We thank Dr. Yajuan Li and Anthony Fung for their technical support, and the Fraley lab for the cell line. We acknowledge the start-up funds from UCSD, NIH U54CA132378, NIH 5R01NS111039, NIH R21NS125395, NIHU54DK134301, NIHU54 HL165443, and Hellman Fellow Award.

1. Media preparation
<ol>
	<li>Prepare 10 mL of control and excess AAAs in Dulbecco&#39;s modified Eagle medium (DMEM) containing 50% D2O.
	<ol>
		<li>For the control media, measure and mix 10 mg of DMEM powder with 4.7 mL of double distilled water (ddH2O) in a 15 mL conical tube. The DMEM powder contains all the amino acids at standard concentrations. Thoroughly vortex and invert the tube to ensure the solution is well mixed. Add 4.7 mL of D2O, 0.5 mL of fetal bovine serum (5% FBS), and 0.1 mL of penicillin/streptomycin (1%). Thoroughly vortex and invert the tube to ensure the solution is well mixed.</li>
		<li>For the 15x Phe treatment medium, measure and mix 10 mg of DMEM powder with 4.7 mL of ddH2O, 0.5 mL of FBS (5%), and 0.1 mL of penicillin/streptomycin (1%) in a 15 mL conical tube. Thoroughly vortex and invert the tube to ensure the solution is well mixed. Add excess Phe powder at a concentration of 924 mg/L to the medium. Thoroughly vortex and invert the tube to ensure the solution is well mixed.</li>
		<li>For the 15x Tryp treatment medium, measure and mix 10 mg of DMEM powder with 4.7 mL of ddH2O, 0.5 mL of FBS (5%), and 0.1 mL of penicillin/streptomycin (1%) in a 15 mL conical tube. Thoroughly vortex and invert the tube to ensure the solution is well mixed. Add excess Tryp powder at a concentration of 224 mg/L to the medium. Thoroughly vortex and invert the tube to ensure the solution is well mixed.</li>
		<li>Add methionine and threonine at 30.0 mg/mL and 95.0 mg/mL, respectively, to all conical tubes of the previous steps. Thoroughly vortex and invert the tube. Sodium pyruvate is not required to be added to the culture media.</li>
		<li>Filter the control and treatment media with a 25 mm syringe filter with 0.22 &#181;m polyethersulfone membrane filters.</li>
		<li>Seal all the media-contained conical tubes with parafilm and store at 4 &#176;C for up to 3 weeks.Prior to treating the cells with media, warm all the media to 37&#176;C. 
		&#8203;NOTE: The powder and liquid reagents should be measured and combined based on the total volume of treatment and control media.</li>
	</ol>
	</li>
</ol>
2. Cell sample preparation
<ol>
	<li>Maintain cervical cancer (HeLa) cells in a culture flask (i.e., T10, T25, etc.) using standard DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 &#176;C and 5% carbon dioxide (CO2).</li>
	<li>Sub-culture the HeLa cells at a split ratio of 1:10 until reaching 80% or higher cell viability.</li>
	<li>Once the cells achieve 80% or higher confluency, proceed to seed 2 x 105 cells per well onto a 24-well plate using DMEM supplemented with 0.5% FBS and 1% penicillin/streptomycin.
	<ol>
		<li>Prior to cell seeding, prepare a 24-well plate with sterilized, poly-d-lysin, laminin-coated, round coverslips, 12 mm in diameter. Submerge the coverslips in 70% ethanol and dry gently using lint-free wipes. Place the clean coverslips in the appropriate wells of the plate.</li>
		<li>Once the cells reach 80% confluency in step 2.2, wash the cells once with 1x phosphate buffered saline (PBS) without magnesium and calcium ions.</li>
		<li>Add 0.25% trypsin to dissociate the adherent cells from the flask and incubate at 37 &#176;C and 5% CO2 for 3 min.</li>
		<li>Add DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, gently pipette up and down, and collect the cells by centrifugation at 300 x g at room temperature (RT) for 3 min.</li>
		<li>Resuspend the cells in DMEM supplemented with 0.5% FBS and 1% penicillin/streptomycin.</li>
		<li>Count the cells using a hemocytometer, light microscope, and trypan blue, and seed a density of 2 x 105 cells per well of a 24-well plate. Alternatively, an automated cell counter can be used to count the cells.</li>
		<li>Return the cells to the incubator at 37 &#176;C and 5% CO2, and gently shake the plate to distribute the cells evenly.</li>
	</ol>
	</li>
	<li>After 8 h, replace the old culture media with 50% D2O/excess Phe or 50% D2O/excess Tryp media. Return the cells to the incubator at 37 &#176;C and 5% CO2 for 36 h.</li>
	<li>After 36 h, fix the cells on microscope slides.
	<ol>
		<li>Prior to fixing, prepare microscope slides with imaging spacers 9 mm in diameter and place 15 &#181;L of 1x PBS supplemented with calcium and magnesium ions.</li>
		<li>Rinse the cells with 1x PBS supplemented with calcium and magnesium ions.</li>
		<li>Add 0.5 mL of 4% methanol-free paraformaldehyde (PFA) solution and leave the plate under the biosafety hood for approximately 15 min.</li>
