Levels Of Isotopic Enrichment Research Articles

A standardized quantitative analysis strategy for stable isotope probing metagenomics.

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, "who is eating what?" and "who is active?" within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism's taxonomic identity and genome composition while providing quantitative estimates of the microorganism's isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

Hydrogeochemical and isotopic evaluation of VOC commingled plumes in a weathered fractured bedrock aquifer treated with thermal and bioremediation

Chlorinated ethanes and ethenes isotopic analyses in groundwater and hydrogeochemical results from a former industrial area in Sao Paulo (Brazil) were used to confirm the existence and allow further characterization of source areas and their commingled plumes, both before and after thermal and bioremediation treatments. Prior to full scale remediation, a recently identified off-site source area with unknown history and limited access for further intrusive works presented lower δ13C values (−6.5‰ to −1.8‰ for 1,2-DCA) than the downgradient on-site source area (+8.6‰ to +20.0‰). Intermediate δ13C values for 1,2-DCA were identified further downgradient from the sources, within commingled plumes patterns. The isotope and concentration results show the typical degradation patterns associated with biotic reductive dechlorination for chlorinated ethenes and dihaloelimination for 1,2-DCA. Results following remediation treatments show further levels of isotopic enrichment, for chlorinated ethenes and chlorinated ethanes in the tropically weathered and deeper fractured bedrock (gneisses) groundwater. Hydrogeochemical results, isotopic mass balance and Carbon-Chlorine isotope slopes data are coherent with remediation treatment and a complex commingled plume setting. The results of this study confirmed the Temporal Conceptual Model proposed by Hart et al. (2021) and identified the need for further studies to evaluate isotopic dynamics under thermal remediation, including thermal-induced hydrolysis processes.

Investigation of phonon properties and thermal behavior of UO2 crystal using density-functional theory

Understanding thermal behavior and processes underlying the heat transport of UO2 nuclear fuel in nuclear reactor plays a key role in predicting the efficiency of the fuel. If the heat transport, which is an important parameter in temperature distribution of the fuel, does not occur properly, the continuous increase of temperature would lead to the melting of the fuel and therefore, environmental hazards. In this work, by using a non-spin-polarized calculation for the simple description of the paramagnetic state and ignoring the Hubbard correction, the thermal properties and phonon properties of bulk UO2 are calculated. These calculations are based on the density-functional theory (DFT) and density-functional perturbation theory (DFPT). To determine the lattice-vibration properties by the finite-displacement method, we have calculated the second-order and third-order force constants based on which such quantities as constant-volume specific heat, Gruneisen parameter, three-phonon scattering rate, scattering rate due to different levels of isotopic enrichment, and cumulative thermal conductivity are calculated. The results of the calculated specific heat based on the harmonic approximation show a good agreement with the experimental values, specifically for temperatures lower than 400 Kelvin. The results obtained for three-phonon scattering rate reveal that the scattering rate increases with temperature, thereby leading to the decrease of thermal conductivity. The results related to different levels of isotopic enrichments do not show any sensible changes in the scattering rates.

Extreme alteration in an acid-sulphate geothermal field: Sulphur Springs, Saint Lucia

