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 s...

<ol>
	<li>Wu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+amino+acids+in+nutrition+and+health.">Functional amino acids in nutrition and health.</a> Amino Acids. 45 (3), 407-411 (2013).</li><li>Wei, Z., Liu, X., Cheng, C., Yu, W., Yi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+amino+acids+in+cancer.">Metabolism of amino acids in cancer.</a> Frontiers in Cell and Developmental Biology. 8, 603837 (2020).</li><li>Parthasarathy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-ring+circus:+Metabolism+of+the+three+proteogenic+aromatic+amino+acids+and+their+role+in+the+health+of+plants+and+animals.">A three-ring circus: Metabolism of the three proteogenic aromatic amino acids and their role in the health of plants and animals.</a> Frontiers in Molecular Biosciences. 5, 29 (2018).</li><li>Wang, 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=l-tryptophan+activates+mammalian+target+of+rapamycin+and+enhances+expression+of+tight+junction+proteins+in+intestinal+porcine+epithelial+cells.">l-tryptophan activates mammalian target of rapamycin and enhances expression of tight junction proteins in intestinal porcine epithelial cells.</a> The Journal of Nutrition. 145 (6), 1156-1162 (2015).</li><li>Saxton, R. 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Cancer. 18 (12), 744-757 (2018).</li><li>Kimura, T., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tryptophan+protects+hepatocytes+against+reactive+oxygen+species-dependent+cell+death+via+multiple+pathways+including+Nrf2-dependent+gene+induction.">Tryptophan protects hepatocytes against reactive oxygen species-dependent cell death via multiple pathways including Nrf2-dependent gene induction.</a> Amino Acids. 48 (5), 1263-1274 (2016).</li><li>Ma, Q., 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=Dietary+supplementation+with+aromatic+amino+acids+decreased+triglycerides+and+alleviated+hepatic+steatosis+by+stimulating+bile+acid+synthesis+in+mice.">Dietary supplementation with aromatic amino acids decreased triglycerides and alleviated hepatic steatosis by stimulating bile acid synthesis in mice.</a> Food and Function. 12 (1), 267-277 (2021).</li><li>Cheng, C., 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=Treatment+implications+of+natural+compounds+targeting+lipid+metabolism+in+nonalcoholic+fatty+liver+disease,+obesity+and+cancer.">Treatment implications of natural compounds targeting lipid metabolism in nonalcoholic fatty liver disease, obesity and cancer.</a> International Journal of Biological Sciences. 15 (8), 1654-1663 (2019).</li><li>Lubes, G., Goodarzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GC-MS+based+metabolomics+used+for+the+identification+of+cancer+volatile+organic+compounds+as+biomarkers.">GC-MS based metabolomics used for the identification of cancer volatile organic compounds as biomarkers.</a> Journal of Pharmaceutical and Biomedical Analysis. 147, 313-322 (2018).</li><li>Di Gialleonardo, V., Wilson, D. M., Keshari, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+metabolic+imaging.">The potential of metabolic imaging.</a> Seminars in Nuclear Medicine. 46 (1), 28-39 (2016).</li><li>Bowman, A. P., 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=Evaluation+of+lipid+coverage+and+high+spatial+resolution+MALDI-imaging+capabilities+of+oversampling+combined+with+laser+post-ionisation.">Evaluation of lipid coverage and high spatial resolution MALDI-imaging capabilities of oversampling combined with laser post-ionisation.</a> Analytical and Bioanalytical Chemistry. 412 (10), 2277-2289 (2020).</li><li>Murphy, R. C., Hankin, J. A., Barkley, R. 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Series A, Mathematical, Physical and Engineering Sciences. 374 (2079), 20150378 (2016).