Numbers of Exchangeable Hydrogens from LC-MS Data of Heavy Water Metabolically Labeled Samples.

Labeling with deuterium oxide (D2O) has emerged as one of the preferred approaches for measuring the synthesis of individual proteins in vivo. In these experiments, the synthesis rates of proteins are determined by modeling mass shifts in peptides during the labeling period. This modeling depends on a theoretical maximum enrichment determined by the number of labeling sites (NEH) of each amino acid in the peptide sequence. Currently, NEH is determined from one set of published values. However, it has been demonstrated that NEH can differ between species and potentially tissues. The goal of this work was to determine the number of NEH for each amino acid within a given experiment to capture the conditions unique to that experiment. We used four methods to compute the NEH values. To test these approaches, we used two publicly available data sets. In a de novo approach, we compute NEH values and the label enrichment from the abundances of three mass isotopomers. The other three methods use the complete isotope profiles and body water enrichment in deuterium as an input parameter. They determine the NEH values by (1) minimizing the residual sum of squares, (2) from the mole percent excess of labeling, and (3) the time course profile of the depletion of the relative isotope abundance of monoisotope. In the test samples, the method using residual sum of squares performed the best. The methods are implemented in a tool for determining the NEH for each amino acid within a given experiment to use in the determination of protein synthesis rates using D2O.

Measurement of Isfahan heavy water zero-power reactor kinetic parameters using advanced pulsed neutron source method in a near critical state.

The pulsed neutron source (PNS) technique was used to determine the prompt neutron decay constant for two different lattice pitches in the HWZPR heavy water zero power reactor. The results were compared to the variance-to-mean ratio (VTM) method. The neutron mean generation time was also calculated for both pitches, and the results were compared to previous Monte Carlo calculations. The findings of this research can be used as a benchmark nuclear codes to validate kinetic parameters.

Heavy water coupling gel for short-wave infrared photoacoustic imaging.

Significance: Changes in lipid, water, and collagen (LWC) content in tissue are associated with numerous medical abnormalities (cancer, atherosclerosis, and Alzheimer's disease). Standard imaging modalities are limited in resolution, specificity, and/or penetration for quantifying these changes. Short-wave infrared (SWIR) photoacoustic imaging (PAI) has the potential to overcome these challenges by exploiting the unique optical absorption properties of LWC>1000 nm. Aim: This study's aim is to harness SWIR PAI for mapping LWC changes in tissue. The focus lies in devising a reflection-mode PAI technique that surmounts current limitations related to SWIR light delivery. Approach: To enhance light delivery for reflection-mode SWIR PAI, we designed a deuterium oxide (D2O, "heavy water") gelatin (HWG) interface for opto-acoustic coupling, intended to significantly improve light transmission above 1200 nm. Results: HWG permits light delivery >1 mJ up to 1850 nm, which was not possible with water-based coupling (>1 mJ light delivery up to 1350 nm). PAI using the HWG interface and the Visualsonics Vevo LAZR-X reveals a signal increase up to 24 dB at 1720 nm in lipid-rich regions. Conclusions: By overcoming barriers related to light penetration, the HWG coupling interface enables accurate quantification/monitoring of biomarkers like LWC using reflection-mode PAI. This technological stride offers potential for tracking changes in chronic diseases (in vivo) and evaluating their responses to therapeutic interventions.

Rapid Detection and Analysis of Raman Spectra of Bacteria in Multiple Fields of View Based on Image Stitching Technique.

BACKGROUND: Due to antibiotic abuse, the problem of bacterial resistance is becoming increasingly serious, and rapid detection of bacterial resistance has become an urgent issue. Because under the action of antibiotics, different active bacteria have different metabolism of heavy water, antibiotic resistance of bacteria can be identified according to the existence of a C-D peak in the 2030-2400 cm-1 range in the Raman spectrum. METHODS: To ensure data veracity, a large number of bacteria need to be detected, however, due to the limitation of the field of view of the high magnification objective, the number of single cells in a single field of view is very small. By combining an image stitching algorithm, image recognition algorithm, and processing of Raman spectrum and peak-seeking algorithm, can identify and locate single cells in multiple fields of view at one time and can discriminate whether they are Antimicrobial-resistant bacteria. RESULTS: In experiments 1 and 2, 2706 bacteria in 9 × 11 fields of view and 2048 bacteria in 11 × 11 fields of view were detected. Results showed that in experiment 1, there are 1137 antibiotic-resistant bacteria, accounting for 42%, and 1569 sensitive bacteria, accounting for 58%. In experiment 2, there are 1087 antibiotic-resistant bacteria, accounting for 53%, and 961 sensitive bacteria, accounting for 47%. It showed excellent performance in terms of speed and recognition accuracy as compared to traditional manual detection approaches. And solves the problems of low accuracy of data, a large number of manual experiments, and low efficiency due to the small number of single cells in the high magnification field of view and different peak-seeking parameters of different Raman spectra. CONCLUSIONS: The detection and analysis method of bacterial Raman spectra based on image stitching can be used for unattended, automatic, rapid and accurate detection of single cells at high magnification with multiple fields of view. With the characteristics of automatic, high-throughput, rapid, and accurate identification, it can be used as an unattended, universal and non-invasive means to measure antibiotic-resistant bacteria to screen for effective antibiotics, which is of great importance for studying the persistence and spread of antibiotics in bacterial pathogens.

