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 imaging methods by eliminating the need for molecule extraction or labeling with cytotoxic reagents and requiring minimal sample preparation. Specifically, DO-SRS microscopy enables us to probe de novo lipogenesis and protein synthesis in animals and cells and visualize their metabolic dynamics in situ23,27,34. 2PEF captures the autofluorescence signals of naturally occurring biomolecules, such as Flavin and NADH, in biological samples35,36. The imaging penetration depth of SRS can achieve up to 200-500 &#181;m in less scattering samples37. With tissue-clearing methods such as urea, the penetration depth can increase more than tenfold37. The coupling of SRS with 2PEF enables us to image endogenous fluorophores such as Flavin and NADH in biological samples, which further eliminates the need for exogenous biomarkers for detection. 2PEF can achieve a penetration depth of 500 &#181;m38. Compared to other multiplex imaging modalities, such as multi-color single-photon confocal fluorescence microscopy, both DO-SRS and 2PEF can achieve a significantly larger penetration depth without cytotoxic exogenous biomarkers.
To increase the spatial resolution of DO-SRS and 2PEF, we developed an adaptive moment estimation (Adam)-based pointillism deconvolution (A-PoD) image post-processing tool34. A-PoD can convert diffraction-limited DO-SRS and 2PEF images to super-resolved ones without the need for any hardware improvements. Additionally, the Raman spectrum contains many specific chemical vibration modes that can be harnessed to increase the number of detectable molecules. Taking this advantage, our lab has further generated a penalized reference matching (PRF) method to distinguish multiple lipid species in cell and tissue models36. These two technical developments open new avenues for studying biological processes in more detail at cellular and sub-cellular levels, which have been used to characterize metabolic changes in brain, cancer, and aging processes26,27,35.
One main limitation of this protocol is the concentration and time incubation of cellular samples with D2O to produce distinct, quantifiable C-D Raman bands. More robust, metabolically active cell lines, such as HeLa cells, can incorporate deuterium atoms into newly synthesized macromolecules at a faster rate than non-diseased mammalian cell lines, such as human embryonic kidney (HEK) cells, leading to various C-D intensities observed for the same concentration and time incubation with D2O in two different cell lines21. Furthermore, the administration of D2O at a concentration of 80% or above for 48 h may introduce toxicity to cellular samples. An optimization experiment to identify the optimal D2O concentration and time incubation is necessary to achieve desirable results.
A critical aspect of the protocol is the integration of DO-SRS and 2PEF imaging. Typically, DO-SRS and 2PEF modalities share the same picosecond excitation laser, as its narrow bandwidth can be optimized with SRS and 2PEF signals. Although our technology is homebuilt, this platform is commercially available39. Additional equipment, such as two synchronized laser pulses with a modulator, photodiode detector, a lock-in amplifier, and a scanning microscope&#39;s joint efforts are required to detect the Raman signal. D2O can be readily purchased from biotechnology vendors. Therefore, researchers can access this technology very easily.