		<li>Aspirate the PFA solution and rinse with 1x PBS supplemented with calcium and magnesium ions twice.</li>
		<li>Add 1.5 mL of 1x PBS supplemented with calcium and magnesium ions in each well.</li>
		<li>Use sterile tweezers to gently grab the coverslips out of each well. Invert and place the cell-containing side of the coverslips onto the imaging spacers to contact the PBS prepared in step 2.5.1. Ensure that the non cell-containing side is exposed to air.</li>
		<li>Using transparent nail polish, seal the outer layer of the coverslip with the imaging spacer.</li>
	</ol>
	</li>
	<li>Store the microscope slides at 4 &#176;C when not imaging.</li>
</ol>
3. Spontaneous Raman spectroscopy measurement (Figure 2)
<ol>
	<li>Use a confocal Raman microscope to obtain spontaneous Raman spectra (Figure 2A).</li>
	<li>Turn on the laser by turning the key to the ON position.</li>
	<li>Double-click on the LabSpec6 software icon to open the acquisition software.</li>
	<li>Load the biological sample onto the sample holder directly underneath the objective lens.</li>
	<li>On the software, click the Camera icon to turn on the video camera.</li>
	<li>Adjust the focus plane to identify a region of interest with the 50x objective lens. Move the joystick to control the planer translation of the XY stage and rotate the joystick to change the depth of focus.</li>
	<li>After choosing the position for measurement, switch to the 100x objective lens with 40 mW of excitation power. Click the red Stop icon to turn off the video camera.</li>
	<li>Select a grating of 1800 lines/mm, and set the acquisition range from 400 cm-1 to 3,150 cm-1.</li>
	<li>Set the Acquisition Time to 90 s, the Accumulation to 3, and the Binning to 4 for the least noise and most accurate spectrum. 
	NOTE: These imaging parameters can be optimized to fit the experiment.</li>
	<li>Click the Arrow icon to turn on the real-time display.</li>
	<li>Tune the Raman shift (cm-1) to a value of choice to fine-tune the focus plane. 
	NOTE: In this experiment, the Raman laser was focused onto the lipid droplets, which contained a high concentration of CH2 bonds. Therefore, a Raman shift of 2,850 cm-1 that matched the stretching modes of the CH2 bond was selected for the purpose.</li>
	<li>Adjust the focus plane with the joystick to optimize the best signal-to-noise ratio.</li>
	<li>After choosing the focus plane, click the red Stop icon for a real-time display.</li>
	<li>Select the Circle icon to start the spectrum acquisition.</li>
	<li>Once the cellular region is taken, use the same focus plane to measure the background with PBS. Subtract the background spectrum from each subcellular target spectrum using a separate computer software application, such as Origin (Figure 1B).</li>
</ol>
4. Imaging experiments with 2PEF and SRS
NOTE: Detailed descriptions of SRS laser alignment can be found in a prior report28. This protocol focuses on the operation of a multimodal SRS and 2PEF imaging system (Figure 2C, D).
<ol>
	<li>Click on Start to warm up the laser.</li>
	<li>Follow the order to set the main switches of the control unit and the monitor on: 1) press the main switch of the control box IX3-CBH to On, 2) press the main switch of the touch panel controller to On, 3) press the main switch of the AC adapter of the power supply for LD OBIS 6 Laser Remote connected to the main laser combiner FV31-SCOMB to On, and 4) press the main switch of the AC adapter of the power supply for LD OBIS 6 Laser Remote connected to the sub laser combiner FV31-SCOMB to On.</li>
	<li>Press the main switches of the Si photodiode detector and lock-in amplifier On.</li>
	<li>Set the Stokes Beam at 1,031 nm, Pulse Width at 6 ps, and Repetition Rate at 80 MHz.</li>
	<li>Coupled into the microscope, set the picoEmerald system supplied with the synchronized pump beam with a Tunable Wavelength from 720-990 nm, a Pulse Width of 5-6 ps, and a Repetition Rate of 80 MHz. The average excitation power of both the pump and Stokes is 450 mW. Optimize the excitation power to minimize photodamage for the sample.</li>
	<li>Use a high-numerical aperture (NA) oil condenser (i.e., 1.4 NA) to collect the Stokes and pump beams where the sample is mounted by emitting a few drops on the condenser.</li>
	<li>Mount the sample on the oil and place a large water droplet on top of the microscope slide where the sample is fixed. This is for the water-immersion objective lens.</li>
	<li>Set the lock-in amplifier to 20 MHz. Process the image now using the super-resolution software module.</li>
	<li>Select 512 pixels x 512 pixels and 80 &#181;s/pixel for the dwell time.</li>
