at 0.60.</li>
	<li>Validate and compare the SC-MIC readout with the MIC determined using conventional broth microdilution assay.</li>
	<li>According to the Clinical and Laboratory Standards Institute (CLSI), the susceptibility category based on the SRS metabolic imaging results for each tested bacterial strain is interpreted as &#34;susceptible&#34;, &#34;resistant&#34;, or &#34;intermediate&#34;.</li>
</ol>

The authors have no conflicts of interest to disclose.

Rapid AST can be obtained by assessing the response of bacterial metabolic activity to antibiotic treatment using single-cell SRS metabolic imaging within 2.5 h from the sample to SC-MIC results. The response of bacterial metabolic activity and antimicrobial susceptibility can be detected by monitoring the metabolic incorporation of D2O for biomolecule synthesis using SRS imaging of C-D bonds. Since water is ubiquitously used in living cells, SRS metabolic imaging provides a universal method for rapid AST. The...

Antimicrobial resistance (AMR) is a growing global threat to the effective treatment of infectious disease1. It is predicted that AMR will cause an additional 10 million deaths per year and $100 trillion global GDP loss by 2050 if no action for combating antibiotic-resistant bacteria is taken1,2. This stresses the urgent need for rapid and innovative diagnostic methods for &#65279;antibiotic susceptibility testing (AST) of infectious bacteria to slow down the emergence of antibiotic-resistant bacteria and reduce the related mortality rate3. To ensure the best possible clinical outcome, it is crucial to introduce effective therapy within 24 h. However, the current gold standard method, like disk diffusion or broth dilution method, usually requires at least 24 h for the preincubation procedure for clinical samples and an additional 16-24 h to obtain the minimal inhibitory concentration (MIC) results. Overall, these methods are too time-consuming to guide an immediate decision for infectious disease treatment in the clinic, which leads to the emergence and spread of antimicrobial resistance4.
Genotypic AST methods, such as polymerase chain reaction (PCR)-based techniques5, have been developed for rapid detection. Such techniques measure the specific resistance genetic sequences in order to provide rapid AST results. They do not rely on time-consuming cell culture; however, only specific known genetic sequences with resistance are tested. Therefore, its application is limited to various bacterial species or different mechanisms of resistance. Also, they cannot provide MIC results for therapy decisions6,7. Besides, novel phenotypic methods for rapid AST are under development to overcome these limitations8, including microfluidic devices9,10,11,12,13, optical devices14,15,16, phenotypic AST quantifying the nucleic acids copy number17,18, and Raman spectroscopic methods19,20,21,22,23,24. These methods reduce time to guide AST results, however, most of them are only applicable to bacterial isolates, not directly to clinical specimens, and still require long-time preincubation.
In this work, we present a method for rapid determination of the susceptibility of bacteria in the urine and whole blood via monitoring of the cellular metabolic activity by SRS imaging. Water (H2O) takes part in the vast majority of essential biomolecular synthesis processes in living cells. As an isotopologue of water, through enzyme-catalyzed H/D exchange reaction between the redox-active hydrogen atom in NADPH and the D atom in D2O, deuterium can be incorporated into biomass inside a cell25,26. A deuterated fatty acid synthesis reaction is mediated by the deuterium labeled NADPH. The D2O incorporation into reactions of amino acids (AAs) results in the deuterated protein production26 (Figure 1). In this way, the newly synthesized C-D bond-containing biomolecules in single microbial cells can be employed as a general metabolic activity marker to be detected. To read out de novo synthesized C-D bonds, Raman spectroscopy, a versatile analytical tool providing specific and quantitative chemical information of biomolecules, is widely used to determine antimicrobial susceptibility and significantly reduce the testing time to a few hours27,28,29,30. However, due to the inherent low efficiency of the Raman scattering process, the spontaneous Raman spectroscopy is of low detection sensitivity. Therefore, it is challenging to obtain real-time image results using spontaneous Raman spectroscopy. Coherent Raman scattering (CRS), including coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has reached high detection sensitivity because of the coherent light field to generate orders of magnitude larger than that of spontaneous Raman spectroscopy, thereby rendering high-speed, specific, and &#65279;quantitative chemical imaging at the single cell level31,32,33,34,35,36,37,38,39.
Here, based on our most recent work40, we present a protocol for rapid determination of the metabolic activity and antimicrobial susceptibility by femtosecond SRS C-D imaging of D2O incorporation of bacteria in the normal medium, urine, and whole blood environment at the single-cell level. Femtosecond SRS imaging facilitates monitoring single cell metabolism inactivation concentration (SC-MIC) against antibiotics at the single bacterium level within 2.5 h. The SC-MIC results are validated by standard MIC test via broth microdilution. Our method is applicable for determining antimicrobial susceptibility of bacteria urinary tract infection (UTI) and bloodstream infection (BSI) pathogens with a much reduced assay time compared to the conventional method, which opens the opportunity for rapid phenotypic AST in the clinic at the single-cell level.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acousto-optic modulation</td>
			<td>Gooch&#38;Housego</td>
			<td>R15180-1.06-LTD</td>
			<td>Modulating stokes laser beam</td>
		</tr>
		<tr>
			<td>Amoxicillin</td>
			<td>Sigma Aldrich</td>
			<td>A8523-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter</td>
			<td>Chroma</td>
			<td>HQ825/150m</td>
			<td>Block the stokes laser beam before the photodiode</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-100G</td>
			<td>Cation adjustment</td>
		</tr>
		<tr>
			<td>Cation-adjusted Mueller-Hinton Broth</td>
			<td>Fisher Scientific</td>
			<td>B12322</td>
			<td>Antimicrobial susceptibility testing of microorganisms by broth dilution methods</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002542</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses</td>
			<td>VWR</td>
			<td>16004-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tube with snap cap</td>
			<td>Fisher brand</td>
			<td>149569B</td>
			<td></td>
		</tr>
		<tr>
			<td>Daptomycin</td>
			<td>Acros</td>
			<td>A0386346</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td></td>
			<td>151882</td>
			<td>Organic solvent to dissolve antibiotics</td>
		</tr>
		<tr>
			<td>Deuterium oxide-d6</td>
			<td>Sigma Aldrich</td>
			<td>156914</td>
			<td>Organic solvent as a standard to calibrate SRS imaging system</td>
		</tr>
		<tr>
			<td>Escherichia coli BW 25113</td>
			<td>The Coli Genetic Stock Center</td>
			<td>7636</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf polypropylene microcentrifuge tubes 1.5 mL</td>
			<td>Fisher brand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>Sigma Aldrich</td>
			<td>G4918</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophilic Polyvinylidene Fluoride filters</td>
			<td>Millipore-Sigma</td>
			<td>SLSV025NB</td>
			<td>pore size 5 &#181;m</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>Version: 2.0.0-rc-69/1.52t</td>
			<td>Image processing and analysis</td>
		</tr>
		<tr>
			<td>Incubating orbital shaker set at 37 &#176;C</td>
			<td>VWR</td>
			<td>97009-890</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Sigma</td>
			<td>BR452201-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>InSight DeepSee femtosecond pulsed laser</td>
			<td>Spectra-Physics</td>
			<td>Model: insight X3</td>
			<td>Tunable laser source and fixed laser source at 1045 nm for SRS imaging</td>
		</tr>
		<tr>
			<td>Lock-in amplifier</td>
			<td>Zurich Instrument</td>
			<td>HF2LI</td>
			<td>Demodulate the SRS signals</td>
		</tr>
		<tr>
			<td>Oil condenser</td>
			<td>Olympus</td>
			<td>U-AAC</td>
			<td>NA 1.4</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa ATCC 47085 (PAO1)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 47085</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Hamamatsu</td>
			<td>S3994-01</td>
			<td>Detector</td>
		</tr>
		<tr>
			<td>Polypropylene conical tube 15 mL</td>
			<td>Falcon</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene filters</td>
			<td>Thermo Scientific</td>
			<td>726-2520</td>
			<td>pore size 0.2 &#181;m</td>
		</tr>
		<tr>
			<td>Sterile petri dishes</td>
			<td>Corning</td>
			<td>07-202-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL</td>
			<td>Fisher brand</td>
			<td>14955459</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Vis Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>Model: DU 530</td>
			<td>Measuring optical density at wavelength of 600 nm</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>Water objective</td>
			<td>Olympus</td>
			<td>UPLANAPO/IR</td>
			<td>60&#215;, NA 1.2</td>
		</tr>
	</tbody>
</table>

rapid antimicrobial susceptibility testing by stimulated raman scattering imaging of deuterium incorporation in a single bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

The effect of incubation time on deuterium incorporation is measured by spontaneous Raman microspectroscopy at the C-D (2070 to 2250 cm-1) and C-H &#65279;(2,800 to 3,100 cm-1) region (Figure 4a). The time-lapse single-cell Raman spectra of P. aeruginosa cultured in 70% D2O containing medium show increasing CD/CH intensity over incubation time from 0 to 180 min. (Figure 4b) The increasing C-D abundance in single microbia...

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Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

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2.6K Views.  Boston University. This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic. 

Video: Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...