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Single Particle Analysis is an electron microscopy method used to determine structures of macromolecules.

What is Single Particle Analysis & How Does it Work?

Single Particle Analysis (SPA) is an electron microscopy method used to determine structures of proteins and other macromolecular complexes. Structures of macromolecules can be determined from cryo-EM images at near-atomic and sometimes true atomic resolution, and are used to investigate biological processes. It is an invaluable tool in multiple scientific fields, and although easy to explain, it is a complex process. This blog post will explain Single Particle Analysis and how it works.

What is Single Particle Analysis?

Single Particle Analysis (SPA) is a technique in structural biology used to determine the three-dimensional structure of proteins and other macromolecular complexes through the use of transmission electron microscopes (TEM). The technique involves the acquisition of high resolution images containing purified and monodisperse macromolecules and computationally extracting the individual particles from the images to reconstruct a final three-dimensional structure. The aim of SPA is to provide deep insights into structure and function of macromolecules, to help understand biological processes at the molecular level, and to help lead drug discovery efforts by providing details of molecular interactions. Therefore, SPA is performed under cryogenic conditions (cryo-EM), where macromolecular complexes are flash-frozen in liquid ethane so as to preserve their native state and kept at low temperature in the electron microscope during data collection. Post-acquisition computer processing involves complex alignments and classification of 2D particle images, which after merging can produce high-resolution 3D density maps , and provide atomic details of macromolecules. Alternatively, SPA can be performed under room temperature conditions on samples treated with negative stain producing lower resolution 3D structures, but which can still provide valuable information such as the shape and size of a macromolecule.

How Does Single Particle Analysis Work?

The working principle of SPA involves several physical and computational techniques that vary depending on the sample and desired outcome of the research. It is typically a complex process due to several factors such as the purity, stability, and heterogeneity of the macromolecular complex under study. The main advantage of cryo-EM and SPA is that it does not require large quantities of a sample. Yet, obtaining a 3D structure is time-consuming and involves processing large quantities of data. Therefore, the four main stages of SPA should be managed efficiently to ensure a successful experiment.

Sample Preparation:
In order to obtain high-resolution structures through cryo-EM, samples must first undergo two fundamental processes: purification and vitrification. Samples are first isolated from biological sources and then further purified to provide highly pure and homogeneous samples in solution. Then the purified samples are frozen in a thin layer of vitreous ice, ready to be imaged in the cryo-electron microscope.
Imaging:
Before data collection, samples are first screened in a cryo-electron microscope to assess qualities such as protein concentration, stability, particle monodispersity and ice thickness. Then, once a suitable sample is identified, thousands of high-resolution images are collected with the help of automatic data collection software and used for further analysis.
Image Processing:
Advanced image-processing software is used to process the images obtained through cryo-EM. The two-dimensional (2D) images are typically first automatically assessed for qualities such as resolution, motion, and ice thickness. Then, 2D images of individual particles are extracted and sorted into 2D classes based on similarity. Finally, these 2D classes are combined to reconstruct a final 3D density map.
Analyzing and Interpreting Data:
Once a 3D density map has been produced, researchers fit the map with the appropriate amino acid chains and build a protein model guided by the map. Higher resolution 3D maps allow researchers to build models with high accuracy and confidence. The completed protein structures and corresponding density maps are then subjected to an analysis to elucidate the function of the complex under study. Researchers can then deposit their data in databases available to the public such as the Protein Data Bank (PDB) or the EMDB (Electron Microscopy Data Bank).

Applications of Single Particle Analysis

Single Particle Analysis is an invaluable tool in many scientific fields. Through the use of Transmission Electron Microscopes, researchers can better understand macromolecules such as membrane proteins, protein complexes, protein-DNA complexes, and nucleic acid structures. The sectors in which Single Particle Analysis is most commonly used include basic, biomedical, and pharmaceutical research.

JEOL and Single Particle Analysis

JEOL is a world leader in supplying electron microscopes, mass spectrometers, NMR spectrometers, and other high-end scientific equipment. Our mission is to offer advanced scientific and metrology products and services to support the development of a sustainable society.

JEOL offers two CRYO ARM™ models - 200kV and 300kV - that achieve unprecedented resolution and stability thus allowing for the automatic and unattended acquisition of image data for Single Particle Analysis. To learn more about what JEOL offers, please contact a member of the team today.