	<li>Once the desired cellular region is focused properly, acquire the image. Using the acquisition software of the microscope, save the images as an Olympus .oir graphic file.</li>
	<li>Acquire a background image at 1,900 cm-1 and subtract it from all cellular images.</li>
	<li>To perform 2PEF imaging, use the same tunable picosecond laser, and set the label-free autofluorescence of Flavin and NADH to 800 nm and 780 nm, respectively.</li>
	<li>Use a 460 nm/515 nm filter cube to collect the Flavin and NADH autofluorescence backscattered emission.</li>
	<li>Adjust the dwell time to 8 &#181;s/pixel and the pixel size to 512 pixels x 512 pixels. Set the laser shutter power parameter to 150 mW.</li>
	<li>For 3D image reconstruction, tune the stimulated Raman loss to 2,850 cm-1 (797 nm).</li>
	<li>Once the desired single cells have been identified, register the laser to scan from the top focus plane to detect the top and bottom layers of those cells. 
	&#8203;NOTE: 3D models are generated and saved as .oir files.</li>
</ol>
5. Spectra and image analysis
<ol>
	<li>Spectra analysis
	<ol>
		<li>Use the Origin software or other relevant applications to subtract the background spectrum from all subcellular target spectra.</li>
		<li>Use MATLAB&#39;s mathematical modeling operations to process the Raman spectra with the software&#39;s built-in functions. Python can also be used for spectra processing.
		<ol>
			<li>Spectral pre-processing begins with converting the files into an array. The spectra are interpolated at every reciprocal centimeter.</li>
			<li>For validation, graph the raw data. Perform the baseline correction on the raw spectra using the built-in function msbackadj in MATLAB. In brief, the built-in function estimates baseline points at separation-unit values identified by users and returns the distance between adjacent windows. From the baseline points, estimate and draw a regression line. Apply Smoothing to smooth the regression line to subtract the baseline from each individual raw spectrum.</li>
			<li>Perform min-max normalization by dividing the Raman intensity of the protein at 2,930 cm-1 by the intensity of each Raman shift. The 2,930 cm-1 signal is typically the highest intensity on the Raman spectrum. With this normalization, the maximum value gets transformed into a 1 while the minimum value gets transformed into a 0. Every other value gets transformed into a decimal between 0 and 1.</li>
			<li>Average the individual spectra of the same condition into a single spectrum to reduce noise.</li>
		</ol>
		</li>
		<li>Once processed, use applications, such as Origin, to display all the spectra simultaneously. The peaks displayed in the spectra represent a specific chemical bond or functional group.</li>
	</ol>
	</li>
	<li>3D image analysis of lipid droplets
	<ol>
		<li>Use FIJI-ImageJ software to process all 3D images</li>
		<li>Perform the functions Bandpass Filters and Smoothing for the 3D image stacks.</li>
		<li>For 3D image analysis, assign each lipid droplet to a spherical score calculated by measuring the distance between its center of mass to the surface. Compare each spherical score with a spherical score of a perfect sphere on the same Euclidean plan. Discard lipid droplets with low spherical scores and evaluate the remaining lipid droplets for their volume and counts.</li>
		<li>Analyze the volume and counts of lipid droplets between different treatments for statistical significance on an appropriate statistical software application. Here, GraphPad Prism was used.</li>
	</ol>
	</li>
	<li>2D hyperspectral image analysis
	<ol>
		<li>Use FIJI-ImageJ software to process all the 2D hyperspectral images.</li>
		<li>Select the 2,930 cm-1 channel to make a mask image that contains 0 and 1 values. Assign the background to 0 and the cellular region to 1.</li>
		<li>Subtract the deuterium-labeled protein channel (2,175 cm-1) to the background channel (1,900 cm-1) to remove the fluorescent effects.</li>
		<li>Divide the resulting deuterium-labeled protein channel by the protein channel (2,930 cm-1) to obtain protein turnover rate ratiometric images.</li>
		<li>Then, multiply the mask image made in step 5.3.2 by the ratiometric images to remove any remaining background noises. As a result, a dark background is generated in each ratiometric image.</li>
		<li>Use the Freehand Selection tool in FIJI-ImageJ to manually segment and calculate pixel intensities displayed on single cells. The pixel intensities correspond to the concentrations of the chemical bond that is being visualized.</li>
		<li>Analyze the pixel intensities for statistical significance on an appropriate statistical software application. Here, GraphPad Prism was used. Repeat this analysis with the remaining ratiometric analysis.</li>
	</ol>
	</li>
</ol>