Sulphur Springs is a vigorous geothermal field associated with the Soufrière Volcanic Centre in southern Saint Lucia. Intensely altered rocks occur over an area of at least 200 × 400 m, together with bubbling hot pools and fumaroles. The pools are sodium‑calcium-sulphate type, with pHs of 3–7 and temperatures of 41–97 °C. Fumaroles have temperatures up to, and at times above 100 °C. Gases collected from both fumaroles and bubbling pools have high contents of CO2 (601–993 mmol/mol) and commonly high H2S (3–190 mmol/mol).Least-altered host rocks surrounding Sulphur Springs comprise calc-alkaline, feldspar-quartz-porphyritic dacites of near-uniform chemical composition. These rocks, which form massive domes and volcaniclastic units, were emplaced 13–30 ka ago (Lindsay et al., 2013). Within the geothermal field, the dacites have been moderately to highly altered to acid-sulphate assemblages of kaolinite, illite, smectite, alunite, natroalunite, cristobalite, opaline silica, native sulphur, jarosite and amorphous compounds. Muddy sediments from grey to blackish thermal pools additionally contain iron sulphides.Despite intense alteration of the volcanic rocks (and derived volcaniclastic sediments), Zr and Ti have remained essentially immobile, which allows identification of the precursors (dacites) and calculation of mass changes. Many of the altered rocks and sediments show major depletions of Fe, Mg, Ca, Na. Extremely altered samples additionally show notable losses of Al, P, K, REE and Y, implying leaching by highly acidic waters. A few of the highly altered rocks, however, have gained Al, together with P, presumably due to neutralization or evaporation of acidic thermal waters. Also present within the geothermal field are metre-scale zones with very high contents of silica and native sulphur, and <1% Al2O3. Based on mass change results, the silica in the silica-sulphur zones, although recrystallized, was mainly inherited from the precursor dacites, rather than added as new hydrothermal precipitates.Surface thermal waters define a linear trend in a plot of δ18O vs. δD, and reach high levels of isotopic enrichment. The enrichment trend is ascribed to progressive evaporation in the thermal pools. The low slope of the isotopic trend (1.4) is interpreted as being due to surficial mixing between meteoric waters and steam condensates derived at about 230 °C from the deep brine that underlies the geothermal field. The whole-rock O-H-isotopic composition of four highly altered samples (kaolinite-smectite-opal-sulphates) can be accounted for if the alteration fluid was a 50:50 mixture of steam and meteoric water, and alteration occurred at 50–125 °C. Whole-rock δ34S values of samples containing variable amounts of sulphates, sulphides and native S are all notably negative (−5 to −16‰), consistent with derivation of sulphur from volcanic gases such as SO2 and H2S. Altered rocks and thermal pool sediments in the geothermal field are commonly enriched in Hg and Se (up to 50 mg/kg).At Sulphur Springs, primary dacitic rocks have been chemically and mineralogically transformed by reaction with acidic waters that formed when uprising volcanic gases condensed into neutral meteoric waters. In many cases, this has led to substantial mass losses or gains in Al, P and REEs. Regardless of the degree of alteration, however, the precursor rocks can still be identified using Ti-Zr relations.

SIPSim: A Modeling Toolkit to Predict Accuracy and Aid Design of DNA-SIP Experiments

DNA Stable isotope probing (DNA-SIP) is a powerful method that links identity to function within microbial communities. The combination of DNA-SIP with multiplexed high throughput DNA sequencing enables simultaneous mapping of in situ assimilation dynamics for thousands of microbial taxonomic units. Hence, high throughput sequencing enabled SIP has enormous potential to reveal patterns of carbon and nitrogen exchange within microbial food webs. There are several different methods for analyzing DNA-SIP data and despite the power of SIP experiments, it remains difficult to comprehensively evaluate method accuracy across a wide range of experimental parameters. We have developed a toolset (SIPSim) that simulates DNA-SIP data, and we use this toolset to systematically evaluate different methods for analyzing DNA-SIP data. Specifically, we employ SIPSim to evaluate the effects that key experimental parameters (e.g., level of isotopic enrichment, number of labeled taxa, relative abundance of labeled taxa, community richness, community evenness, and beta-diversity) have on the specificity, sensitivity, and balanced accuracy (defined as the product of specificity and sensitivity) of DNA-SIP analyses. Furthermore, SIPSim can predict analytical accuracy and power as a function of experimental design and community characteristics, and thus should be of great use in the design and interpretation of DNA-SIP experiments.

Electron Spin Coherence of Shallow Donors in Natural and Isotopically Enriched Germanium.