References

1. Dang, SY., and X., YX. (2022) Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM. Frontiers in Molecular Biosciences. Available from: https://www.frontiersin.org/articles/10.3389/fmolb.2022.892459
2. Sundaram, J. (20218) Single Particle Analysis Techniques. News Medical https://www.news-medical.net/life-sciences/Single-Particle-Analysis-Techniques.aspx
3. Weissenberger, G., Henderikx, R. J. M., and Peters, P. J. (2021). Understanding the Invisible Hands of Sample Preparation for Cryo-EM. Nat. Methods 18, 463–471. doi:10.1038/s41592-021-01130-6. Available from: https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=Understanding+the+Invisible+Hands+of+Sample+Preparation+for+Cryo-EM&btnG=

Student presentations at past M&M conferences

STEM Students from Massachusetts Heading to M&M 2023

A group of hard-working middle and high school students in northeast Massachusetts have conducted research during this school year that has earned them a spot at the prestigious Microscopy & Microanalysis (M&M) annual conference. Four of the young authors of two papers are headed to the major conference to be held in Minneapolis this July. There they will present their work in an interdisciplinary (cross-cutting) symposium, focusing on “expanding engagement to build a bigger, better future for the M&M community.”

This is the fourth year that local teachers’ guidance has brought them into the world of professional research and to M&M.

Encouraged, inspired, and lead by science and engineering teachers Saman Abbas and Doug Shattuck, the students who attend St. Joseph’s School and Malden Catholic High School are engaged in the outreach program of Professor Marcus Buhler, head of the Massachusetts Institute of Technology (MIT) Laboratory for Atomistic and Molecular Mechanics. Students also have access to the impressive lab resources at Malden Catholic. Additionally, they tap into the microscopy resources and expertise at JEOL USA in Peabody, Mass. JEOL’s Vern Robertson provides high magnification SEM images and X-ray microanalysis as well as lively talks during STEM fairs and conference presentations.

This year, the two research projects the students decided to pursue are 1) exploring lunar resources for human habitation on the moon, and 2) analyzing the natural radiation resistance of a microorganism called Tardigrades that could be applied to sunscreen.

Potential lunar resources for construction contain basalt, which happens to be used in building materials on earth. They are light but strong. “Basalt fibers are spun through a die and look like cotton candy, or human hair” says Mr. Shattuck. As for its stability “You can do magic with it. Kits are sold to patch concrete, and it is much stronger than steel rebar which is typically used in Portland cement.” Multiple tests on the fibers included the use of electron microscope data and EDS analyses provided by Mr. Robertson.

As for the sunscreen research, students looked to Tardigrades, invertebrate microscopic animals that are difficult to see with the human eye. They are found in mosses and lichens, soils and sand covered in water. Tardigrades have a special ability to resist radiation. Shattuck said that fact led students to the question, “How could the Damage Suppressor Protein (DSUP) that naturally shields Tardigrades become an active ingredient in a protective sunscreen for humans?” In their research, students applied the protein to clear plastic beads and exposed them to UV light. The application shows promise for preventing exposure to radiation from sunlight.

With research ranging from Martian and lunar regolith for extraterrestrial building materials, to spider webs for tensile strength (a research project a few years ago), and Tardigrade proteins for sunscreen, Shattuck and Robertson have certainly enjoyed learning along with the STEM students. “This is a real-world opportunity for students to conduct research,” said Robertson. JEOL is pleased to be part of this work and we hope to share some of the students’ work in our booth #706 at M&M 2023.

Also see:

In-source fragmentation part one. “Breaking up is (not so) hard to do.”

Introduction

In the preceding segment of this series, we found out that our “unknown” has the elemental composition C₈H₁₁N₄O₂. However, that’s not sufficient to identify the compound because there are many possible chemical structures (isomers) that can have the same elemental composition. We can use fragment ions to distinguish many isomers. Here, let’s discuss in-source fragmentation in depth.

Distinguishing Between Isomers

We can’t base a chemical analysis of a pure compound on elemental composition alone. Let’s suppose that we determine that a sample found in a suspect’s car contains a molecule with the elemental composition C₁₇H₁₉NO₃. It could be morphine – but let’s not break out the handcuffs yet! It could be one of several drug isomers that have the composition C₁₇H₁₉NO₃ such as hydromorphone or norcodeine. It could also be another isomer – piperine, the spicy molecule in black pepper! Clearly, we need a way to distinguish between isomers.

In traditional gas chromatography coupled with mass spectrometry (GC-MS) the electron ionization (EI) mass spectra have fragment-ion peaks that we can search against databases to identify compounds. How can we get fragment-ion data for compounds analyzed with soft ionization methods like DART-MS?

Collision-induced dissociation

The most common way to break apart ions in a mass spectrometer (in-source fragmentation) is to use an electric field to accelerate the ions and collide them with gas molecules in a partial vacuum. This is called collision-induced dissociation (CID) which is the basis for tandem mass spectrometry (MS/MS) in mass spectrometers like the JEOL TQ4000 triple quadrupole mass spectrometer system.

