Oregon State University
Electron Microscope Facility

USER INFORMATION AND FACILITY POLICIES

PROTOCOLS

Powders And Suspensions For "Particle Size" Microscopy  

"Those who wish to succeed must ask the right preliminary questions." 
Aristotle, from Metaphysics, II 

The Electron Microscope Facility receives many requests for work intended to size and otherwise characterize particles which are presented as dry powders or liquid suspensions.  Typical specimens are delivered in glass or plastic ware labeled with a letter or number code or a run or sample identification designation.  When questioned about the contents, data needed, and how the data needed relates to an experiment or technical process, clients seldom provide useful information.  We may be told a vial contains a "drug" or "phosphor", "clay", or "silica".  We're most frequently told the information needed is "particle size", with an occasional "porosity" or "contaminant" statement added.  Rarely are details about the relationships between this data and how it connects to function provided. 

The goals and intent of our services are to be helpful to clients, and although needs for proprietary or unbiased results are understandable, our abilities to meet a client's needs as well as our concerns for personal safety and microscope care are closely tied to the quality of information provided with specimens.  Taking the last two issues mentioned, specimens need to be prepared for microscopy and are examined under high vacuum and ionizing radiation conditions, so questions about health, handling, and material stability and safety are not inconsequential.  Providing Materials Safety Data Sheets (MSDS) with specimens, or reasonable written similar information for experimental substances, is appreciated. 

Clients want accurate and useful information.  They also want the results quickly and cheaply.  Both expectations are compromised by the form in which the specimens are presented.  Because the client has much greater knowledge and insight about their specimens and how the specimens relate to their application(s), and usually have access to the equipment needed to collect and manipulate their unique specimens, it is critical that the client invest the effort required to fracture, grind, disrupt, sort, fractionate, centrifuge, filter, suspend in appropriate solvent, disperse, or otherwise pre-process particle specimens prior to submitting them for microscopy.  Doing so shortens data return time, permits us to provide more useful answers to your specific questions, and may appreciably reduce the charges that are applied if Facility labor is needed to do pre-processing manipulations necessary to obtain meaningful specimens.  When the fundamental particles remain in aggregates, it is the client's responsibility to disrupt those aggregates by means compatible with the specimens and investigative requirements so as to render specimen submissions that are compatible and appropriate to the analytical goals. 

Regarding the data requested and how that information relates to larger issues, typical submissions contain particles ranging in size from several millimeters to fractions of a nanometer, and not uncommonly, ultimately down to the size of the molecules, unit cell crystals, or other atomic condensations that comprise the material(s).  This is especially common in dry power submissions because particles clump together or were scraped from filters or surfaces on which they were concentrated by adhesion.  What are acceptable methods to reduce clumped aggregates to fundamental particles?  What mechanical disruptive process(es) or solvent(s) are acceptable to use with what's submitted to break clumps down to fundamental particles?  Are the aggregates, the fundamental particles, or an intermediate condition critical to understanding the dynamics of these particles relative to the application? 

In routine practice, the results provided when particle size information is requested will be a series of pictures taken at increasingly higher magnification which visually "average" and document an "as received" specimen.  Clumped powders may be spread to a thin layer in which particles smaller than the macroscopically visible clumps are possibly revealed. 

The Electron Microscopy Facility at O.S.U. offers plant virus assay services to horticulturalists.  The assay performed is most valuable when used for detection and diagnosis of plant infecting rod‑like viruses such as lily symptomless virus, potato viruses X and Y, or tobacco mosaic virus.  The test is not recommended for detection or diagnosis of spherical viruses like apple mosaic, cucumber mosaic, or tobacco ringspot viruses, and is NEVER guaranteed to prove a plant free of an infectious agent. 

Before submitting samples, it should be understood that we provide a diagnostic analysis; we are not in the business of certifying plants as being specific pathogen free for commercial or research purposes.  Clients are cautioned not to misuse, misinterpret, or otherwise extend our findings beyond the specific plant examined at a specific point in time.  Furthermore, although we are willing to discuss our client's specific plant disease problems, we are not a horticultural consulting service and do not make recommendations about management, marketing, laboratory, greenhouse, nursery, or field practices. 

Data generated for a customer, and customer information of a proprietary nature is treated as confidential. 

THE TEST: MATERIALS AND METHODS 

Fresh, fully hydrated leaves are preferred samples.  A small piece of leaf tissue is macerated in a water‑based electron dense stain on a TEM specimen grid, excess fluid is blotted away, and the dried sap/stain residue examined by transmission electron microscopy. 