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R., Dugail, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplet-membrane+contact+sites+-+from+protein+binding+to+function.">Lipid droplet-membrane contact sites - from protein binding to function.</a> Journal of Cell Science. 132 (12), (2019).</li><li>Schott, M. 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=Lipid+droplet+size+directs+lipolysis+and+lipophagy+catabolism+in+hepatocytes.">Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes.</a> The Journal of Cell Biology. 218 (10), 3320-3335 (2019).</li><li>Hoang, K., 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=Subcellular+resolution+DO-SRS+and+2PEF+imaging+of+metabolic+dynamics+regulated+by+L-methionine+in+amyotrophic+lateral+sclerosis.">Subcellular resolution DO-SRS and 2PEF imaging of metabolic dynamics regulated by L-methionine in amyotrophic lateral sclerosis.</a> Optical Biopsy XXI: Toward Real-Time Spectroscopic Imaging and Diagnosis. SPIE. 1237303, 6-13 (2023).</li><li>Jang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+stimulated+Raman+scattering+microscopy+with+A-PoD.">Super-resolution stimulated Raman scattering microscopy with A-PoD.</a> bioRxiv. , (2022).</li><li>Li, 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=Optical+metabolic+imaging+uncovers+sex-+and+diet-dependent+lipid+changes+in+aging+drosophila+brain.">Optical metabolic imaging uncovers sex- and diet-dependent lipid changes in aging drosophila brain.</a> bioRxiv. , (2022).</li><li>Zhang, W., 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=Multi-molecular+hyperspectral+PRM-SRS+imaging.">Multi-molecular hyperspectral PRM-SRS imaging.</a> bioRxiv. , (2022).</li><li>Wei, 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=Volumetric+chemical+imaging+by+clearing-enhanced+stimulated+Raman+scattering+microscopy.">Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.</a> Proceedings of the National Academy of Sciences. 116 (14), 6608-6617 (2019).</li><li>Chang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+monitoring+of+cell+metabolism+and+lipid+production+in+3D+engineered+human+adipose+tissues+using+label-free+multiphoton+microscopy.">Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy.</a> Biomaterials. 34 (34), 8607-8616 (2013).</li><li>Leica TCS SP8 CARS CARS Microscope - Label Free Imaging. Leica Microsystems Available from: <a target="_blank" href="https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-cars/downloads/">https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-cars/downloads/</a> (2023)</li></ol>

The authors have no competing financial interests or other conflicts of interest.

DO-SRS and 2PEF imaging have been applied to investigate metabolic dynamics in various ex vivo models, including Drosophila and human tissues21,22,23,24,26,27,33. The imaging modality used in this study integrates DO-SRS and 2PEF microscopy, which can outpace other molecule-specific...

Being essential aromatic amino acids (AAAs), phenylalanine (Phe) and tryptophan (Tryp) can be absorbed by the human body to synthesize new molecules for sustaining normal biological functions1. Phe is needed for the synthesis of proteins, melanin, and tyrosine, while Tryp is required for the synthesis of melatonin, serotonin, and niacin2,3. However, excess consumption of these AAAs can upregulate the mammalian target of the rapamycin (mTOR) pathway, inhibit AMP-activated protein kinase, and interfere with the mitochondrial metabolism, collectively altering macromolecule biosynthesis and leading to the production of malignant precursors, such as reactive oxygen species (ROS) in healthy cells4,5,6. Direct visualization of altered metabolic dynamics under excess AAA regulation is essential to understand AAAs&#39; roles in promoting cancer development and the growth of healthy cells7,8,9.
Traditional AAA studies rely on gas chromatography (GC)10. Other methods, such as magnetic resonance imaging (MRI), have limited spatial resolutions, making it hard to perform cellular and sub-cellular analysis of biological samples11. Recently, matrix-assisted laser desorption/ionization (MALDI) has been developed to elucidate the role of AAAs in lipid and protein syntheses in cancer proliferation with noninvasive biomarkers12,13,14. However, this technique still suffers from shallow imaging depths, poor spatial resolution, and extensive sample preparation. At the cellular level, nontoxic stable isotopes, such as nitrogen-15 and carbon-13, can be traced with multi-isotope imaging and nanoscale secondary ion mass spectrometry to understand their incorporation into macromolecules. However, these methods are destructive to living biological samples15,16. Atomic force microscopy (AFM) is another powerful technique that can visualize metabolic dynamics17. The slow rate of scanning during AFM imaging, on the other hand, may cause image distortion of the result from thermal drift.
We developed a noninvasive biorthogonal imaging modality by coupling deuterium-oxide (D2O) probed stimulated Raman scattering (DO-SRS) microscopy and label-free two-photon excitation fluorescence microscopy (2PEF). This modality achieves a high spatial resolution and chemical specificity when imaging biological samples18,19,20,21,22,23,24. This protocol introduces the applications of DO-SRS and 2PEF to examine the metabolic dynamics of lipids, protein, and redox ratio changes during cancer progressions. With D2O being a stable isotope form of water, cellular biomolecules can be labeled with deuterium (D) due to its quick compensation with total body water in cells, forming carbon-deuterium (C-D) bonds through enzymatic exchange21. The C-D bonds in newly synthesized macromolecules, including lipids, proteins, DNA/RNA, and carbohydrates, can be detected in the cell silent region of the Raman spectrum20,21,22,25,26,27. With two synchronized laser pulses, C-D bonds of newly synthesized lipids and proteins can be displayed on single cells via hyperspectral imaging (HSI) without extracting or labeling them with cytotoxic agents. In addition, SRS microscopy has the capability to construct three-dimensional (3D) models of selected regions of interest in biological samples by capturing and combining a set of cross-sectional images22,26. With hyperspectral and 3D volumetric imaging, DO-SRS can obtain spatial distributions of newly synthesized macromolecules in single cells, along with the type of organelles that facilitate the process of promoting cancer growth under AAA regulation22. Furthermore, using 2PEF, we can obtain autofluorescence signals of Flavin and nicotinamide adenine dinucleotide (NADH) with high resolution, deep penetration depth, and low-level damage in biological samples21,23,24. Flavin