People with obesity exhibit losses in muscle proteostasis that are partly improved by exercise training.

This pilot experiment examines if a loss in muscle proteostasis occurs in people with obesity and whether endurance exercise positively influences either the abundance profile or turnover rate of proteins in this population. Men with (n = 3) or without (n = 4) obesity were recruited and underwent a 14-d measurement protocol of daily deuterium oxide (D2 O) consumption and serial biopsies of vastus lateralis muscle. Men with obesity then completed 10-weeks of high-intensity interval training (HIIT), encompassing 3 sessions per week of cycle ergometer exercise with 1 min intervals at 100% maximum aerobic power interspersed by 1 min recovery periods. The number of intervals per session progressed from 4 to 8, and during weeks 8-10 the 14-d measurement protocol was repeated. Proteomic analysis detected 352 differences (p < 0.05, false discovery rate < 5%) in protein abundance and 19 (p < 0.05) differences in protein turnover, including components of the ubiquitin-proteasome system. HIIT altered the abundance of 53 proteins and increased the turnover rate of 22 proteins (p < 0.05) and tended to benefit proteostasis by increasing muscle protein turnover rates. Obesity and insulin resistance are associated with compromised muscle proteostasis, which may be partially restored by endurance exercise.

Flexible Quality Control for Protein Turnover Rates Using d2ome.

Bioinformatics tools are used to estimate in vivo protein turnover rates from the LC-MS data of heavy water labeled samples in high throughput. The quantification includes peak detection and integration in the LC-MS domain of complex input data of the mammalian proteome, which requires the integration of results from different experiments. The existing software tools for the estimation of turnover rate use predefined, built-in, stringent filtering criteria to select well-fitted peptides and determine turnover rates for proteins. The flexible control of filtering and quality measures will help to reduce the effects of fluctuations and interferences to the signals from target peptides while retaining an adequate number of peptides. This work describes an approach for flexible error control and filtering measures implemented in the computational tool d2ome for automating protein turnover rates. The error control measures (based on spectral properties and signal features) reduced the standard deviation and tightened the confidence intervals of the estimated turnover rates.

Simple Self-Powered Sensor for the Detection of D2O and Other Isotopologues of Liquid Water.

Distinguishing between heavy water and regular water has been a continuing challenge since these isotopologues of water have very similar physical and chemical properties. We report the development and evaluation of a simple, inexpensive sensor capable of detecting liquid D2O and other isotopologues of liquid water through the measurement of electrical signals generated from a nanoporous alumina film. This electrical output, consisting of a sharp voltage pulse followed by a separate broad voltage pulse, is present during the application of microliter volumes of liquid. The amplitude and temporal characteristics of these pulses have been combined to enable four diagnostic parameters for sensing D2O and H218O. The sensing mechanism is based on different modification effects on the alumina surface by H2O and D2O, spatially localized variations in the surface potential of alumina induced by isotopically substituted water molecules, combined with the effect of isotopic composition on charge transfer. As a proof-of-concept demonstration, a sensing system has been developed that provides real-time detection of liquid D2O in a stand-alone system.

Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

Imaging ocular water inflow in the mouse with deuterium oxide MRI.