In preparing the sample, fresh stain made from powdered reagents and sterile, distilled water is used.  The stain is filter sterilized into a sterile glass serum bottle and thereafter handled in a sterile syringe.  The work counter is surface sterilized with 70% ethanol before each group of samples is prepared.  New TEM support grids, coated with polyvinyl formal, are fastened to clean glass microscope slides with adhesive tape.  Forceps used to obtain and macerate the leaf tissue are alcohol flame sterilized both before and after preparing each individual sample to prevent contamination of samples by our tools.  Blotting of liquids is done with fresh, clean chromatography paper pieces; one or more pieces are used to blot each grid. 

Only one leaf sample is removed from its shipping container and prepared at a time to prevent possibilities of either sample cross‑contamination or mislabeling.  The unused leaf material is returned to its shipping container after the grid has been prepared.  A stain blank is prepared for every group of specimens made as a control to prove the stain free of contaminating material.  Each grid is examined by microscopy.  If virus is detected, examination may be for under a minute.  However, if virus is not detected, examination is continued for at least ten minutes, after which a second sample is prepared, again in accordance with the above methodology, and that grid also examined for at least ten minutes.  Occasionally even a third sample will be tried if there is reason to suspect the presence of a low level infection. If virus is not detected by repeated examinations, the result is reported as "virus not detected." 

RESULTS 

Virus Detection:  A simple "detected/not detected" answer is often the only concern of a client.  Results of our assay will be reported by letter, or in some cases, by telephone followed by confirming correspondence.  Verbal reports may be supplemented with micrographs to further document the nature of detected virus.  Micrographs will be provided to show any virus detected in a sample.  Obviously, if virus is not detected, micrographic evidence cannot be obtained.  

When virus is detected, the verbal report will read "virus detected," or, when virus cannot be found, will read "virus not detected."  Even if we find but one virus in a sample, we report the plant as "virus detected."  Occasionally incomplete virus‑associated structures may be detected.  These have been reported in scientific literature as "associated proteins," "fibers," and "coated lines."  We detect this condition both with and without the presence of actual virus, and report this condition as "incomplete particles detected." 

Sizing:  We can provide data about the size of detected virus.  This information is useful in ascertaining the specific virus(es) involved in the infection and determining possible corrective or preventative management practices.  Precise sizing is possible, but must be done on photographic images to be accurate, and, to be statistically meaningful, many particles should be available for measurement.  It is necessary that the customer advise us that size information is needed at the time samples are submitted.  Both the size information and the documenting photograph will be included in our report. 

Multiple Infection:  It is possible to detect multiple infection by more than one type of virus or by virus and bacteria or mycoplasma.  Our report will indicate this condition and the nature of the infectious agents whenever it is applicable. 

Relative Concentration:  An estimate of relative levels of virus(es) detected can be made from the microscopic examination.  This is a subjective visual impression based on what the microscopist sees.  There are statistical limitations to both visual estimates and photographic techniques, but either one can give the client a feeling for the relative concentration of virus in a particular sample.  The report will express relative virus concentrations as "low," "moderate," or "high" concentration.  When knowledge about relative virus concentration is required, the client should relay that information to us when submitting samples. 

RELIABILITY 

Through the stained sap technique and our quality assurance procedures herein described, we endeavor to give clients accurate, reliable information that if properly understood and used can be valuable for their production, management, and marketing decisions.  However, because these procedures assay only one very small piece of tissue from one plant at one point in time, we cannot unequivocally guarantee a particular plant is specific pathogen free.  OUR ASSAY IS TO BE CONSIDERED AS A DIAGNOSTIC TOOL - IT IS A MUCH BETTER ASSAY TO PROVE THE PRESENCE OF A PATHOGEN THAN TO PROVE ITS ABSENCE. 

After selecting the sample, any subsequent handling of a plant by anyone can potentially cause introduction of a pathogen to a clean plant.  Consequently, although we diagnose plants to the best of our abilities and the limits of specimen preparation technique, we cannot guarantee nor in any other way certify a plant is disease free.  Any material cloned or cultured from a checked, apparently clean plant should not be assumed to be virus free by our client.  Our role is diagnostic, and is most useful in the early stages of producing virus free plants or when a disease outbreak is noted.  Quality assurance of healthy plants is the responsibility of the grower and is not assumed by the Electron Microscope Facility. 