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><tbody><tr><td>10 mL Serological Pipettes&amp;nbsp;</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 &amp;mu;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&amp;nbsp;</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&amp;nbsp;</td><td>196055</td><td>https://www.mpbio.com/0219605525-dimethyl-sulfoxide-cf</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&amp;rsquo;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&lt;br/&gt; With Sodium Bicarbonate and without Methionine, Threonine, and Sodium Pyruvate&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;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&#039;s%20Modified%20Eagle&#039;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&lt;br/&gt; zce15A5FIy0WS7I6ec2KVk&lt;br/&gt; QPXVMEqlAnYis_bKB6P6lr&lt;br/&gt; SIZ-wAXOyAELIaAhhEEAL&lt;br/&gt; w_wcB&amp;amp;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&amp;nbsp;</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 &amp;amp; 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.&amp;nbsp; 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&amp;ndash;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&amp;nbsp;</td><td>https://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11363</td></tr><tr><td>NA Oil Condenser</td><td>Olympus&amp;nbsp;</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&lt;br/&gt; 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&#039;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, &amp;Oslash;1&quot; 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&amp;nbsp; 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)&amp;nbsp;</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&quot;</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&quot;</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&amp;nbsp;</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 (LDs), we noticed that both 15x Phe and 15x Tryp induced newly synthesized lipids and protein signals at 2,143 cm-1 and 2,172 cm-1, respectively. DO-SRS was subsequently used to visualize the spatial distribution of C-D signals on single cells (Figure 3). Using ImageJ, pixel intensities of individual cells were manually segmented and calculated, as indicated by dotted-white borders in Figure 3A. The control cells display moderate C-D lipid and protein bands; however, the 15x Phe and 15x Tryp display stronger C-D lipid and protein bands. Quantitative analysis indicates that excess AAAs may upregulate lipid synthesis by 10%-17% but downregulate protein synthesis by 10% (Figure 3C,D). The results infer the possibility of a lack of autophagy that accumulates newly synthesized lipids, promotes mitochondrial dysfunction, and induces oxidative imbalance under excess AAA regulation7.
Label-free multimodal SRS and 2PEF imaging of unsaturated lipid (~3,011 cm-1), saturated lipid (~2,880 cm-1), and NADH, Flavin were acquired to understand the effects of AAAs on cancer metabolism. Similarly, pixel intensities of individual cells were manually segmented and calculated using ImageJ, as indicated by dotted-white borders in Figure 3B. Ratiometric analysis of AAA-treated cells displays a 10% increase in unsaturated lipid/saturated lipid and a 50% increase in Flavin/Flavin + NADH (Figure 3E,F). In the electron transport chain of mitochondria, ROS can be generated from the transfer of electrons from NADH and FADH2 to molecular oxygen species. The accumulation of ROS in many cancer cells results in an oxidative imbalance that oxidizes unsaturated fatty acids, promotes saturated fatty acid synthesis, and depletes NADH autofluorescence signals. Therefore, the observed increase in Flavin/Flavin + NADH is an indicator of accumulated ROS that reduces NADH autofluorescence signals. In response to oxidative imbalance, HeLa cells may upregulate their unsaturated lipid synthesis to replace the oxidized ones. This response is not observed in other cancer cell lines29, which signifies the metabolic heterogeneity of cancer cells under an excess AAA diet30.
In addition to multimodal imaging, SRS can reconstruct label-free 3D images of LDs in control and AAA-treated HeLa cells. In brief, microscopy generates a set of cross-sectional images throughout a selected region of interest. In this study, the stimulated Raman loss (SRL) was tuned to 2,845 cm-1 and scanned from the top layer to the bottom layer with a step size of 1 &#181;m (Figure 4A). Quantitative analyses of 3D lipid droplets reveal that LDs reduced in size but increased in counts in AAA-treated cells compared to the control (Figure 4B,C). The increased presence of bulky hydrophobic amino acids, such as Tryp and Phe may impair the function of LD-coating proteins. This ultimately reduces lipolysis, which accumulates numerous small LDs. The label-free 3D SRS volumetric imaging results of this study corroborate with previous studies by visualizing that excess AAA-treated cells exhibit numerous smaller LDs31,32.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65121/65121fig01v2.jpg" /> 
Figure 1: An illustration of the image acquisition and analysis with DO-SRS and 2PEF. (A) A 3D image reconstruction and analysis to acquire lipid droplet number, volume, and spherical score. (B) Hyperspectral imaging and analysis of deuterium-labeled lipid (CDL; 2,145 cm-1), lipid (2,845 cm-1), deuterium-labeled protein (CDP; 2,175 cm-1), protein (2,940 cm-1), unsaturated lipid (3,011cm-1), saturated lipid (2,880 cm-1), NADH, and Flavin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65121/65121fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65121/65121fig02v2.jpg" /> 
Figure 2: Physical setup of spontaneous Raman spectroscopy and stimulated Raman scattering microscopy. (A) Spontaneous Raman spectroscopy setup used for this study. (B) Sample spontaneous Raman spectra for CD label (red), without CD label (black), control group (blue, solid line), 15x Phe-treated group (red, dotted line), and 15x Tryp-treated group (pink, dotted line). (C) Schematic diagram of the stimulated Raman scattering microscopy setup used for this investigation. (D) Stimulated Raman scattering microscopy setup used as the DO-SRS and 2PEF platform. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65121/65121fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65121/65121fig03v2.jpg" /> 
Figure 3: Visualizing metabolic dynamics in HeLa cells using DO-SRS and 2PEF microscopy. (A) Deuterium-labeled lipid (2,145 cm-1), lipid (2,845 cm-1), deuterium-labeled protein (2,175 cm-1), protein (2,940 cm-1) visualized in HeLa cells under control (Ctrl), 15x phenylalanine (15x Phe), and 15x tryptophan (15x Tryp) with the DO-SRS platform. Lipid turnover rate and protein turnover rate were calculated as <img alt="Equation 1" src="/files/ftp_upload/65121/65121eq01.jpg" style="margin: auto;" /> and <img alt="Equation 2" src="/files/ftp_upload/65121/65121eq02.jpg" style="margin: auto;" />. Raw images were first subtracted by the PBS signal to remove background intensity and masked to remove intensity outside of the cells using ImageJ. Pixel-wise division was performed for the ratiometric analysis. (B) NADH and Flavin channels visualized with 2PEF microscopy, and unsaturated lipid (3,011 cm-1) and saturated lipid (2,880 cm-1) visualized with label-free SRS microscopy. Optical redox ratio and saturation ratio were calculated with <img alt="Equation 3" src="/files/ftp_upload/65121/65121eq03.jpg" style="margin: auto;" /> and <img alt="Equation 4" src="/files/ftp_upload/65121/65121eq04.jpg" style="margin: auto;" />. (C-F) Quantification of ratiometric intensities for each HeLa cell under control, 15x Phe, and 15x Tryp conditions. The statistical difference was used to compare excess AAA conditions with the control conditions. ****p &#60; 0.0001, ***p &#60; 0.001, **p &#60; 0.01, *p &#60; 0.05 were calculated from a two-way ANOVA test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65121/65121fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65121/65121fig04.jpg" /> 
Figure 4: Visualization of 3D lipid droplet distribution of a single HeLa cell using a label-free SRS microscope. (A) SRS 3D lipid droplet volume projection in HeLa cells under control and excess AAA conditions. The threshold was defined prior to the analysis, revealing lipid droplet signal distribution. (B,C) Quantification of lipid droplet volume and counts within individual HeLa cells under control group and excess AAA conditions using the two-way ANOVA test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65121/65121fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Cancer Research

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Biochemistry

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting endogenous fluorescence. Viscosity is measured using a fluorescent molecular rotor.

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Biology

Autofluorescence Imaging to Evaluate Cellular Metabolism

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.

This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced ...

Comprehensive Protocol for Cell Identification and Metabolic Analysis Using OPTIR-FISH

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

Author Spotlight: Integrated OPTIR-FISH for Single-Cell Metabolic and Identity Analysis in Complex Environments

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

Expansion Microscopy for Visualizing Intracellular Sialylated Glycans via MOE

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
</div>
<div class="chapter_text" id="script_2">
The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
</div>
<div class="chapter_text" id="script_3">
In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
</div>

Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

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Utilizing ¹⁸F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer