ISO/TR 4550

FINAL DRAFT
Technical
Report
ISO/DTR 4550
ISO/TC 201
Surface chemical analysis — Surface
Secretariat: JISC
chemical analysis of bacteria and
Voting begins on:
biofilms
2025-10-13
Voting terminates on:
2025-12-08
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Reference number
ISO/DTR 4550:2025(en) © ISO 2025

FINAL DRAFT
ISO/DTR 4550:2025(en)
Technical
Report
ISO/DTR 4550
ISO/TC 201
Surface chemical analysis — Surface
Secretariat: JISC
chemical analysis of bacteria and
Voting begins on:
biofilms
Voting terminates on:
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
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Published in Switzerland Reference number
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ii
ISO/DTR 4550:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms. 1
5 X-ray photoelectron spectroscopy . 2
5.1 General .2
5.2 XPS on freeze-dried bacteria .2
5.3 Bacterial surface characterization by Cryo-XPS .4
5.4 Bacterial and biofilm surface characterisation by near-ambient pressure XPS .4
6 Fourier-transform infrared spectroscopy . 5
6.1 General .5
6.2 State of the art .6
6.3 Beyond state of the art .6
6.4 Quantitative analysis of triclosan uptake in E. coli biofilms by FTIR .6
7 X-ray fluorescence spectroscopy and related X-ray spectroscopy . 8
7.1 General .8
7.2 Calibration of quantitative XRF measurements .9
7.3 XRF analysis of bacteria and biofilms .9
8 Secondary ion mass spectrometry .11
8.1 General .11
8.2 SIMS imaging of dehydrated biofilms .11
8.3 SIMS imaging of frozen-hydrated bio-samples . 12
9 Raman-spectroscopy .13
9.1 General . 13
9.2 Dielectrophoresis-Raman analysis of bacteria in a liquid matrix .14
9.3 Raman biofilm analysis . 15
9.4 Quantitative Raman spectroscopy on freeze-dried bacteria .16
10 Super-resolution microscopy . 17
10.1 General .17
10.2 Single-molecule tracking techniques . .17
10.3 Single-molecule localization microscopy techniques .18
10.4 Highly inclined and laminated optical sheet microscopy . 20
11 Concluding remarks .21
Bibliography .25

iii
ISO/DTR 4550:2025(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 201, Surface chemical analysis.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO/DTR 4550:2025(en)
Introduction
Biofilms represent a predominant form of microbial life on our planet. These are aggregates of
microorganisms, which are embedded in a self-produced matrix formed by extracellular polymeric
substances (EPS). Biofilms are capable of forming on virtually every surface in aqueous, humid and non-
sterile environments. They can adapt to the most extreme environments from hot springs to frozen
glaciers, from very acidic to very alkaline environments. Biofilms can colonise nearly all interfaces and
affect many fields of life. Therefore, detailed knowledge of microorganisms enclosed in biofilms as well
as the chemical composition, structure, and functions of the complex biofilm matrix and their changes at
different stages of the biofilm formation and under various physical and chemical conditions is necessary.
Globally, a significant drive for research in microbial studies comes from the healthcare and biomedical
sectors, including diagnostics and pharmaceutical industries. Biofilm characterization is also relevant to the
needs of the medical devices industry by providing understanding of the barriers that need to be overcome.
Biofilm formation creates a problem in water and oil pipelines as well as microbial-induced corrosion in
marine environment. Biofilm characterization is the most important and fundamental activity both for
understanding the risk associated with the accumulation of reservoirs of antimicrobial resistant pathogen
build-up within biofilms that can affect healthcare (e.g. on indwelling devices such as catheters), domestic
(e.g. washing machines) and commercial (e.g. in food industry pipelines) environments. Consultations have
been carried with experts from major European initiatives, specifically New Drugs for Bad Bugs (ND4BB),
a large consortium funded by the Innovative Medicine Initiative (IMI), as well as the Joint Programming
Initiative on Antimicrobial Resistance (JPIAMR). From these discussions, the following key needs for
advancing measurement capability and metrology have been identified:
— well-controlled model systems to allow cross-platform measurement of bacterial components and
bacterial processes in single cells, suspended cellular aggregates and in biofilm communities;
— metrology for 3D chemical imaging of microbial samples allowing measurements with high sensitivity,
with high-spatial resolution at a single cell level and allowing qualitative measurements and sample
components detection and identification;
— reliable, reproducible, and traceable quantitative measurements of the vertical concentration profiles of
antibacterial agents in bacteria and biofilms;
— measurements to be performed in liquid and near ambient pressure necessitating innovation in
instrumentation;
— methodology, based on cryogenic preparation methods, to enable analysis of hydrated samples in the
vacuum of high-performance metrology instruments without ultrastructural reorganisation and
translocation of exogenous and endogenous molecules;
— advancements in measurement capabilities and metrology to image surface macromolecules, such as
porins or metal-transport proteins, to study the efflux mechanisms in bacteria and to give real-time
quantitative measurements of drug-uptake in bacteria and biofilms;
— signal enhancement strategies, such as surface nanofabrication, to aid the applicability of existing
analytical methods to the analysis of microbial samples;
— numerical modelling and algorithms to support measurement in complex biological environments.
This document is based on work of the 15HLT01 MetVBadBugs project funded by the European Metrology
Programme for Innovation and Research (EMPIR) under Horizon 2020. The MetVBadBugs project was
formulated in response to global challenge of antimicrobial resistance and the objectives of the project were
to advance the measurement capability by providing urgently needed essential metrology to quantitatively
measure and image the localisation of antibiotics and to understand the antibiotic penetration and efflux
processes in bacteria and biofilms. The project tested, advanced and developed a range of physical methods
and techniques with a focus on spectroscopical methods. This report gives an overview of these methods
and summarises their applicability to measurement of microbial samples.

v
FINAL DRAFT Technical Report ISO/DTR 4550:2025(en)
Surface chemical analysis — Surface chemical analysis of
bacteria and biofilms
1 Scope
This document gives an overview of a variety of physical and analytical methods by which bacteria, biofilms,
and the interaction of those with antimicrobial compounds can be analysed. For each technique a general
overview is given and its current state of the art. The strengths and limitations of each technique to measure
microbial samples are given alongside suggestions for future developments.
This document is intended as a guide and a starting point to more specific activities of ISO/TC 201, Surface
chemical analysis, in the future, which end in standardized procedures for measurements.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 18115-1, Surface chemical analysis — Vocabulary — Part 1: General terms and terms used in spectroscopy
ISO 18115-2, Surface chemical analysis — Vocabulary — Part 2: Terms used in scanning-probe microscopy
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 18115-1 and ISO 18115-2 apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Symbols and abbreviated terms
AFM atomic force microscopy
AST antibiotic susceptibility test
CLSM confocal laser scanning microscopy
DEP dielectrophoresis
EPS extracellular polymeric substances
FTIR fourier-transform infrared spectroscopy
HILO highly inclined and laminated optical sheet
MALDI matrix assisted laser desorption/ionization
MIC minimum inhibitory concentration

ISO/DTR 4550:2025(en)
MIR mid-infrared spectral window
NAP-XPS near-ambient pressure X-ray photoelectron spectroscopy
PALM photoactivated localization microscopy
SEIRA surface-enhanced infrared absorption spectroscopy
SIMS secondary ion mass spectrometry
SMT single molecule tracking
SNOM scanning near-field optical microscopy
STORM stochastic optical reconstruction microscopy
TOF-SIMS time of flight secondary ion mass spectrometry
UHV ultra-high vacuum
XAFS X-ray absorption fine structure
XRF X-ray fluorescence
XRS X-ray spectrometry
XPS X-ray photoelectron spectroscopy
5 X-ray photoelectron spectroscopy
5.1 General
X-ray photoelectron spectroscopy (XPS) is a widely used technique in surface analysis, providing both
qualitative and quantitative information of the sample surface. The probing depth is typically 5 nm to 10 nm
for XPS-measurements with a standard aluminium Kα X-ray source. The information depth is on the same
scale as the phospholipid-membrane and the length of exopolysaccharides anchored to the bacterial surface.
[1]
The relevant elements (typically carbon, oxygen, nitrogen and phosphorous) are easily detected and high-
resolution core level spectra reveal details about the binding environment of the atoms. Typical XPS high
resolution spectra of nitrogen, carbon and oxygen from bacterial samples are shown in Figure 1.
To prevent the emitted photoelectrons being scattered by gas molecules, XPS is traditionally performed
in ultra-high vacuum (UHV). This enables a controlled sample environment with minimal contamination,
but it puts restrictions on the type of samples that can be measured. Bacteria and biofilms are inherently
in a hydrated state, and therefore are only compatible with ultra-high vacuum after extensive sample
preparation, usually involving freeze drying or fast-freezing. With the development of near-ambient pressure
XPS (NAP-XPS), bacterial samples can be characterised with minimal sample preparation in various gas