Abnormal intraocular fluid flow or clearance is involved with a variety of eye diseases such as glaucoma and diabetic retinopathy, but measurement of water exchange dynamics in the vitreous and aqueous remain challenging. 2H MRI can be used to image deuterium oxide (D2O) as a tracer, but the signal-to-noise ratio for deuterium is low due to its low concentration, which has hampered its application to imaging the eye. To overcome this challenge, we investigated the feasibility of direct D2O MRI to measure water dynamics in the mouse eye. The balanced steady-state free precession (bSSFP) sequence provided substantially higher signal-to-noise ratio for imaging D2O in fluid compared to standard gradient echo and spin echo sequences. bSSFP allowed dynamic imaging of intraocular water inflow in the mouse with 41 s temporal resolution. The inflow rate in the vitreous was found to be faster than in the aqueous. These studies demonstrate the feasibility of in vivo imaging of water inflow dynamics into the both the vitreous and aqueous in mice, which could be useful in studies of abnormal fluid exchange in rodent models of eye disease.

Replacing Foods with a High-Glycemic Index and High in Saturated Fat by Alternatives with a Low Glycemic Index and Low Saturated Fat Reduces Hepatic Fat, Even in Isocaloric and Macronutrient Matched Conditions.

BACKGROUND: Current guidelines aim to limit the dietary glycemic index (GI) and intake of saturated fatty acids (SFA). Several studies have shown favorable effects of low-GI or low-SFA diets on intrahepatic lipid content (IHL), but these studies were performed under overfeeding conditions or extreme differences in GI or SFA to maximize the contrast between diets. By combining changes in GI and SFA, we can mimic how people can improve their diet in a realistic setting. OBJECTIVES: We investigated the effect on liver fat content and substrate metabolism of both reducing GI and replacing SFA with polyunsaturated fat in practically realistic amounts under isocaloric conditions. DESIGN AND METHODS: In a randomized crossover study, thirteen overweight participants consumed two diets, one high in GI and SFA (high GI/SFA) and one low in GI and SFA (low GI/SFA) with identical macronutrient composition, for two weeks each. Diets were equal in caloric content, consisted of habitual food items, and had a macronutrient composition that can be easily achieved in daily life. At the end of each intervention, IHL content/composition and liver glycogen were measured by magnetic resonance spectroscopy. Additionally, fasted and postprandial hepatic de novo lipogenesis and glycemic and metabolic responses were investigated. RESULTS: IHL was significantly lower (-28%) after the two-week low-GI/SFA diet (2.4 ± 0.5% 95% CI [1.4, 3.4]) than after the two-week high-GI/SFA diet (3.3 ± 0.6% 95% CI [1.9, 4.7], p < 0.05). Although hepatic glycogen content, hepatic de novo lipogenesis, hepatic lipid composition, and substrate oxidation during the night were similar between the two diets, the glycemic response to the low-GI/SFA diet was reduced (p < 0.05). CONCLUSIONS: Changes in macronutrient quality can already have drastic effects on liver fat content and postprandial glycemia after two weeks and even when energy content and the percentage of total fat and carbohydrate remains unchanged.

Measurement of protein in vivo turnover rate with metabolic labeling using LC-MS.

Understanding the protein dynamics of a drug target is important for pharmaceutical research because it provides insight into drug design, target engagement, pharmacodynamics and drug efficacy. Nonradioactive isotope labeling has been the method of choice for protein turnover measurement thanks to the advancement of high-resolution mass spectrometry. While the changes in proteome in cell cultures can be monitored precisely, as the culture media can be completely replaced with 2 H-, 15 N- or 13 C-labeled essential amino acids, quantifying rates of protein synthesis in vivo is more challenging. The amount of isotope tracer that can be administered into the body is relatively small compared with the existing protein, thus requiring more sensitive detection, and the precursor-product labeling relationship is more complicated to interpret. The purpose of this review is to provide an overview of the principles of in vivo protein turnover studies using deuterium water (2 H2 O) with an emphasis on targeted protein analysis by hybrid LC-MS assay platforms. The pursuit of these opportunities will facilitate drug discovery and research in preclinical and clinical stages.

Retention Time Alignment for Protein Turnover Studies Using Heavy Water Metabolic Labeling.

Retention time (RT) alignment has been important for robust protein identification and quantification in proteomics. In data-dependent acquisition mode, whereby the precursor ions are semistochastically chosen for fragmentation in MS/MS, the alignment is used in an approach termed matched between runs (MBR). MBR transfers peptides, which were fragmented and identified in one experiment, to a replicate experiment where they were not identified. Before the MBR transfer, the RTs of experiments are aligned to reduce the chance of erroneous transfers. Despite its widespread use in other areas of quantitative proteomics, RT alignment has not been applied in data analyses for protein turnover using an atom-based stable isotope-labeling agent such as metabolic labeling with deuterium oxide, D2O. Deuterium incorporation changes isotope profiles of intact peptides in full scans and their fragment ions in tandem mass spectra. It reduces the peptide identification rates in current database search engines. Therefore, the MBR becomes more important. Here, we report on an approach to incorporate RT alignment with peptide quantification in studies of proteome turnover using heavy water metabolic labeling and LC-MS. The RT alignment uses correlation-optimized time warping. The alignment, followed by the MBR, improves labeling time point coverage, especially for long labeling durations.