One of the simplest ways to implement CID is to fragment the ions in the interface between atmospheric pressure and vacuum, referred to as “in-source CID” or “cone voltage CID”. To understand how this works, let’s take a closer look at how ions in a gas stream at atmospheric pressure are introduced into the vacuum system of the mass spectrometer. There are many different designs for atmospheric pressure interfaces for mass spectrometers. The AccuTOF-DART mass spectrometer has one of the simplest and most robust designs.

The figure below shows a schematic of atmospheric pressure interface for the AccuTOF-DART mass spectrometer. The DART gas stream passes through a small hole in the first skimmer (“Orifice 1”) into a region that is evacuated by a mechanical roughing pump. Ions are directed by the electric field through a ring lens and into a small hole in a second skimmer (“Orifice 2”). A bent quadrupole ion guide transports the ions further into the vacuum system and the time-of-flight mass analyzer. The off-axis skimmer design blocks contamination from entering the ion guide and mass analyzer.

The important parameter is the potential difference between Orifice 1 and Orifice 2. If this value (the “Orifice 1 Potential” or “cone voltage”) is small – for example 20 Volts – then ions will drift through the partial vacuum region without undergoing strong collisions with gas molecules. If we increase the potential difference to say, 90 Volts, then ions will collide with gas molecules with enough kinetic energy to form fragment ions.

How about the morphine/black pepper example? The 90V fragmentation patterns for the two isomers are quite different!

Coming back to our caffeine example, here are the positive-ion DART-MS mass spectra for two different Orifice 1 potentials. The in source fragmentation of Orifice 1 = 90V spectrum are distinctive and can be related to the structure of caffeine.

Conclusion

In-source collision-induced dissociation (CID) is a simple way to generate fragment ions that can distinguish between many isomers. In the next segment of this discussion, we will consider the effect of changing the collision energy on the measured mass spectra.

To learn more about JEOL mass spectrometers and the AccuTOF-DART system, please visit us here.

How DART isotope measurements assist in elemental composition measurements

Previously, we found 4 possible elemental compositions for even-electron ions in our example of a DART-MS spectrum with a peak at m/z 195.0899. If we have a time-of-flight mass spectrometer such as the AccuTOF-DART that can provide accurate isotope measurements, we can use that information to determine the most likely elemental composition.

Isotope measurements

The elements have characteristic stable isotope patterns. Elements such as carbon, hydrogen, oxygen, and nitrogen have more than one stable isotope. For example, on earth, the isotope carbon-13 (¹³C) has an average relative abundance of 1.082, normalized to a relative abundance of 100% for ¹²C. Elements such as fluorine and phosphorus have only one stable isotope. A table of the average relative abundances of the isotopes for these elements on earth is available from the International Union of pure and Applied Chemistry (IUPAC). Using this information, software can calculate the exact masses and relative abundances for any given elemental composition.

Our example DART-MS positive-ion mass spectrum showed the most abundant peak (“base peak”) at m/z 195.0899, but there are also two smaller isotope peaks detected. The isotope measurements are shown below with measured m/z and relative abundances shown below.

m/z	Rel. abundance (%)
195.089905	100
196.090607	10.4671
197.095093	0.91

We can let our elemental composition software calculate the average relative abundances for each of the four possible compositions we determined for our m/z 195.0899 peak and compare the calculated values to the measured values. Now we can rank the elemental compositions based on how well the measured isotope m/z values and relative abundances match the calculated values. Here are the results and the constraints specified for elemental composition determination:

Elemental Compositions
Element Limits: C 0/16 H 0/34 O 0/12 N 0/14
Tolerance: 5 mmu
Even or odd electron ion or both: Even
Electron correction: None
Charges: 1
Minimum unsaturation: -1
Maximum unsaturation: 100

Calc. m/z	Abund %	mmu	Peaks	Score	DBE	Composition	NIST
195.088199	0.01	0.98	3	0.000266	5.5	C8H11O2N4	4
195.086863	0.15	1.76	3	0.008828	0.5	C7H15O6	0
195.085512	0.14	2.55	3	0.012170	6.5	C4H7N10	0
195.092223	0.27	1.37	3	0.012287	9.5	C13H11N2	0

The score multiplies the relative abundance match and the accurate-mass (m/z) match and divides by the number of matching isotopes detected. A smaller score denotes a better match, with a perfect score being zero. The NIST column shows the number of entries in the selected NIST-formatted database that have that elemental composition. The correct composition for protonated caffeine C₈H₁₁N₄O₂ has the best matching score, and it is the only one of the four compositions that has matches compounds in the NIST 20 Mass Spectral Database.

Here is a labeled screen shot from the Mass Mountaineer program of the complete elemental composition determination including the measured mass spectrum, limits and constraints, results, and the matching isotope measurement data for the selected (best matching and correct) elemental composition. The calculated isotope pattern is shown in red in the isotope match tile, and the measured isotope pattern is shown in blue with a slight offset to make it possible to compare the calculated and measured relative abundances of each peak.

Now we have determined the most likely elemental composition for our “unknown” compound from its positive-ion DART-MS spectrum. However, we still have a problem! There are 16 isomers in the NIST 20 Mass Spectral Database. Is there anything we can do to increase our confidence that this is caffeine? Yes, there is! In the next series of articles, I’ll be discussing the role of collision-induced dissociation and fragment ions.

If you have a time-of-flight mass spectrometer that measures accurate masses and provides accurate isotope measurements, you can compare the calculated isotope patterns for candidate elemental compositions with the measured isotope patterns for a chemical compound. You can read more about determining elemental compositions here. A free Periodic Table app that lists the m/z and average relative abundance data for stable isotopes is available for iPhone and Android users. A free MS Calculator app that can calculate isotope patterns for elemental compositions is available for iPhone users. To learn more about JEOL mass spectrometers and the AccuTOF-DART system, please visit us here.

Note

¹There are slight variations in the isotopic abundances for molecules depending on factors like geographic origin. Measuring these slight variations with high precision and accuracy requires special isotope-ratio mass spectrometers. For the purpose of determining elemental compositions, we can ignore those slight variations and just use the average relative abundances.

ENC 2023 Conference Notes & Mini Symposium

The Experimental Nuclear Magnetic Conference (ENC) celebrated its 64^th conference April 16-21, 2023 at the Asilomar Conference Center in Pacific Grove, CA. Beginning in 1959 and established as a 501(c)(3) non-profit in 1987, the conference was organized with the following stated goals:

Promote the interest of molecular spectroscopy in general and NMR spectroscopy, in particular;
Organize an international meeting for the exchange of state-of-the-art information with special attention on experimental aspects of NMR spectroscopy.

Although ENC is not hosted in a single location, the conference has a long history with the Asilomar Hotel and Conference Grounds. The conference grounds are situated along Asilomar State Beach, which is part of the Asilomar Marine Reserve and boasts a rich and biodiverse ecosystem, and close to the Asilomar Dunes Natural Preserve, which features 25 acres of pedestrian boardwalk through the sand dune ecosystem.

As a conference, ENC is excellent for networking with the thought leaders of today’s NMR community in company-hosted hospitality suites. JEOL hosted their events in the Fred Farr Forum, including the JEOL Announcement Night, NMR Mini Symposium, iPad Raffle, and more.

JEOL Announcement Night

JEOL’s Announcement Night kicked off Tuesday, 4/18. Highlights of the presentation included the following:

New ECZL System – Our new NMR ECZL G series is a flagship model for cutting-edge NMR methods. The footprint of the spectrometer has been reduced to less than 60% of ECZR, while maintaining the expandability needed to support a wide range of applications. It is flexible in terms of expansion, with support for three or more channels, high-power amplifiers, and high-output magnetic field gradients, allowing for future functional expansion even when installed in the minimum configuration
ROYALPROBE™ HFX – The ROYALPROBE™ HFX is the world's first liquid NMR probe with the capability to switch between single tune and dual tune modes on the high frequency coil. This expands the capability of a standard workhorse NMR, making it an excellent choice for scientists looking to achieve the maximum impact of their instrument.
Cryogenic Probes for NMR – JEOL offers two ultrahigh sensitivity autotune probes using cryogenic probe technology: The SuperCOOL Cryogenic Probe and the UltraCOOL Cryogenic Probe.

The SuperCOOL Probe features significantly improved sensitivity as its thermal noise is reduced by cooling of both the detection coil and preamplifier. The SuperCOOL probe reduces measurement times to up to 75% to enable many more samples to be measured in a single day.
The UltraCOOL Probe achieves more than 4 times the sensitivity of conventional probes while thermal noise is reduced by cooling of both the detection coil and preamplifier. Measurement times using the UltraCOOL probe are only 1/16 that of a conventional probe.

Cryogen Reclamation System – JEOL’s new Cryogen Reclamation System offers a convenient, reliable, and highly effective solution to managing and maintaining your NMR instrument’s cryogen levels. This system maximizes NMR instrument uptime and reduces the risk of shutdown due to cryogen supply issues by substantially reduces the evaporation of liquid helium and liquid nitrogen from the superconducting magnet.