environments such as air and water vapour. Subclauses 5.2, 5.3 and 5.4 provide a brief overview of surface
characterisation of biological samples by XPS.
5.2 XPS on freeze-dried bacteria
Freeze drying of bacterial cells is the most common sample preparation method to make bacteria compatible
with ultra-high vacuum. Bacterial cells in suspension are typically centrifuged and rinsed with water, then
[2]
cooled in liquid nitrogen and freeze-dried. Pembrey et al. investigated how sample preparation protocols
affect the cell surface properties. Parameters such as centrifugation speed, type of culture media and rinsing
media were varied. Physicochemical properties such as hydrophobicity were also investigated. For example,
it was found that physicochemical properties varied when using high-salt buffers compared to low-salt
buffers for washing or resuspension. The review article by van der Mei and Busscher also discusses how each
[3]
step of the sample preparation affects the surface composition. The authors conclude that although the

ISO/DTR 4550:2025(en)
extensive sample preparation causes variations in the obtained surface composition, the inherent biological
properties are the main cause for variations between bacterial strains.
Quantitative XPS-analysis reveal the elemental composition (excluding hydrogen and helium), which for
bacterial samples consists of mainly carbon, oxygen, phosphorous and nitrogen. The elemental composition
can be used to estimate the amount of the three main organic compounds found on the bacterial surface:
lipids/hydrocarbon-like compounds (CH), polysaccharides (PS) and proteins/peptides (Pr). The atomic
fractions for nitrogen and oxygen relative to carbon are calculated from the XPS-results and related to
the fraction of carbon from each of the compounds (CCH/C, CPS/C, CPr/C) by Formula (1), Formula (2) and
Formula (3):
[N/C]=0,279(CPr/C) (1)
[O/C]=0,325(CPr/C)+0,833(CPS/C) (2)
[C/C]=1=(CPr/C)+ (CPS/C)+(CCH/C) (3)
The fractions are derived from model constituents: glucan (C H O ) as a model for polysaccharides,
6 10 5
hydrocarbon (CH )n as model for hydrocarbon-lice compounds and a membrane protein of a Pseudomonas
[4]
fluorescens strain as model for protein/peptides . Since these formulae are based on dehydrated model
compounds, they are only valid for freeze-dried samples.
Key
X E ev
b/
Y intensity (arb. units)
Figure 1 — Representative oxygen 1 s, nitrogen 1 s, and carbon 1 s core level spectra of P.
fluorescens
[3]
In the review article by van der Mei and Busscher, the cell surface composition of 210 different strains are
listed: 131 Gram-positive, 39 Gram-negative and 40 yeast trains. To find out if species, strains or the Gram-
character was related to the surface composition, hierarchical cluster analysis was performed on the data
set. 31 of 33 staphylococci-strains were located in a distinct group, and yeast strains were distinguished
from the other groups by a low P/C and N/C followed by a high O/C surface concentration ratio. The Gram-
character of the bacterial strains was generally not revealed by the cluster analysis.
The review also summarises how the elemental composition determined by XPS is linked to biochemical
and physical properties such as surface charge and hydrophobicity. In general, it is found that the elemental
composition of Gram-positive bacteria correlates well with hydrophobicity, electrophoretic mobility,