Deuterium brain imaging at 7T during D2 O dosing.

PURPOSE: To characterize the (2 H) deuterium MR signal measured from human brain at 7T in participants loading with D2 O to ˜1.5% enrichment over a six-week period. METHODS: 2 H spectroscopy and imaging measurements were used to track the time-course of 2 H enrichment within the brain during the initial eight-hour loading period in two participants. Multi-echo gradient echo (MEGE) images were acquired at a range of TR values from four participants during the steady-state loading period and used for mapping 2 H T1 and T2 * relaxation times. Co-registration to higher resolution 1 H images allowed T1 and T2 * relaxation times of deuterium in HDO in cerebrospinal fluid (CSF), gray matter (GM), and white matter (WM) to be estimated. RESULTS: 2 H concentrations measured during the eight-hour loading were consistent with values estimated from cumulative D2 O dose and body mass. Signal changes measured from three different regions of the brain during loading showed similar time-courses. After summing over echoes, gradient echo brain images acquired in 7.5 minutes with a voxel volume of 0.36 ml showed an SNR of ˜16 in subjects loaded to 1.5%. T1 -values for deuterium in HDO were significantly shorter than corresponding values for 1 H in H2 O, while T2 * values were similar. 2 H relaxation times in CSF were significantly longer than in GM or WM. CONCLUSION: Deuterium MR Measurements at 7T were used to track the increase in concentration of 2 H in brain during heavy water loading. 2 H T1 and T2 * relaxation times from water in GM, WM, and CSF are reported.

Software Tool for Visualization and Validation of Protein Turnover Rates Using Heavy Water Metabolic Labeling and LC-MS.

Metabolic stable isotope labeling followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein turnover studies of individual proteins on a large scale and with high throughput. Turnover rates of thousands of proteins from dozens of time course experiments are determined by data processing tools, which are essential components of the workflows for automated extraction of turnover rates. The development of sophisticated algorithms for estimating protein turnover has been emphasized. However, the visualization and annotation of the time series data are no less important. The visualization tools help to validate the quality of the model fits, their goodness-of-fit characteristics, mass spectral features of peptides, and consistency of peptide identifications, among others. Here, we describe a graphical user interface (GUI) to visualize the results from the protein turnover analysis tool, d2ome, which determines protein turnover rates from metabolic D2O labeling followed by LC-MS. We emphasize the specific features of the time series data and their visualization in the GUI. The time series data visualized by the GUI can be saved in JPEG format for storage and further dissemination.

Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Research Progress in the Medical Application of Heavy Water, Especially in the Field of D2O-Raman Spectroscopy.

Heavy water is an ideal contrast agent for metabolic activity and can be adapted to a wide range of biological systems owing to its non-invasiveness, universal applicability, and cost-effectiveness. As a new type of probe, the heavy isotope of water has been widely used in the study of cell development, metabolism, tissue homeostasis, aging, and tumor heterogeneity. Herein, we review findings supporting the applications of and research on heavy water in monitoring of bacterial metabolism, rapid detection of drug sensitivity, identification of tumor cells, precision medicine, and evaluation of skin barrier function and promote the use of heavy water as a suitable marker for the development of detection and treatment methodologies.

Free-energy differences of OPC-water and SPC/HW-heavy-water models using the Bennett acceptance ratio.

Surface tension, vapor density of OPC-water and SPC/HW-heavy-water models have been estimated at low temperatures using the scaled model. The free-energy difference, - Δ F , of n-molecules and (n-1)-molecules plus a free probe has been calculated using the Bennett acceptance ratio with the aid of Monte-Carlo simulations. Our results show that the relation between the free-energy difference divided by k B T and the number of molecules to the power minus one-third is linear for n > 6 . Consequently, the surface tension can be extracted from the straight line slope, whereas the vapor density can be extracted from the intercept, which is proportional to the logarithmic ratio of liquid density to that of vapor density. By scaling the free-energy differences, for at least three different temperatures, to T C T - 1 , we estimated the critical temperature and hence the surface tension and the vapor density at a wide range of temperatures. The free-energy differences have been calculated at 240K, 260K, and 280K for OPC-water, and at 260K, 280K, and 300K for the SPC/HW-heavy water model.