Many clients have confidence in statistical methods for screening their plants for disease agents.  We do not recommend reliance on statistics.  We examine the specific plant(s) of interest for a client and report our findings about that specific plant at one specific point in time.  To have continuing confidence in a pathogen‑free plant or a disease treatment program, parent material and offspring and/or clones, or additional samples of treated plants, should be checked at intervals not exceeding 180 days. 

Minimum detectable infection and reliability of EM on stained sap as a diagnostic tool for detection of rod-like plant viruses is comparable to the detection and reliability obtained by the ELISA method.  By immuno-electron microscopy, the minimum detectable infection level is lower due to the greater sensitivity of that procedure. 

ALTERNATIVE METHODS 

ELISA:  Enzyme-linked immunosorbent assay (ELISA) is an immunological test with minimum detectable infection accuracy and reliability comparable to examination of stained sap by electron microscopy.  It is not a microscopical technique and is not available through our Facility.  ELISA is a relatively rapid assay recommended for large batch checking.  It is favored as a quality assurance assay by growers in extensive production or marketing situations.  Virus concentration and multiple infection (especially with bacteria) information can be missed by ELISA unless specially designed into the test. 

Immuno-Electron Microscopy:  Immuno-EM, also called immunosorbent electron microscopy, combines an ELISA-like immunological test with the electron microscopic examination of stained sap.  This procedure is at least 4 orders of magnitude more sensitive in detecting a low level virus infection than either ELISA or the conventional stained sap EM assay used alone.  Although immuno-EM testing can be performed by the Electron Microscope Facility, it costs 5 times as much as a conventional stained sap assay, and the customer must supply at least 10 square centimeters of leaf for each plant to be tested and the antiserum(a) against the virus(es) of interest.  There are few commercial sources of antisera to common viruses of interest.  Clients must either produce their own antisera or obtain it through special arrangements with a university or biochemical supply house.  Specific activity and quality assurance of antisera from diverse and unreliable sources is a problem and may influence accuracy of results. 

Immuno-EM is recommended when information about very low levels of infection is needed.  Additional to its greater sensitivity, it retains the variety of data available by electron microscopy.  It is slow, costly, and not suited for routine or batch checking.

Please avoid submitting specimens which are in a condition which compromises our abilities to provide quality specimen preparation.  

Biological specimens which have been through one or more freeze-thaw cycles, transported under conditions where high or sub-zero temperatures or desiccation might be expected (e.g.; long car trips unprotected in the trunk or back seat), have been improperly fixed or washed prior to receipt, have been over or under centrifuged, or suspensions pelleted in deep glass or plastic (ETFE, styrene, polypropylene, polysulfone, polycarbonate) tubes will have been compromised before reaching us.  

Specimens may be submitted in a vigorous living condition, in fixative, or fixed and moved into a wash buffer. Fixed specimens should never be washed nor stored in either water or saline! Samples should be fixed a minimum of 2 hours and generally not more than 24 hours. Fixed specimens may be kept in buffer up to a week. Tissues with trapped air spaces (stems, leaves, muscle, etc) need to be fixed in a modest (20-30 inch) vacuum.  

If you do the fixation, use of an appropriate EM fixative is imperative to quality EM results. The organic fixation chemicals glutaraldehyde, formaldehyde, or acrolein must be highest purity EM grade materials from freshly opened ampoules, not from pint, quart, or gallon sized stocks or previously opened supplies. Sorensens monobasic/dibasic sodium phosphate or sodium cacodylate are the recommended buffer options. Use of ion or tonicity adjusters (NaCl, MgCl, KCl, sucrose, etc.) in the fixative is acceptable.  

Suspension specimens should be submitted as centrifuged pellets in polyethylene microfuge tubes. Excessively centrifuged materials may be damaged or the resulting pellets too firm for processing chemicals to penetrate. Too lightly pelleted specimens resuspend, disperse, and result in sections with few cells. Recovering pellets from deep tubes or tubes of plastics other than polyethylene drastically compromises the integrity of the pellet. The EM Facility does not have a high speed centrifuge and may not be able to pellet (or re-pellet) specimen suspensions.  

Finally, if a sample is submitted, it's important we know the sample arrived, what the sample is, and at what processing step we have obtained the sample. Things left on our bench or in our fridge with little or no information might become compromised through communications oversights or presumptions.  

Immunological methods used in conjunction with microscopy are used to visualize location and distribution of antigens, antibodies, or site-specific proteins on cells or subcellular structures. The following comparison summarizes the common methods.  