ISO/DTR 4550:2025(en)
[5] [6]
isoelectric point and infra-red spectroscopy. , On the other hand, such relationships are not apparent
[7]
for Gram-negative bacteria. One reason for this can be that the surface of Gram-negative bacteria are in
general more complex than Gram-positive bacteria, so that various effects cancel each other out. In addition,
[2]
Gram-negative bacteria are more sensitive to sample preparation, since they lack a rigid cell membrane.
[8]
Generally, Gram-positive bacteria are more widely studied than Gram-negative bacteria. Rouxhet et. al.
review the literature available on XPS-analysis of bio-organic systems including food products, extracellular
polymer substances and bacteria. The methodology related to sample preparation and data analysis is also
discussed.
[9]
Ojeda et al. combined XPS, FTIR spectroscopy and potentiometric titrations to investigate the cell
surface of Aquebacterium commune, a common drinking water bacteria. Although FTIR measurements in
transmission mode also probe the interior of the cell, the cell wall constitutes 60 % to 70 % of the total
[3]
bacterial weight in a hydrated state . Further studies comparing FTIR-spectra from isolated cell walls and
[10]
whole cells also show that both samples mainly reflect the properties of the cell surface .
5.3 Bacterial surface characterization by Cryo-XPS
As an alternative to freeze-dry the bacteria, the sample can instead be fast-frozen and kept under cryo-
conditions during measurements. The rapid freezing causes the water in the sample to vitrify instead of
forming ice glass-crystals, which preserves the spatial structure of the cells. Since water is still present in
[4]
the sample, the Formulae (1) to (3) developed by Rouxhet et al are not valid. As an alternative approach
to obtain the amount of lipids, polysaccharides and proteins/peptide on the sample surface, an approach
[11]
using multivariate analysis has been developed by Ramstedt et al. The C 1 core level spectrum can be
described as a linear combination of three substance spectra in various ratios, one for polysaccharides, one
for peptides/proteins and one for hydrocarbon-like-compounds.
The protonation/deprotonation of nitrogen on the surface of Bacillus subtilis as a function of pH was tracked
[12]
by Leone et al. It was found that the N/P ratio had a maximum at pH 7, while the amount of protonated
nitrogen to neutral nitrogen decreased by increasing pH. It is possible that this sign of increased metabolic
activity at the physiological pH is related to the production of extracellular polymeric substances. In a follow-
2+
up publication, the surface composition of Bacillus subtilis is traced as a function of pH and Zn -exposure.
At higher pH, the peptide and polysaccharide-content decreases while the lipid-content increases. It is also
2+
found that the accumulation of Zn is stronger at higher pH. For a more complete overview concerning
[13]
Cryo-XPS measurements of biological, see the review by Shchukarev and Ramstedt .
5.4 Bacterial and biofilm surface characterisation by near-ambient pressure XPS
The focus has mainly been on planktonic bacteria harvested from solution for both freeze dried and cryo-
preserved bacteria. However, with NAP-XPS, not only vacuum-compatible samples can be investigated. This
provides more flexibility in sample preparation. Figure 2 shows a schematic of a NAP-XPS instrument. As a
regular XPS-instrument it has an X-ray anode as the radiation source and a hemispherical electron analyser.
To enable samples to be analysed in near-ambient pressure, while the sensitive instruments are still
under ultra-high vacuum, the X-ray source is separated from the analysis compartment by a silicon nitride
window. Further, the first aperture is only 300 µm in diameter and a three-stage differential pumping
system separates the sample environment and the electron analyser. With this set-up, planktonic bacteria
or biofilms grown on a substrate can be transferred directly from buffer or water-solution to measurement
chamber and characterised in air or water vapor. Measurements of Escherichia coli reveal that the carbon-
1) [14]
spectrum changes when the condition changes from 11 mbar water vapor to 1 mbar air . The carbon
components associated with single and double bonds to oxygen and nitrogen increased relative to aliphatic
carbon, which is probably due to a combined effect of drying and reorganisation on the cell surface. The
trend in the carbon spectrum is opposite to what is expected if it were due to radiation damage of the sample,
where one expects breakage of C-O and C-N bonds and therefore a decrease of intensity in the associated
[4]
spectra. Water absorption on phospholipid molecules representing a model biological membrane was also
[15]
studied in NAP-conditions. The effect of water absorption was mainly seen as a shift in binding energy
for the nitrogen and phosphorous photoelectron peak. Radiation damage is also addressed, which seems to
be a higher concern when using synchrotron radiation compared to X-ray anodes as the radiation source.
1) 1 bar = 0,1 MPa = 105 Pa; 1 MPa = 1 N/mm .