The Hydration State of Bone Tissue Affects Contrast in Neutron Tomographic Images.

Neutron tomography has emerged as a promising imaging technique for specific applications in bone research. Neutrons have a strong interaction with hydrogen, which is abundant in biological tissues, and they can penetrate through dense materials such as metallic implants. However, in addition to long imaging times, two factors have led to challenges in running in situ mechanical characterization experiments on bone tissue using neutron tomography: 1) the high water content in specimens reduces the visibility of internal trabecular structures; 2) the mechanical properties of bone are dependent on the hydration state of the tissue, with drying being reported to cause increased stiffness and brittleness. This study investigates the possibility of improving image quality in terms of neutron transmission and contrast between material phases by drying and rehydrating in heavy water. Rat tibiae and trabecular bovine bone plugs were imaged with neutron tomography at different hydration states and mechanical testing of the bone plugs was carried out to assess effects of drying and rehydration on the mechanical properties of bone. From analysis of image histograms, it was found that drying reduced the contrast between bone and soft tissue, but the contrast was restored with rehydration. Contrast-to-noise ratios and line profiles revealed that the contrast between bone tissue and background was reduced with increasing rehydration duration but remained sufficient for identifying internal structures as long as no free liquid was present inside the specimen. The mechanical analysis indicated that the proposed fluid exchange protocol had no adverse effects on the mechanical properties.

Degradation of ribosomal and chaperone proteins is attenuated during the differentiation of replicatively aged C2C12 myoblasts.

BACKGROUND: Cell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and 'proteostasis collapse'. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age-related declines in muscle homeostasis. METHODS: The Absolute Dynamic Profiling Technique for Proteomics (Proteo-ADPT) was used to investigate proteostasis in young (passage 6-10) and replicatively aged (passage 48-50) C2C12 myoblast cultures supplemented with deuterium oxide (D2 O) during early (0-24 h) or late (72-96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. RESULTS: Young cells exhibited a consistent ~25% rise in protein accretion over the 96-h experimental period. In aged cells, protein accretion increased by 32% (P < 0.05) during early differentiation, but then fell back to baseline levels by 96-h. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P < 0.05, FDR < 5% interaction between age × differentiation stage) different changes in abundance between young and aged cells at early and later periods of differentiation, including proteins associated with translation, glycolysis, cell-cell adhesion, ribosomal biogenesis, and the regulation of cell shape. During early differentiation, heat shock and ribosomal protein abundances increased in aged cells due to suppressed degradation rather than heightened synthesis. For instance, HS90A increased at a rate of 10.62 ± 1.60 ng/well/h in aged which was significantly greater than the rate of accretion (1.86 ± 0.49 ng/well/h) in young cells. HS90A synthesis was similar in young (21.23 ± 3.40 ng/well/h) and aged (23.69 ± 1.13 ng/well/h), but HS90A degradation was significantly (P = 0.05) greater in young (19.37 ± 2.93 ng/well/h) versus aged (13.06 ± 0.76 ng/well/h) cells. During later differentiation the HS90A degradation (8.94 ± 0.38 ng/well/h) and synthesis (7.89 ± 1.28 ng/well/h) declined and were significantly less than the positive net balance between synthesis and degradation (synthesis = 28.14 ± 3.70 ng/well/h vs. degradation = 21.49 ± 3.13 ng/well/h) in young cells. CONCLUSIONS: Our results suggest a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts. The quality of key chaperone proteins, including HS90A, HS90B and HSP7C was reduced in aged cells and may account for the disruption to cell signalling required for the later stages of differentiation and fusion.

Deuterium Raman imaging for lipid analysis.

Raman microscopy has been used to deduce information about the distributions of endogenous biomolecules without exogenous labeling. Several functional groups, such as alkynes (CC), nitriles (CN), and carbon-deuterium (C-D) bonds, have been employed in recent years as Raman tags to detect target molecules in cells. In this article, we review some recent advances in applications using deuterated fatty acids for lipid analysis, such as investigation of tumor-selective cytotoxicity of γ-linolenic acid (GLA), simultaneous two-color imaging of stearate and oleate using deuterated and protonated alkynes, Raman hyperspectral imaging, and analyses of the physical properties of lipids through spectral unmixing of the C-D vibrational frequencies. In addition, we review some advanced methods for observing intracellular metabolic activities, such as de novo lipogenesis from deuterium-labeled precursors.