NMR Mini Symposium

The JEOL NMR Mini Symposium included six scientific presentations covering a range of NMR topics. Speakers included:

Paul Ellis, Daniel Arcos, and F. David Doty, Doty Scientific - Spin Echoes, Sensitivity, Wurst (Adiabatic) Pulses, and Artifact Suppression Utilizing Modern ssProbes
Professor Federico Del Rio, UNAM, Mexico - Structure of Arachnid Toxin by NMR
Ronald Crouch, JEOL USA - Balancing the Robust and Convenient with the Challenging
Yusuke Nishiyama, JEOL LTD - 14N Solid State NMR at Fast MAS
Peter Kirali, JEOL UK - The Hidden Gem of Data Processing in JASON
Manuel Perez, JEOL UK - JASON: Advanced Functionality, Enabling Automaton

In case you missed it, you can view our NMR Mini Symposium on-demand.

NMR Technical Presentations

In addition to our Mini Symposium, JEOL scientists contributed to two technical talks. The first took place on Monday 4/17 and was presented by Genevieve Seabrook:

“Small GTPases are regulators mediating important cellular functions. These sGTPases are often mutated in human cancers. We have developed a real-time multiplex NMR assay allowing the following of several sGTPases nucleotide exchange in a single experiment. Along with sGTPase proteins strategically selectively labeled, time-shared NMR methodology was used to reduce acquisition time. Analysis of sGTPases amides chemical shift changes, allowed us to identify residues that have been perturbed during the nucleotide exchange and the resulting structural changes within the sGTPases. A mixture of six sGTPases was used to assay GEF activities present in cells lysates and in organoids lysates. A combination of selective isotopic labeling and real-time, time-shared NMR experiments can be extended to other biological processes”

The second, 1H CSA: Friend or Foe?, was presented by Frederic A. Perras:

“Despite the high sensitivity, and recent resurgence, of 1H solid-state NMR, measurements of 1H chemical shift anisotropy (CSA) have remained rather niche. In many instances, we would even consider it a nuisance that leads to decoherence and t1 noise in 1H dipolar recoupling. This presentation will cover the development of highly stable dipolar recoupling methods that decouple the 1H CSA in addition to new 1H CSA recoupling schemes that enable the measurement of tensor skew, and small anisotropies. Lastly, the utility and limitations of 1H CSA for the measurement of dynamic information in low sensitivity samples, such as heterogeneous catalysts, will be discussed.”

NMR Technical Posters

JEOL presented four technical posters at ENC 2023. The first, titled “Simplifying triple resonance experiment for high quality NMR spectra with Multi Frequency Drive System” and presented by Hiroaki Sasakawa, explored our new Multi Frequency Drive System, which is available on our ECZ Luminous NMR console:

“Organic compounds with phosphorus and boron nuclei often exhibit spectral complexity and reduced sensitivity in NMR analysis due to J couplings between hydrogen and carbon with these nuclei. We developed a triple resonance system called Multi Frequency Drive System (MFDS) to address this issue, enabling triple resonance experiments with a standard 2-channel NMR system. Using a JEOL JNM-ECZL600G spectrometer equipped with the ROYALPROBE™ P+, we conducted various solution NMR measurements. We present examples of signal enhancement and spectrum simplification achieved by triple resonance measurements of 1H, 31P, and 11B collected with a 2-channel NMR instrument.”

The second poster focused on our new 1.01 GHz NMR system. Titled “Development and applications of a 1.01 GHz (23.7 T) NMR system,” this poster was presented by Yoshitaka Ishii:

“We discuss development of an ultra-compact 1.01 GHz NMR magnet, and its preliminary NMR applications. The new ultra-compact 1GHz NMR magnet utilizes high-temperature superconducting (HTS) coils made of bismuth-based cuprates besides conventional low temperature superconducting coils. Because of the high current density of the HTS coil, the magnet weighs only 1.6 tons and its footprint is the smallest among the existing 1 GHz NMR systems. The cryogenic refrigerator mounted on the magnet eliminates needs of regular liquid-helium refilling. We have successfully collected multi-dimensional solution NMR and solid-state NMR data for proteins at a 1H frequency of 1.01 GHz. The quality of the NMR data and other research progress from the ongoing project to develop 1.3 GHz NMR will be also discussed.”