and NADH autofluorescence signals have been used to characterize redox homeostasis and lipid peroxidation in cancer cells22,26. As such, not only does the coupling of DO-SRS and 2PEF provide subcellular analysis of AAA-regulated metabolic dynamics in cancer cells with high spatial distribution, chemical specificity information, and minimal sample preparation, but the method also reduces the need to extract or label endogenous molecules with toxic reagents. In this protocol, we first present the procedures of D2O and amino acid preparation, as well as cancer cell culture. Then, we show the protocols of DO-SRS imaging and 2PEF imaging. Finally, we present the representative results of SRS and 2PEF imaging, which demonstrate AAA-regulated metabolic changes of lipids and protein, and redox ratio changes in cancer cells. A detailed illustration of the process is highlighted in Figure 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological Pipettes&#160;</td>
			<td>Avantor (by VWR)</td>
			<td>75816-100</td>
			<td>https://us.vwr.com/store/product?keyword=75816-100</td>
		</tr>
		<tr>
			<td>15 mL Conical Centrifuge Tube</td>
			<td>VWR</td>
			<td>89039-664</td>
			<td>https://mms.mckesson.com/product/1001859/VWR-International-89039-664</td>
		</tr>
		<tr>
			<td>16% Formaldehyde, Methanol-free</td>
			<td>ThermoFisher Scientific</td>
			<td>28906</td>
			<td>https://www.thermofisher.com/order/catalog/product/28906</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Fisherbrand</td>
			<td>FB0112929</td>
			<td>https://www.fishersci.com/shop/products/24-well-tc-multidish-100-cs/FB012929#?keyword=FB012929</td>
		</tr>
		<tr>
			<td>25 mm Syringe Filter, 2 &#956;m PES</td>
			<td>Foxx Life Sciences</td>
			<td>381-2216-OEM</td>
			<td>https://www.foxxlifesciences.com/collections/pes-syringe-filters/products/381-2216-oem?variant=16274336003</td>
		</tr>
		<tr>
			<td>460 nm Filter Cube</td>
			<td>Olympus</td>
			<td></td>
			<td>OCT-ET460/50M32</td>
		</tr>
		<tr>
			<td>AC Adapters of the Power Supply for LD OBIS 6 Laser Remote</td>
			<td>Olympus</td>
			<td></td>
			<td>Supply power to the laser</td>
		</tr>
		<tr>
			<td>Band-pass Filter</td>
			<td>KR Electronics</td>
			<td>KR2724</td>
			<td>8 MHz</td>
		</tr>
		<tr>
			<td>BNC 50 Ohm Terminator&#160;</td>
			<td>Mini Circuits</td>
			<td>STRM-50</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC Cable</td>
			<td>Thorlabs</td>
			<td>2249-C</td>
			<td>Coaxial Cable, BNC Male/Male</td>
		</tr>
		<tr>
			<td>Broadband Dielectric Mirror</td>
			<td>Thorlabs</td>
			<td>BB1-E03</td>
			<td>750 - 1100 nm</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Condenser</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Corning</td>
			<td>2850-25</td>
			<td>https://ecatalog.corning.com/life-sciences/b2b/NL/en/Glassware/Cover-Glass/Corning%C2%AE-Square-%231%C2%BD-Cover-Glass/p/2850-25</td>
		</tr>
		<tr>
			<td>DC power supply</td>
			<td>TopWard</td>
			<td>6302D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic Mount</td>
			<td>Thorlabs</td>
			<td>KM100CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide Cell Culture Reagent</td>
			<td>mpbio&#160;</td>
			<td>196055</td>
			<td>https://www.mpbio.com/0219605525-dimethyl-sulfoxide-cf</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#8217;s Medium without Methionine, Threonine, and Sodium Pyruvate</td>
			<td>MilliporeSigma</td>
			<td>38210000</td>
			<td>https://www.usbio.net/media/D9800-22/dulbeccorsquos-mem-dmem-wsodium-bicarbonate-wo-methionine-threonine-sodium-pyruvate-powder 
			With Sodium Bicarbonate and without Methionine, Threonine, and Sodium Pyruvate&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Corning</td>
			<td>MT10027CV</td>
			<td>https://www.fishersci.com/shop/products/dmem-dulbecco-s-modified-eagle-s-medium-4/MT10027CV#:~:text=Dulbecco&#39;s%20Modified%20Eagle&#39;s%20Medium%20</td>
		</tr>
		<tr>
			<td>FIJI ImageJ</td>
			<td>ImageJ</td>
			<td>Version 1.53t 24 August 2022</td>
			<td>https://imagej.net/software/fiji/downloads</td>
		</tr>
		<tr>
			<td>Heavy Water (Deuterium Oxide)</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>7732-18-5</td>