</li><li>Wang, T., Shogomori, H., Hara, M., Yamada, T., Kobayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+recognition+of+sphingomyelin-rich+membrane+domains+by+atomic+force+microscopy.">Nanomechanical recognition of sphingomyelin-rich membrane domains by atomic force microscopy.</a> Biochemistry. 51 (1), 74-82 (2012).</li><li>Fung, A. A., Shi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+and+tissue+imaging+using+Raman+and+coherent+Raman+microscopy.">Mammalian cell and tissue imaging using Raman and coherent Raman microscopy.</a> Wiley Interdisciplinary Reviews. Systems Biology and Medicine. 12 (6), e1501 (2020).</li><li>Shi, L., Fung, A. 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A., Hussain, S., Shi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+cancer+cell+metabolic+dynamics+regulated+with+aromatic+amino+acids+using+DO-SRS+and+2PEF+microscopy.">Visualizing cancer cell metabolic dynamics regulated with aromatic amino acids using DO-SRS and 2PEF microscopy.</a> Frontiers in Molecular Biosciences. 8, 779702 (2021).</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=Direct+imaging+of+lipid+metabolic+changes+in+drosophila+ovary+during+aging+using+DO-SRS+microscopy.">Direct imaging of lipid metabolic changes in drosophila ovary during aging using DO-SRS microscopy.</a> Frontiers in Aging. 2, 819903 (2022).</li><li>Li, Y., Zhang, W., Fung, A. A., Shi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DO-SRS+imaging+of+metabolic+dynamics+in+aging+Drosophila.">DO-SRS imaging of metabolic dynamics in aging Drosophila.</a> Analyst. 146 (24), 7510-7519 (2021).</li><li>Zhang, L., 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=Spectral+tracing+of+deuterium+for+imaging+glucose+metabolism.">Spectral tracing of deuterium for imaging glucose metabolism.</a> Nature Biomedical Engineering. 3 (5), 402-413 (2019).</li><li>Fung, A. 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(182), e63547 (2022).</li><li>Rysman, E., 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=De+novo+lipogenesis+protects+cancer+cells+from+free+radicals+and+chemotherapeutics+by+promoting+membrane+lipid+saturation.">De novo lipogenesis protects cancer cells from free radicals and chemotherapeutics by promoting membrane lipid saturation.</a> Cancer Research. 70 (20), 8117-8126 (2010).</li><li>Lisec, J., Jaeger, C., Rashid, R., Munir, R., Zaidi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lipid+class+homeostasis+is+altered+under+nutrient-deprivation+but+stable+under+hypoxia.">Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia.</a> BMC Cancer. 19 (1), 501 (2019).</li><li>Thiam, A. 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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>
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		<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>
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		<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>
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		<tr>
			<td>460 nm Filter Cube</td>
			<td>Olympus</td>
			<td></td>
			<td>OCT-ET460/50M32</td>
		</tr>
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			<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>
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			<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>
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			<td>BNC Cable</td>
			<td>Thorlabs</td>
			<td>2249-C</td>
			<td>Coaxial Cable, BNC Male/Male</td>
		</tr>
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			<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>
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			<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>
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			<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, ...