The third technical poster, presented by Takuya Matsumoto and title “Cryogen Reclamation System for NMR Magnets” offers a solution for evaporation of cryogens:

“NMR magnets are usually cooled by two kinds of cryogens, ie.liquid helium and liquid nitrogen. The boil-off rates of the cryogen in general NMR magnets are typically around 20 cc/h for liquid helium and 200 cc/h for liquid nitrogen. We have developed new cryogen reclamation system that can greatly suppress the evaporation both of liquid helium and liquid nitrogen. The system was tested with an NMR magnet, and it was confirmed that the noise generated by system vibration was at a level that would not interfere with NMR measurements. It has also confirmed that the magnet maintained stable zero boil-off status for more than 6 months.”

The fourth technical poster was presented by Yutaro Ogaeri and titled “Internuclear Distance Measurements between 1H and 14N in Multi-Component Rigid Solids at Fast MAS”:

“1H-14N internuclear distances are readily and accurately measured using the symmetry-based phase modulated resonance-echo saturation-pulse double-resonance (PM-S-RESPDOR) method in rigid solids. Analytical equation of the fraction curve easily provides 1H-14N couplings. However, this treatment is only applicable when NH proton resonance is well separated from the other proton peaks, which is not necessarily satisfied even at fast MAS >60kHz, especially in multi-component systems. To overcome this problem, THMQC filtering is applied to suppress the 1H signals other than NH proton prior to the PM-S-RESPDOR experiments. The method is well demonstrated on two components acetaminophen-oxalic acid (APAP-OXA) systems.”

New ECZ Luminous NMR Console

Our new NMR ECZL G series is a flagship model for cutting-edge NMR methods. The footprint of the spectrometer has been reduced to less than 60% of ECZR, while maintaining the expandability needed to support a wide range of applications. It is flexible in terms of expansion, with support for three or more channels, high-power amplifiers, and high-output magnetic field gradients, allowing for future functional expansion even when installed in the minimum configuration. Learn more about the ECZL.

How do Ion Milling Systems Work?

Ion milling systems designed for microscopy are used to prepare samples for scanning electron microscope (SEM) or transmission electron microscope (TEM) analysis. This is done by removing an outer layer of a sample, creating a cross section, a polished surface or preparing an ultrathin sample. The result is a clean, undamaged surface ideal for high-resolution imaging. This post will provide an overview of how ion milling systems work and their applications.

How Does an Ion Milling System Work?

Ion milling systems for sample preparation in microscopy can be broken down into two categories: Broad Ion Beam and Focused Ion Beam (FIB). In each case, ions are used to mill away the surface of a sample exposed to the ion beam. Broad Ion Beam milling systems are popular for use in SEM sample preparation where they create a pristine surface or cross section for SEM observation and analysis. In these systems, a sample is protected by a shield plate and placed in the beam path of a broad Argon ion beam. Only the area of the sample exposed (protruding from the shield plate) gets milled away. In contrast, FIB milling systems use a finely focused beam of ions (typically gallium ions) to mill away a sample surface. FIB milling systems are used for both SEM and TEM sample preparation. With FIB, successive milling in very small, targeted areas can be accomplished to view the three dimensional subsurface of a sample or to create ultra-thin samples suitable for TEM.

The Advantages of Ion Milling

Why invest in an ion milling system? Ion milling eliminates artifacts associated with traditional mechanical preparation methods and prepares surfaces with minimal strain or distortion. It is often the only way to create high quality surfaces or thin sections that are required in high resolution imaging and analysis in SEM and TEM. Materials that are difficult to handle such as: brittle materials, multilayer samples with differences in hardness or thermal expansion, fragile materials, thermally sensitive or air sensitive samples are easily managed in ion milling systems.

Having an undamaged sample is crucial for accurate analysis.

Broad Ion Milling or FIB? How do I Choose?

Choose FIB for TEM thin-film sample preparation. It is also perfect for creating cross sections where precise positioning of very small features is required. In addition, FIB is great for three-dimensional visualization of images or analysis data (such as EDS or EBSD) by successive milling and imaging. A broad ion milling system is an economical choice if cross sections for SEM observation and analysis is all that is required. These systems also provide cross sections over a much wider area when compared with FIB. Cross sections in the range of 1mm to 8mm wide can be achieved with Broad Ion Beam milling.

JEOL USA: Ion Milling Systems

JEOL offers a range of sample preparation tools from Focused Ion Beam (FIB) systems to our Cross Section Polisher, a benchtop, broad ion beam instrument. These instruments are ideal for preparing battery materials, ceramics, multilayer coatings, polymers, environmentally sensitive materials, semiconductor devices and more.