Germanium is a widely used material for electronic and optoelectronic devices and recently it has become an important material for spintronics and quantum computing applications. Donor spins in silicon have been shown to support very long coherence times (T_{2}) when the host material is isotopically enriched to remove any magnetic nuclei. Germanium also has nonmagnetic isotopes so it is expected to support long T_{2}'s while offering some new properties. Compared to Si, Ge has a strong spin-orbit coupling, large electron wave function, high mobility, and highly anisotropic conduction band valleys which will all give rise to new physics. In this Letter, the first pulsed electron spin resonance measurements of T_{2} and the spin-lattice relaxation (T_{1}) times for ^{75}As and ^{31}P donors in natural and isotopically enriched germanium are presented. We compare samples with various levels of isotopic enrichment and find that spectral diffusion due to ^{73}Ge nuclear spins limits the coherence in samples with significant amounts of ^{73}Ge. For the most highly enriched samples, we find that T_{1} limits T_{2} to T_{2}=2T_{1}. We report an anisotropy in T_{1} and the ensemble linewidths for magnetic fields oriented along different crystal axes but do not resolve any angular dependence to the spectral-diffusion-limited T_{2} in samples with ^{73}Ge.

Evidence that 2-hydroxyglutarate is not readily metabolized in colorectal carcinoma cells.

BackgroundTwo-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action.ResultsBy applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly 13C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in 13C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from 13C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period.ConclusionsTaken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products.

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Microbiological Synthesis of 2Н- and 13С-Labeled Amino Acids and Proteins With Various Levels of Isotopic Enrichment

Microbiological Synthesis of 2Н- and 13С-Labeled Amino Acids and Proteins With Various Levels of Isotopic Enrichment

Russian

With using of a strain of of L-phenylalanine secreted Gram-positive aerobic facultative methylotrophic bacteria Brevibacterium methylicum В-5652, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of carbon assimilation it was carried out the preparative microbiological synthesis of phenylalanine and metabolically related amino acids (alanine, valine, leucine/isoleucine in the amount of 5–6 mmol/l), labelled with deuterium (2H). The data on adaptation of L-phenylalanine secreted methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in minimal growth medium M9 with 98 % 2Н2O and 2 % [2Н]methanol, and data on biosynthesis of deuterium labelled L-phenylalanine with different levels of enrichment are submitted. The developed method for biosynthesis allows to obtain [2Н]phenylalanine with different levels of isotopic enrichment, depending on 2Н2O concentration in growth media M9, from 17 % (2 deuterium atoms) (on growth medium with 24,5 % 2Н2О) right up to 75 % (6 deuterium atoms) (on growth medium with 98 % 2Н2О) with introduction of deuterium to benzyl С6Н5СН2-fragment of molecule that is confirmed with the data of the electron impact (EI) mass-spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5- 25 European Journal of Molecular Biotechnology, 2014, Vol.(3), № 1 sulfochloride (dansyl) derivatives of [2Н]phenylalanine, isolated from the liquid culture after its separation by RP HPLC

Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon

The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.

Regioselective and Stereospecific Deuteration of Bioactive Aza Compounds by the Use of Ruthenium Nanoparticles

An efficient H/D exchange method allowing the deuteration of pyridines, quinolines, indoles, and alkyl amines with D2 in the presence of Ru@PVP nanoparticles is described. By a general and simple procedure involving mild reaction conditions and simple filtration to recover the labeled product, the isotopic labeling of 22 compounds proceeded in good yield with high chemo- and regioselectivity. The viability of this procedure was demonstrated by the labeling of eight biologically active compounds. Remarkably, enantiomeric purity was conserved in the labeled compounds, even though labeling took place in the vicinity of the stereogenic center. The level of isotopic enrichment observed is suitable for metabolomic studies in most cases. This approach is also perfectly adapted to tritium labeling because it uses a gas as an isotopic source. Besides these applications to molecules of biological interest, this study reveals a rich and underestimated chemistry on the surface of ruthenium nanoparticles.

Microbial synthesis of 2H-labelled L-phenylalanine with different levels of isotopic enrichment by a facultative methylotrophic bacterium Brevibacterium methylicum with RuMP assimilation of carbon

Microbial synthesis of 2H-labelled L-phenylalanine with different levels of isotopic enrichment by a facultative methylotrophic bacterium Brevibacterium methylicum with RuMP assimilation of carbon

Synthesis of isotopomers of N-(tert-butoxycarbonyl)-4-cyano-l-phenylalanine methyl ester: choice of cyanation solvent

N-(tert-butoxycarbonyl)-4-cyano-l-phenylalanine methyl ester and three isotopomers (C15N, 13CN, and 13C15N) were successfully synthesized in two steps to expand the utility of the nitrile symmetric stretch vibration of this modified amino acid as a vibrational reporter of local environments. The choice of cyanation solvent directly impacted the level of isotopic enrichment of the isotopomers. The commonly used solvent acetonitrile resulted in an isotopic enrichment of only ∼80% with a cyanation reaction time of 4.5h, however, the cyanation solvent N,N-dimethylformamide afforded the isotopomers with >98% isotopic enrichment.