Requirements for high immunological sensitive, high spatial resolution, and high magnification frequently necessitate EM immuno methods, especially those using TEM. These technically complex procedures involve many steps where materials, direct action, and critical decisions are responsibilities of the research scientist. The EM Facility staff can assist in immuno-EM experiments with specimen processing steps involving sample fixation, dehydration, embedding, sectioning, and examination. All specimen immunochemistry details remain the responsibility of the research scientist. The following points may be helpful to planning immuno-EM studies.  

Critical considerations in immuno-EM experiments include the tissue type(s) to be immuno-assayed; when and how to fix, dehydrate, embed, and immuno-label samples; and reaction time, temperature, and reagent concentration factors. As an aid to experiment design, review of literature reporting immuno-EM experiments similar to those proposed is recommended.  

The goals in immuno-EM are to preserve biological structure while simultaneously retaining immunochemical reactivity. EM preparation procedures can significantly alter cellular chemistry, so compromises among goals and technique limitations which retain adequate immunological activity within adequately preserved structures must be obtained. Central to this compromise is the necessary crosslinking of proteins needed to stabilize structure in ways that keep the crosslinking minimal so the critical proteins remain immunologically recognizable.  

Tissues may be fixed before or after the immuno-labeling reaction(s). Most procedures endorse fixation, embedding and sectioning prior to labeling. Tissue "fixation" may be accomplished by chemical or cryogenic methods.  

CHEMICAL METHODS  

Chemically fixed specimens are dehydrated with organic solvents, embedded in plastic resin, and then sectioned. The embedded tissue and the sections are permanently stable, giving the advantages of repeatable immuno-testing and examination. However, the extensive chemical processing required may reduce or block immunological activity.  

The acrylic embedding resin LR WHITE has advantageous characteristics for post-embedding immuno-labeling. This resin sections easily, has low bonding affinities with cell proteins, readily accepts immuno-labeling reaction methods, and has stability in an electron beam.  

CRYOGENIC METHODS  

Cryogenically fixed specimens may be cryo sectioned, with the sections examined by cryo microscopy or after freeze drying. Cryogenic methods maximize retention of immunological competency but frozen tissues and cryo sectioned materials examined by cryo microscopy must be forever maintained at cryogenic temperatures to preserve structure. Freeze drying gives permanency to the cryo sections but may significantly disrupt structure.  

FREEZE SUBSTITUTION METHODS  

Cryogenically fixed specimens may be embedded in plastic resin (freeze substituted), then sectioned, thus combining the advantages of minimized chemical modification and specimen permanency with the disadvantages of more extensive chemical modification of immunological activity. LOWICRYL resins are the embedding media of choice in freeze substitution methods.  

LABELING  

Macro-molecular markers for immuno-ultrastructural studies must be visible (ie, contrast against) biological structures and be suitable for specimen labeling, preservation, and examination methods. A variety of markers are available, including latex, ferritin, hemocyanin, and colloidal gold. The common marker in immuno-EM methods is colloidal gold, which is nontoxic, available in a variety of particle sizes (3-40 nm), offers high contrast, has recognizable, well defined shape, and is available commercially prepared for immuno- applications.  

Direct labeling conjugates the labeling marker to the antigen. The conjugate, incubated with tissue, reacts with antibody. Sites where immunological binding reactions occur complex the conjugate and antibody. The marker reveals these reaction sites. 

Indirect labeling requires antibody and antigen be reacted together. Independently, the labeling marker is conjugated with a second protein complex. When the marker conjugate is then reacted with the bonded antibody/antigen, the complex formed, which incorporates the labeling marker, reveals the reaction sites.  

Please request additional information if you are interested in using these techniques.  

The EM Facility can assist with chemical or freeze subsitution preparation of biological specimens for immuno-cytochemical studies but does not have equipment for cryo-ultramicrotomy. Special pricing, timing, or procedural situations may be needed, so PLEASE discuss your research need(s) with the Facility Manager well in advance of an experiment. 

X-ray spectroscopy is accomplished through collection of x-rays produced from a sample bombarded with an electron beam.  X-ray energies emitted from the sample are dependent on the element(s) from which the x-rays originate.  Differences in x-ray energies are distinguished and graphed as an x-ray spectrum for the specimen under examination.  X-ray spectra contain both qualitative and quantitative information.  

QUALITATIVE ANALYSES  

Information contained in the x-ray spectrum is used to determine detectable elements present in a specimen.  A detectable element must have an atomic number greater than 5 (boron), and must be present in the specimen at a concentration greater than one to five atoms per thousand atoms of sample, i.e., 1000 to 5000 ppm, or 0.1 to 0.5 atomic percent.  Elements with atomic numbers less than 6, (carbon) or elements present at concentrations under one to three atoms per thousand atoms of specimen cannot be detected.  Computerized aids simplify the identification of detected elements.  

In some instances x-ray energies of two elements overlap and may appear as a single spectral peak.  Specialized routines may help in discovering what elemental components may be contributing information into these peaks.  Other artifacts, called ESCAPE PEAKS, occur frequently in x-ray spectra.  These peaks must be correctly identified and removed from the analysis.  

ELEMENT DISTRIBUTION  

Once the detectable elements in a sample are identified, it may be possible to determine spatial distribution of constituent elements, provided a specimen matrix is NOT chemically homogenous.  Element distribution information may be formatted as LINE PROFILES or as ELEMENT MAPS.  

Line Profiles report relative x-ray intensity detected from a designated element as the electron beam traces a single line (i.e. transects) over the specimen detail of interest.  The result is a visual, semi quantitative comparison of element concentration at all points along the scanned line.  Line profiles are used to show changes in element concentration associated with diffusion or with boundaries such as structural layers, inclusions, or zones, layers, pockets or particles of contaminant.  

Element Maps document the pattern of a designated element's distribution over an area of specimen.  The "map" consists of an image formed by dots which correspond to x-rays collected from that element.  The dots are positioned spatially on the image so as to represent or "image" those locations in the specimen which contain detectable concentrations of the selected element.  Element map information is primarily yes/no data about the presence of an element in a given area of a specimen.  

QUANTITATIVE ANALYSES  

Quantitative x-ray spectroscopy suggests amounts or percentages of elements detected within a specimen.  Depending on characteristics of the specimen, an analysis can have anywhere from only "ball park" to very high accuracy.  

Specimen characteristics have profound effects on the validity of any quantitative result. The following questions are an aid in determining what kind of accuracy might be expected from a particular sample.  

1.   What elements do you expect to find  

•       The x-ray detector will not recognize any element below a specified atomic number.  Analysis can be performed only on those elements "seen", and those elements are assumed to make up 100% of the specimen.  This means the presence of lighter elements prevents accurate results as a consequence of their omission from the analysis.  
•       Specimens must be examined at high vacuum and will be bombarded with an electron beam.  Some elements may be volatilized under these conditions.  Volatilized elements may not be detected and cannot be quantified.  
•       X-ray emission from certain elements have overlapping energies.  When overlap occurs it may not be possible to distinguish which of two elements is present or if both are present.  A deconvolution algorithm may be employed which attempts to extract and separate information from two different elements forming a single peak. These are theoretical calculations and may not reflect the actual element concentrations involved. 

2.   What are the supposed concentrations of the elements in the sample?  

•       This is an important factor when information for trace elements is required.  To detect an element there must be one to five atoms of that element per thousand atoms of sample (0.1 to 0.5 atomic percent) or the element must be present at a concentration of at least 1.0% by weight.  Even if minimal requirements are met, all results will be governed by the rule that the lower the concentration of an element, the greater the error/uncertainty associated with its concomitant analysis figure.  

3.   On an elemental level, how homogeneous is the sample?  

•       With non-homogeneous specimens, information acquired over several sampling areas cannot be extrapolated to be representative of the whole.  Only when the specimen is homogeneous on a scale below 1 um in the x-y (surface) plane and 2 um in the z (depth) direction is it possible to approach a fully accurate analysis.  

4.   What is the sample's physical topography?  

•       Rough topography causes x-ray scattering and absorption to be unpredictable and results in loss of quality x-ray information.  High quantitative accuracy cannot be approached unless the sample is polished perfectly flat (surface roughness less than +/-1 um).  
•       Computations can be used which make some corrections for particulate specimens.  Since these corrections are derived by theoretical and modeling methods, results can carry an inherent uncertainty.  

5.   Do you have standards, or are standards available, for your sample?  

•       Standards are specimens similar to the sample being analyzed but which have a precisely known chemistry.  All elements expected in the analysis should exist in the standard at levels at least equal to those present in the specimen.  When a single standard is not available, it may be possible to use several standards to cover a range of elements.  It is also possible to complete an analysis without a standard for one constituent element.  
•       Quantitative analysis without a standard is equivalent to running an experiment without controls.  One might obtain useful information, but the conclusions drawn may not hold much validity and are difficult to defend.  