			<td>https://shop.isotope.com/productdetails.aspx?itemno=DLM-4-1L</td>
		</tr>
		<tr>
			<td>Hela Cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>https://www.atcc.org/products/ccl-2</td>
		</tr>
		<tr>
			<td>Hemocymeter</td>
			<td>MilliporeSigma</td>
			<td>Z359629-1EA</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/z359629?gclid=Cj0KCQiA37KbBhDgARIsAI 
			zce15A5FIy0WS7I6ec2KVk 
			QPXVMEqlAnYis_bKB6P6lr 
			SIZ-wAXOyAELIaAhhEEAL 
			w_wcB&#38;gclsrc=aw.ds</td>
		</tr>
		<tr>
			<td>High O.D. Bandpass Filter</td>
			<td>Chroma Technology</td>
			<td>ET890/220m</td>
			<td>Filter the Stokes beam and transmit the pump beam</td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum (FBS)</td>
			<td>Cytiva&#160;</td>
			<td>SH300880340</td>
			<td>https://www.fishersci.com/shop/products/hyclone-fetal-bovine-serum-u-s-standard-4/SH300880340</td>
		</tr>
		<tr>
			<td>HyClone Trypsin 0.25% (1x) Solution</td>
			<td>Cytiva</td>
			<td>SH30042.02</td>
			<td>https://www.cytivalifesciences.com/en/us/shop/cell-culture-and-fermentation/reagents-and-supplements/cell-disassociation-reagents/hyclone-trypsin-protease-p-00445</td>
		</tr>
		<tr>
			<td>Integrated SRS Laser System</td>
			<td>Applied Physics &#38; Electronics, Inc.</td>
			<td>picoEMERALD</td>
			<td>picoEMERALD provides an output pulse at 1031 nm with 6-ps pulse width and 80-MHz repetition rate, which serves as the Stokes beam.&#160; The frequency doubled beam at 532 nm is used to synchronously seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with five~6 ps pulse width (the idler beam of the OPO is blocked with an,interferometric filter). The output wavelength of the OPO is tunable from 720&#8211;950 nm, which serves as the pump beam. The intensity of the 1031 nm Stokes beam is modulated sinusoidally by a built-in EOM at 8 MHz with a modulation depth of more than 90%. The pump beam is spatially overlapped with the Stokes beam by using a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulses are achieved with a built-in delay stage and optimized by the SRS signal of pure D2O at the microscope.</td>
		</tr>
		<tr>
			<td>Inverted Laser-scanning Microscope</td>
			<td>Olympus</td>
			<td>FV1200MPE</td>
			<td></td>
		</tr>
		<tr>
			<td>IX3-CBH Control box</td>
			<td>Olympus</td>
			<td></td>
			<td>Control the laser-scanning microscope</td>
		</tr>
		<tr>
			<td>Kinematic Mirror Mount</td>
			<td>Thorlabs</td>
			<td>POLARIS-K1-2AH</td>
			<td>2 Low-Profile Hex Adjusters</td>
		</tr>
		<tr>
			<td>L-Phenalynine</td>
			<td>Sigma</td>
			<td>P5482-25G</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/p5482</td>
		</tr>
		<tr>
			<td>L-Tryptophan</td>
			<td>Sigma</td>
			<td>T8941-25G</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/t8941</td>
		</tr>
		<tr>
			<td>LabSpec 6</td>
			<td>Horiba XploRA</td>
			<td>N/A</td>
			<td>https://www.horiba.com/gbr/scientific/products/detail/action/show/Product/labspec-6-spectroscopy-suite-software-1843/</td>
		</tr>
		<tr>
			<td>Lock-In Amplifier</td>
			<td>Zurich Instruments</td>
			<td>N/A</td>
			<td>https://www.zhinst.com/americas/en/products/shfli-lock-in-amplifier</td>
		</tr>
		<tr>
			<td>Long-pass Dichroic Beam Splitter</td>
			<td>Semrock</td>
			<td>Di02-R980-25x36</td>
			<td>980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>Version: R2022b</td>
			<td>https://www.mathworks.com/products/new_products/latest_features.html</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-003</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-selectfrost-microscope-slides-9/12550003#?keyword=12-550-003</td>
		</tr>
		<tr>
			<td>Microscopy Imaging Software</td>
			<td>Olympus</td>
			<td>FluoView</td>
			<td></td>
		</tr>
		<tr>
			<td>MPLN 100x, Olympus</td>
			<td>Olympus</td>
			<td>MPLAPON</td>
			<td>https://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11364</td>
		</tr>
		<tr>
			<td>MPLN 50x, Olympus</td>
			<td>Olympus</td>
			<td>MPLAPON&#160;</td>
			<td>https://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11363</td>
		</tr>
		<tr>
			<td>NA Oil Condenser</td>
			<td>Olympus&#160;</td>
			<td>6-U130</td>