Habitat contrasts reveal a shift in the trophic position of ant assemblages

1. Trophic structure within a guild can be influenced by factors such as resource availability and competition. While ants occupy a wide range of positions in food webs, and ant community composition changes with habitat, it is not well understood if ant genera tend to maintain their position in the trophic structure, or if trophic position varies across habitats. 2. We used ratios of stable isotopes of carbon and nitrogen to test for differences in the trophic structure and position of assemblages of ants among habitat types. We tested for differences between assemblages in replicate sites of the land use categories: (i) pastures with old large trees; (ii) recently revegetated pastures with small young trees; and (iii) remnant woodlands. Known insect herbivores and predatory spiders provided baselines for herbivorous and predaceous arthropods. Soil samples were used to correct for the base level of isotopic enrichment at each site. 3. We found no significant interactions between land use and ant genus for isotope enrichment, indicating that trophic structure is conserved across land use categories. The fixed relative positions of genera in the trophic structure might be re-enforced by competition or some other factor. However, the entire ant assemblage had significantly lower δ(15) N values in revegetated sites, suggesting that ants feed lower down in the food chain i.e. they are more 'herbivorous' in revegetated sites. This may be a result of the high availability of plant sugars, honeydew and herbivorous arthropod prey. 4. Surprisingly, ants in remnants and pastures with trees displayed similar isotopic compositions. Interactions within ant assemblages are thus likely to be resilient to changes in land use, but ant diets in early successional habitats may reflect the simplicity of communities, which may have comparatively lower rates of saprophagy and predation.

A Simple and Economical Method for the Production of 13C,18O-Labeled Fmoc-Amino Acids with High Levels of Enrichment: Applications to Isotope-Edited IR Studies of Proteins

Isotope-edited IR of proteins has generated considerable interest. Double labeling with 13C and 18O with high levels of isotopic enrichment is required for residue-specific resolution. Current methods for the preparation of doubly labeled amino acids give modest 18O enrichment, limiting the utility of the approach. We report a simple and economical method for preparing 13C,18O-doubly labeled N-(9-fluorenylmethoxycarbonyl)amino acids with high levels of enrichment for residues that do not require acid-labile side-chain protecting groups.

Assessing the fate and transformation of plant residues in the terrestrial environment using HR-MAS NMR spectroscopy

Plant litter decomposition plays a fundamental role in carbon and nitrogen cycles, provides key nutrients to the soil environment and represents a potentially large positive feedback to atmospheric CO 2. However, the full details of decomposition pathways and products are unknown. Here we present the first application of HR-MAS NMR spectroscopy on 13C and 15N labeled plant materials, and apply this approach in a preliminary study to monitor the environmental degradation of the pine and wheatgrass residues over time. In HR-MAS, is it possible to acquire very high resolution NMR data of plant biomass, and apply the vast array of multidimensional experiments available in conventional solution-state NMR. High levels of isotopic enrichment combined with HR-MAS significantly enhance the detection limits, and provide a wealth of information that is unattainable by any other method. Diffusion edited HR-MAS NMR data reveal the rapid loss of carbohydrate structures, while two-dimensional (2-D) HR-MAS NMR spectra demonstrate the relatively fast loss of both hydrolysable and condensed tannin structures from all plant tissues studied. Aromatic (partially lignin) and aliphatic components (waxes, cuticles) tend to persist, along with a small fraction of carbohydrate, and become highly functionalized over time. While one-dimensional (1-D) 13C HR-MAS NMR spectra of fresh plant tissue reflect compositional differences between pine and grass, these differences become negligible after decomposition suggesting that recalcitrant carbon may be similar despite the plant source. Two-dimensional 1H– 15N HR-MAS NMR analysis of the pine residue suggests that nitrogen from specific peptides is either selectively preserved or used for the synthesis of what appears to be novel structures. The amount of relevant data generated from plant components in situ using HR-MAS NMR is highly encouraging, and demonstrates that complete assignment will yield unprecedented structural knowledge of plant cell components, and provide a powerful tool with which to assess carbon sequestration and transformation in the environment.