      To summarize:  If concentration accuracy approaching 100% +/-2% is required from an x-ray spectroscopic analysis, the sample must  

•       contain only detectable elements;  
•       be homogeneous at a scale below 1 um in the x-y (surface) plane and 2 um in the z (depth) direction.  
•       have a flat, polished surface;  
•       have appropriate standards which also meet all specified conditions.  

Within the levels of existing technology, these specifications hold true no matter what x-ray spectroscopy equipment is used or what operator does the analysis.  

Many samples may not conform to the strict limitations necessary for a "fully" quantitative analysis, so it is important to ask if a "fully" quantitative analysis is essential to answer the question one has about a sample.  If absolute numbers are required, then the sample must meet stated requirements or another method of assessing the chemistry must be employed.  If exact accuracy is not essential, specimen specifications can be relaxed and a number of analysis routines employed that can give good quantitative approximations from non-ideal specimens.  Since many SEM specimens will not be ideal candidates for x-ray spectrochemical accuracy, these analytical procedures are often most beneficial.  They include:  

RATIOS  

Ratios may be computed between peaks within a spectrum or between spectral peaks and background.  Ratio data may suggest compounds formed between elements.  Furthermore, ratio data permits comparative statements to be made, such as:  Sample B has twice as much barium as is contained in sample A, even if we do not know the exact amount of barium in either specimen.  Whether B contains 20% barium to A's 10%, or 80% to A's 40%, the proportion is the same.  

SPECTRAL MATCHING  

A spectrum acquired from a sample may be matched to other spectra stored in memory.  A qualitative match can be made either between only designated peaks or across the complete spectrum using a Chi Square analysis.  The result obtained is a list of "best fit" spectral matches with their associated Chi Square "goodness of fit" values. 

When standards with precisely known chemistry are available, their spectra and chemical composition data can be stored in memory.  The known concentrations of elements in the standard spectra may then be used to estimate element concentrations in the analyzed specimen using an algorithm which constructs curves from which the quantitative results can be derived.  This algorithm does not use matrix effect corrections employed in more "quantitative" algorithms, assuming instead that matrix effects are similar if the specimens are similar.  

STANDARDLESS ANALYSES  

Semi-quantitative algorithms may be used when standards are not available or do not exist. Computer generated model spectra of ideal specimens having identical chemistry as the sample, and examined under identical operating conditions, are constructed, ratioed to data acquired from the analyzed specimen, and element concentration values estimated. Matrix effect corrections are then applied to derive the result.  

ANALYSIS FROM STANDARDS  

Analysis against standards of exactly known chemistry is also possible and provides the best qualitative and quantitative accuracy. However, working against standards takes the greatest amount of analysis time. Spectra from the standards and the unknowns must be collected, stored, and analyzed under identical conditions of excitation, geometry, and data reduction. IT IS THE CLIENT'S RESPONSIBILITY TO PROVIDE BOTH THE "UNKNOWN" AND THE STANDARD SPECIMENS. The following criteria relate to the selection of standards.  

A.   Every standard must have exact and completely known chemistry.  

B.   Each standard specimen matrix must be a completely homogeneous mix of all elements in that standard.  

C.  You should have a minimum of three (3) standards for each element you may want to analyze for:  

      1.   At least one should be a pure element or compound standard.

      2.   At least one should probably be in the form of a powder.

      3.   At least one should approximate the matrix of the unknowns you plan to analyze (ie, metal, geological, powder, semiconductor, etc).  

D.  Concentration of the elements in the standard should be no less than 5 to 10%. We normally do not use standards with concentrations below 5%.  

E.   At least one standard should have a low concentration of the element of interest (ie, 5 to 10%). The other standards should have concentrations of the element equal to or greater than the concentration you expect in the unknowns.  

F.   All standards and unknowns should be analyzed at the same geometry and excitation to the extent the specimens will permit.  

G.  Avoid standards with peak overlaps unless you expect to analyze unknowns with similar peak overlaps. If your unknowns will have peak overlaps, at least one standard with similar peak overlaps will be very helpful.  

H.   You should have two spectra saved on disc for every standard. One should be the "raw" spectrum acquired from the standard. The other should be the "analyzed" version of the "raw" spectrum.  

I.    Clients are responsible for obtaining appropriate standards and controls and for obtaining and providing the fully and correct chemical analysis for each standard or control submitted. Analysis must be by a method(s) other than XES. EMF will reject inappropriate standards or controls.