Cross Section Polisher (CP)

Our Cross Section Polisher is used for preparing samples for imaging and analysis via SEM. It creates polished cross sections of a sample and is suitable for a wide range of hard and soft materials such as battery components, ceramics, metals, polymers, composites, and electronics etc. Key features of the CP include easy set-up, high-speed, and intermittent milling for temperature sensitive samples and the ability to create wide area cross sections (up to 8 mm). In addition, our cooling model is ideal for temperature or air sensitive samples as might be found with biological samples, polymers, catalysts or lithium-ion battery materials. An air isolation transfer vessel is included with our cooling model and this vessel is compatible with our SEMs.

Focused Ion Beam

JEOL’s new focused ion beam instrument, the JIB-PS500i FIB-SEM, is a hybrid of FIB technology and SEM designed for high-resolution analysis and high throughput specimen milling, observation, and transfer to the TEM. With the ability to prepare samples thinner than 30nm, the FIB-SEM enables superior atomic resolution imaging and analysis with STEM and TEM. The fine milling capabilities of the FIB-SEM are essential for quality lamella preparation for imaging, EDS analysis, and 3D microscopy. The high current (up to 100 nA) FIB column is especially effective for large-area milling and analysis, and is ideal for semiconductor samples.

A deeper dive into elemental composition determination with DART

In the last article, we found 8 possible elemental compositions for a protonated molecule having a positive-ion DART-MS peak at m/z 195.0899 +/- 0.005 and elements C, H, N, and/or O. We can use constraints such as unsaturation (double bond equivalents) to eliminate many of the incorrect compositions.

Electron configuration

Ions in a mass spectrum fall into two categories: even-electron ions and odd-electron ions (radical ions). Odd-electron ions have an unpaired electron. An example of an odd-electron ion is the molecular ion M^+• in electron ionization (EI) mass spectra. Protonated molecules [M + H]⁺ that are formed in positive-ion DART-MS are even-electron ions. DART-MS produces primarily even-electron ions¹. Even-electron ions are also found in other mass spectra from many other ionization methods, such as chemical ionization (CI) and electrospray ionization (ESI) and as fragment ions in EI mass spectra.

Even-electron ions (half integer unsaturation):

Protonated molecule [M+H]⁺
Deprotonated molecule [M+H]^-
Chloride adduct [M+Cl]^-
Ammoniated molecule [M+NH₄]⁺
Fragment F⁺

Even-electron ions (exact integer unsaturation):

Molecular radical cation M⁺
Molecular radical anion M^-
Fragment F⁺

Examples of even-electron and odd-electron ions

Double bond equivalents

For any given elemental composition, we can calculate a number called Double Bond Equivalents (DBE, or unsaturation) that represents the number of rings and sites of unsaturation (double and triple bonds) that would occur in molecules with the given composition. This is calculated using a formula based on the number and valence of each element in the composition. For compounds with a composition C_xH_yN_zO_n, the formula is:

The more general formula² is:

where DBE is the unsaturation, i_max is the total number of different elements in the composition, N_i the number of atoms of element i, and V_i is the valence of atom i.

If we have additional information about the unknown molecule, such as NMR or FTIR data, we might be able to set limits on the range of DBE values for our unknown.

Using the DBE value to distinguish even-electron and odd-electron ions

Assuming that we don’t have additional information, why is this value is still useful? Simple! The calculated double bond equivalents for odd-electron ions are whole integers, but the calculated double bond equivalents for even-electron ions are half-integer values.

Application to our accurate-mass DART-MS example

Our elemental composition software calculates a DBE for each candidate composition determined from the accurate-mass data. Because we are using DART-MS, an ionization method such as that produces primarily even-electron ions, we can display only compositions with a half-integer DBE to eliminate all but four compositions for our peak at m/z 195.0899.

Constraining our elemental compositions to even-electron ions eliminated half of the compositions. We happen to know that the correct composition is in this list, but in practice we would have to choose between the four remaining compositions. The next article in this series will explain how to use isotopic data to rank the compositions.

If you know what kind of ions are formed by an ionization method such as DART-MS, you may be able to eliminate many incorrect elemental compositions determined from accurate-mass spectra. You can read more about determining elemental compositions here. To learn more about JEOL mass spectrometers and the AccuTOF-DART system, please visit us here.

Notes

¹Odd-electron ions are rare in DART-MS, but they can occur in certain circumstances. This will be the subject of a future post

²This formula is only going to give the correct number of double bond equivalents for elements in their lowest valence state. Be careful when you are interpreting DBE values for compositions with elements such as S and P that have more than one common valence state.

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How DART mass spectrometry uncovers elemental compositions

If we have accurate mass data, DART-MS can tell us more than the molecular weight of a molecule. Let’s see how to determine the elemental composition from accurate-mass DART-MS data.