Noninvasive In vivo Detection of Glutathione Metabolism in Tumors

Magnetic resonance spectroscopic imaging has been used to follow glutathione metabolism and evaluate glutathione heterogeneity in intact tumor tissue. Stable isotope-labeled glutathione was detected in s.c. implanted fibrosarcoma tumors in anesthetized rats following infusion of [2-13C]glycine. Using 1H-decoupled 13C magnetic resonance spectroscopy, the appearance of [2-13C]glycine at 42.4 ppm and the subsequent incorporation of this isotope label into the glycyl residue of glutathione at 44.2 ppm can be detected. The identity and relative concentrations of labeled metabolites observed in the in vivo spectrum were confirmed in studies of tissue extracts. The high level of isotopic enrichment and the concentration of glutathione in tumor tissue allow for collection of spatially localized spectra using 13C chemical shift imaging methods. These data provide the first direct images of glutathione in intact tumor tissue and show metabolic heterogeneity. This method may lead to the ability to monitor changes in tumor tissue redox state that may ultimately affect diagnosis, monitoring, and treatment.

High-performance liquid chromatography/inductively coupled plasma mass spectrometry and tandem mass spectrometry for the detection of carbon-containing compounds.

High-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICPMS) has been studied as a means for the detection of carbon to provide a 'universal' method for detecting organic compounds in chromatographic eluents. Carbon is particularly difficult to ionise and the amount of carbon present in normal chromatographic systems leads to high backgrounds, making detection a challenge. Novel separation approaches were therefore employed, using either entirely aqueous eluents (at temperatures of 60 and 160 degrees C, dependent on the column used) to eliminate the organic modifier completely, or isotopically enriched solvents. For the aqueous eluents, detection limits for sulphanilamide were found to be 2.26 microg, corresponding to 1.13 micromol (0.47 micromol of carbon), injected on a conventional 4.6 mm i.d. column. The use of a narrow bore column with highly isotopically enriched 12C-methanol (99.95 atom%) as organic modifier for the mobile phase enabled the detection of 86 micromol for 13C-triple-labelled caffeine and 79 micromol for 13C-double-labelled phenacetin. The sensitive detection of 12C-compounds with 13C-enriched methanol as organic modifier proved impractical due to a lower level of isotopic enrichment (99 atom%) of this solvent, with the residual 12C-methanol resulting in significant interference.

Isotopically Selective Infrared Multiphoton Dissociation of Vibrationally Excited SiH4

We use mass-resolved 2+2 resonantly enhanced multiphoton ionization (REMPI) of silicon atoms to detect isotopically selective infrared multiphoton dissociation (IRMPD) of both ground-state and vibrationally pre-excited SiH4. To demonstrate that the detection scheme provides a faithful monitor of the isotopic composition of the primary dissociation products, this approach was first tested on the products generated by both IRMPD and UV photodissociation of phenylsilane. It was then employed to evaluate the degree of isotopic enrichment achieved by IRMPD of ground-state silane using an ammonia laser for dissociation. In a second type of experiment, silane molecules are pre-excited to the first overtone of the Si−H stretch, and then dissociated selectively with the ammonia laser. We find that pre-excitation increases the dissociation cross section by a factor of 230. Tuning the overtone pre-excitation laser while collecting the mass-resolved silicon ion signal generates an overtone excitation spectrum of SiH4 that is sorted by the silicon isotope. Certain combinations of overtone pre-excitation and ammonia laser dissociation frequencies lead to a high level of isotopic enrichment in the dissociation products: >99% in 28Si or 29Si, and >96% in 30Si. We evaluate the practicality of an overtone-pre-excitation/IRMPD scheme for silicon isotope separation on a macroscopic scale.