We showcase a new imaging technique with label-free capability, high chemical specificity, and subcellular resolution. Our methodology can answer a questions about metabolic dysfunction in aging and diseases such as cancer Raman imaging, coupled with two photon fluorescence and second harmonic generation, offers a non-destructive and label-free imaging technology that requires minimal sample preparation and achieves high subcellular resolution without damaging the cell sample. Several imaging modalities are operated as a part of our platform.Individuals should follow our protocol to the best of their abilities, noting that our system is home-built. Along the way they should develop their own practice to ensure success. In two separate tubes begin by measuring and mixing 10 milligrams of DMEM powder with 4.7 milliliters of double distilled water in a 15 mL conical tube.Thoroughly vortex and invert the tube to ensure the solution is well mixed. For both tubes, add 4.7 mL of deuterium oxide, 0.5 mL of FBS, and 0.1 mL of penicillin and streptomycin before vortexing and inverting the tube. Next, add excess phenylalanine to one tube and excess tryptophan powder to the second tube and mix again.Add methionine and threonine at 30 and 95 mg per mL, respectively, to both tubes, followed by vortexing and inverting the tube. Afterward, filter the prepared media with a 25 millimeter syringe filter with 0.22 micrometer filters. Seal all the media contained conical tubes with Parafilm.Store prepared media at four degrees Celsius. Maintain healer cells in a culture flask using standard DMEM supplemented with 10%FBS, 1%penicillin and streptomycin. Subculture the healer cells at a split ratio of one to 10 until reaching 80%or higher cell viability.Before seeding the cells, submerged the sterilized Poly-D-Lysine, laminin-coated, round 12 mm diameter cover slips in 70%Ethanol. Dry the cover slips using lint free wipes, and placed the clean cover slips in the appropriate wells of the plate. Wash the cells once with PBS without magnesium and calcium ions.Add 0.25%trypsin to dissociate the adherence cells from the flask and incubate at 37 degrees Celsius and 5%carbon dioxide for three minutes. Then add DMEM supplemented with 10%FBS, 1%penicillin, and streptomycin to the cells. Gently pipette up and down.Collect the cells by centrifugation at 300 G for three minutes at room temperature. Next, re-suspend the cells in DMEM supplemented with 0.5%FBS and 1%penicillin and streptomycin. Count the trypan blue stain cells using a hemocytometer under a light microscope and seed at a density of two times 10 to the fifth cells per well of a 24 well plate.Distribute the cells evenly by shaking the plate gently. Incubate at 37 degrees Celsius and 5%carbon dioxide. After eight hours, replace the old culture media with 50%deuterium oxide and excess phenylalanine or 50%deuterium oxide and excess tryptophan media.Return the cells to the incubator at 37 degrees Celsius and 5%carbon dioxide for 36 hours. Now prepare microscope slides with imaging spaces nine mm in diameter. Place 15 microliters of PBS, supplemented with calcium and magnesium ions and rinse the cells with the same solution.Add 0.5 mL of 4%methanol-free paraformaldehyde solution and leave the plate under the biosafety hood for 15 minutes. Aspirate the paraformaldehyde solution and rinse it with PBS, supplemented with calcium and magnesium iron twice. Add one mL of PBS, supplemented with calcium and magnesium ions to each well.Utilize sterile tweezers to remove the cover slips from each well carefully. Invert them and place the cell containing side onto the imaging spaces in contact with the PBS. Seal the outer layer of the cover slip with the imaging spacer using transparent nail polish.Load the biological sample onto the sample holder directly underneath the objective lens. Then click the camera icon on the software to turn on the video camera and adjust the focus plane to identify a region of interest with the 50X objective lens. Switch to the 100X objective lens.Click the stop icon to turn off the video camera. Then select a grating of 1, 800 lines per mm and set the the acquisition range from 400 to 3, 150 centimeters inverse. Lastly, set the acquisition time to 90 seconds the accumulation to three and the binning to four for the least noise and most accurate spectrum.Click on start to warm up the laser. Start by pressing the main switch of the control box IX3CBH to On, followed by the main switch of the touch panel controller. Then turn on the main switch of the AC adapter of the power supply for LDOBIS6 laser remote connected to the main laser combiner FV31S comb.Also switch on the AC adapter of the LDOBIS6 laser remote power supply connected to the sub laser combiner FV31S comb. After that, press the main switches of the SI photodiode detector and lock-in amplifier on. Set the laser IR at 1031 nanometers Set the pico emerald system to at tunable wavelength between 720 to 990 nm.Mount the sample on the oil and place a large water droplet on the microscope slide where the sample is fixed. Select scan size as 512 by 512 pixels. Acquire and save the images as an Olympus.OIR graphic file. After setting the signal to 862.0, acquire a background image and save it. Adjust the dwell time to eight microseconds per pixel and the pixel size to 512 by 512 pixels.Set the laser shutter power parameter to 150 milliwatts For 3D image reconstruction, tune the stimulated Raman loss to 2, 850 cm inverse. 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. Deuterium oxide, probed, stimulated Raman scattering was used to visualize the spatial distribution of CD signals on single cells.The control cells displayed moderate CD, lipid and protein bands. However, the 15 x v and 15 x trip displayed stronger CD lipid and protein bans. Quantitative analysis indicated that excess aromatic amino acids may upregulate lipid synthesis by 10 to 17%but downregulate protein synthesis by 10%Ratio metric analysis of aromatic amino acids treated cells showed a 50%increase in flavin divided by flavin plus NADH and a 10%increase in unsaturated lipid divided by saturated lipid.The stimulated Raman 3D lipid droplet volume projection in healer cells under control and excess aromatic amino acids conditions are shown. Quantitative analyses of 3D lipid droplets showed an increase in counts, but a reduction in size in aromatic amino acids treated cells compared to the control. Imaging parameters for Raman and two photon fluorescent imaging should be optimized from one sample to the next to ensure best imaging quality.Individuals can couple our system with second harmonic generation to study the collagen structure, which elucidates early metabolic changes in aging and diseases, and understanding the progression of neurodegeneration or cancer. Our technology is the first of this kind to visualize lipid and protein synthesis in biological systems with high subcellular resolution. The technology can also be translated into clinic for non-invasive disease detection.

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

Research

JoVE Journal

Bioengineering

951 visualizações.  University of California San Diego. Apresentamos uma nova técnica de imagem com capacidade livre de marcação, alta especificidade química e resolução subcelular. Nossa metodologia pode responder a uma pergunta sobre disfunção metabólica no envelhecimento e doenças como câncer A imagem Raman, juntamente com fluorescência de dois fótons e segunda geração harmônica, oferece uma tecnologia de imagem não destrutiva e livre de rótulos que requer preparação mínima da amostra e alcança alta resolução subcelular ...