Note: The next few articles focus on the interpretation of accurate-mass spectra. Although I am using a DART-MS example, the information here applies to any data from an accurate-mass mass spectrometer, such as the JEOL AccuTOF GC-Alpha. We’ll come back to DART-MS shortly.

Elemental composition determination from an accurate mass

The first step in determining an elemental composition from an accurate mass is to specify a set of elements that might be present and set some limits on the minimum and maximum number of each element that might be present. We’ll use the caffeine example from the previous posts. Let’s assume that we know that we don’t know that the compound is caffeine, but that the compound only contains carbon, hydrogen, nitrogen and oxygen. Because accurate mass measurements are not always exactly correct, let us also assume that the measured m/z is 195.0899 instead of the exact calculated value of 195.0882. We can set the element limits based on the maximum number of each element that could give a molecular weight less than 195.

Element	Symbol	Maximum
Carbon	C	16
Hydrogen	H	34
Oxygen	O	12
Nitrogen	N	14

Next, we specify an error tolerance of 5 millimass units (5 mmu) for the accurate mass measurement. That’s 0.005 u, a bit wider than the error we expect, but wide enough that we won’t miss any possible compositions. Software then calculates the weight (actually, the m/z) for all possible combinations of the elements within the specified limits and only reports those elemental compositions that have a calculated m/z within 5 mmu. There are nine compositions that fit those constraints.

Calc. m/z	mmu	DBE	Composition
195.085512	-4.39	6.5	C₄ H₇ N₁₀
195.08552	-4.38	1	C₅ H₁₃ O₅ N₃
195.086856	-3.04	6	C₆ H₉ O₁ N₇
195.086863	-3.04	0.5	C₇ H₁₅ O₆
195.088199	-1.7	5.5	C₈ H₁₁ O₂ N₄
195.089543	-0.36	5	C₁₀ H₁₃ O₃ N₁
195.092223	2.32	9.5	C₁₃ H₁₁ N₂
195.094065	4.17	2	H₉ O₂ N₁₁

Here’s a summary of the process:

Elemental composition determination from an accurate mass

What does DBE mean, and how do we know that the correct elemental composition for the m/z 195.0899 peak is C₈H₁₁N₄O₂ instead of one of the other 8 compositions? Stay tuned for the next piece in this series!

Accurate mass information obtained with DART-MS can be used with to determine the elemental composition of molecules. You can read more about how elemental compositions are determined from accurate-mass data here. To learn more about JEOL mass spectrometers and the AccuTOF-DART system, please visit us here.

How DART mass spectrometry uncovers molecular weight information

This post describes the first piece of information we get from ambient ionization and DART-MS: the molecular weight.

How DART examines molecular weight

In the previous blog, I explained how positive-ion DART-MS can form protonated molecules [M + H] ⁺ for compounds like caffeine. Here is the DART-MS caffeine mass spectrum again, with the caffeine structure and elemental composition:

Positive-ion DART mass spectrum of caffeine measured on the JEOL AccuTOF-DART system

Mass spectrometers measure the mass-to-charge ratio (symbol “m/z”). DART-MS only produces single-charge ions, so for the purpose of this discussion we can ignore the charge.

The exact masses in unified atomic mass units (u) of the most abundant stable isotopes of the elements carbon, hydrogen, oxygen, and nitrogen are:

¹²C: 12.000000
¹H: 1.007825
¹⁶O: 15.994915
¹⁴N: 14.003074

We measured the protonated molecule, so the peak at m/z 195.088 tells us that the molecular weight of the compound must be m/z 195.088 minus the mass of the proton (1.0078), or 194.080. Caffeine has the elemental composition C₈H₁₀N₄O₂, so the molecular weight of caffeine is 194.080, which matches what we measured.

If we only have a low-resolution mass spectrometer (such as a quadrupole MS), we will only be able to measure the molecular weight to about one decimal place, but the JEOL AccuTOF-DART measures accurate masses with errors in the 3^rd or 4^th decimal place. Accurate molecular weight determination is important information for synthetic chemists.

If we want to identify an unknown compound, the molecular weight is useful information, but not enough to uniquely identify a molecule. Knowing the accurate mass is considerably more selective than just having an integer mass. If we search the NIST 20 mass spectral database for compounds with an integer molecular weight of 194, we find 1277 compounds. Limiting the search to 194.080 with an error tolerance of +/- 0.001 u returns 37 compounds.

Fortunately, an accurate-mass system like the AccuTOF-DART with DART-MS provides more information than just the molecular weight. In the next few articles, we’ll see how to determine the elemental composition from accurate-mass DART-MS data.

DART-MS can be used to determine the molecular weight of molecules. To learn more about JEOL mass spectrometers and the AccuTOF-DART system, please visit us here.