			<td>https://www.hitechinstruments.com/Product-Details/olympus-achromatic-aplanatic-high-na-condneser</td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Wet n Wild</td>
			<td>B01EO2G5O4</td>
			<td>https://www.amazon.com/dp/B01EO2G5O4/ref=cm_sw_r_api_i_E609VVDWW 
			HHQP38FXXDC_0</td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>OriginLab</td>
			<td>Origin 2022b (9.95)</td>
			<td>https://www.originlab.com/index.aspx?go=PRODUCTS/Origin</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>S37440</td>
			<td>https://www.fishersci.com/shop/products/parafilm-m-wrapping-film-3/p-2379782</td>
		</tr>
		<tr>
			<td>PBS 1x (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Thermofischer - Gibco</td>
			<td>14040117</td>
			<td>https://www.thermofisher.com/order/catalog/product/14040117?SID=srch-hj-14040117</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermofischer - Gibco</td>
			<td>15140122</td>
			<td>https://www.thermofisher.com/order/catalog/product/15140122</td>
		</tr>
		<tr>
			<td>Periscope Assembly</td>
			<td>Thorlabs</td>
			<td>RS99</td>
			<td>Includes the top and bottom units, &#216;1&#34; post, and clamping fork.</td>
		</tr>
		<tr>
			<td>picoEmerald System</td>
			<td>A.P.E</td>
			<td>N/A</td>
			<td>https://www.ape-berlin.de/en/cars-srs/</td>
		</tr>
		<tr>
			<td>Shielded Box with BNC Connectors</td>
			<td>Pomona Electronics</td>
			<td>2902</td>
			<td>Aluminum Box with Cover, BNC Female/Female</td>
		</tr>
		<tr>
			<td>Si Photodiode Detector</td>
			<td>Home Built</td>
			<td>N/A</td>
			<td>DYI series</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>Grace Bio-Labs</td>
			<td>654008</td>
			<td>https://gracebio.com/product/secureseal-imaging-spacers-654008/</td>
		</tr>
		<tr>
			<td>Spontaneous Raman spectroscopy</td>
			<td>Horiba XploRA</td>
			<td>N/A</td>
			<td>https://www.horiba.com/int/products/detail/action/show/Product/xploratm-plus-1528/</td>
		</tr>
		<tr>
			<td>Stimulated Raman Scattering Microscopy</td>
			<td>Home Built</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Touch&#160; Panel Controller</td>
			<td>Olympus</td>
			<td></td>
			<td>Control the X-Y direction of the laser-scanning microscope</td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4% (0.85% NaCl)&#160;</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>https://bioscience.lonza.com/lonza_bs/US/en/Culture-Media-and-Reagents/p/000000000000181876/Trypan-Blue%2C-0-4%25-Solution&#34;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Kaverme - Amazon</td>
			<td>B07RNVXXV1</td>
			<td>https://www.amazon.com/Precision-Anti-Static-Electronics-Laboratory-Jewelry-Making/dp/B07RNVXXV1&#34;</td>
		</tr>
		<tr>
			<td>Two Photon Excitation Fluorescence Microscopy</td>
			<td>Home Built</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing Paper&#160;</td>
			<td>VWR</td>
			<td>12578-165</td>
			<td>https://us.vwr.com/store/product/4597617/vwr-weighing-paper</td>
		</tr>
		<tr>
			<td>Zurich LabOneQ Software</td>
			<td>Zurich Instruments</td>
			<td></td>
			<td>Control the Zurich lock-in amplifier</td>
		</tr>
	</tbody>
</table>

bioorthogonal chemical imaging of cell metabolism regulated by aromatic amino acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

The addition of excess AAAs at 15x concentrations to the 50% D2O-containing cell culture media produced distinct C-D Raman bands of newly synthesized lipids and proteins in HeLa cells (Figure 2B). Previous experiments were performed with different concentration levels, such as 2x and 5x, and although the data is not presented, the 15x concentration produced the most distinct C-D Raman bands of newly synthesized lipids and proteins. Specifically, by investigating lipid droplets (LD...

Watch this Scientific Journal Video about Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids at JoVE.com

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

bioorthogonal-chemical-imaging-cell-metabolism-regulated-aromatic

Research

JoVE Journal

Bioengineering

1.0K Views.  University of California San Diego. We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF). 

Video: Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.