ISO/DTR 4550:2025(en)
Key
1 silicon nitride window
2 working distance ~ 300-600 μm
3 first aperture ⌀ 300 μm
4 electron analyser
5 detector
6 electron path focusing mirror
7 X-ray beam
8 sample
9 X-ray anode
SOURCE SPECS Surface Nano Analysis GmbH, adapted with permission from the authors.
Figure 2 — Schematic of an XPS with the typical modifications to adapt it to near-ambient pressure
measurements
6 Fourier-transform infrared spectroscopy
6.1 General
Fourier-transform infrared spectroscopy (FTIR) is a surface-analytical methodology for biochemical
[16]
analysis of tissues and other biological matrices (i.e. cells, viruses, bacteria) . As a non-invasive, non-
ionising and low-level readout technique, FTIR is considered to be highly specific for the biocompatible
-1 -1
analysis of biological specimens. Specifically, the mid-infrared spectral window (4 000 cm to 400 cm )
can be exploited for analysing molecular fingerprints. Characteristic energies and intensities of absorbance
[17]
bands of cellular macromolecules and other functional groups can be assigned, including carbohydrates,
[18] [19] [20],[21] [21] [21]
lipids, proteoglycans, collagens, nucleic acids and proteins . They can be used as markers
for probing the biochemical response of cells and tissues to different treatments and pathologies.
A targeted enhancement for efficient and sensitive detection of IR-active modes in the DNA-, phosphodiester-
and Amide I- and Amide II region, in conjunction with plasmonic sensing platforms, is enabled by surface-
[22]
enhanced infrared absorption spectroscopy (SEIRA) .
Combined with optical microscopy, hyperspectral chemical imaging can be conducted for correlating the
molecular IR response with the region of interest. FTIR-based hyperspectral imaging permits spectra to be
obtained from very small sample regions of interest, with a resolution down to less than 2 µm, laterally, and
is limited to a penetrative depth of approximately 10 µm due to detector saturation. Measurements can be
conducted either on solids, liquids, or on gaseous samples. Experiments preserving native conditions can

ISO/DTR 4550:2025(en)
be realised in a liquid cell or in a "near-native" fashion through cryo-sectioned or freeze-dried biological
material. Typical thicknesses of biomaterials to be investigated are monolayer cell cultures or 5 µm to 10 µm
[23]
thick tissues . Sample thickness, hydration and the interference caused by external substances such
as culture media can have an effect on the spectral output. Moreover, infrared spectra of cells and tissue
samples are prone to baseline distortions and mode shifts from light scattering which is a result of sample
[24],[25]
morphology .
6.2 State of the art
Infrared micro-spectroscopy has emerged as a rapid and inexpensive technique and has been used
[26]
successfully for the detection and identification of various biological specimens over decades . However,
its applicability in routine clinical diagnostics has not yet been established. Evaluative approaches on the
potential of this technique for rapid identification of bacterial susceptibility to specific antibiotics are under
[27]
debate, based on a qualitative biological assessment supported by computational classification methods .
6.3 Beyond state of the art
The determination of bacteria pathogeny and resistance to antibiotics is crucial and can be assessed by FTIR
spectroscopic analysis on the biological assessment (i.e. dose response) of micro-organisms. Antibiotics
inhibit the growth of harmful microorganisms and themselves can be harmful to health. Therefore, their
dose and the treatment duration requires careful judgement. To lay the foundation for a well-tolerated
and targeted medical therapy, quantitative measurement of antibacterial agents is needed to obtain the
minimum inhibitory concentration and exact point of action. Computational classification methods and
multivariate statistics tools can support reliable pattern-recognition combined with highly specific and
selective spectroscopies tools in correlation with structure-function.
6.4 Quantitative analysis of triclosan uptake in E. coli biofilms by FTIR
[28]
Within the scope of the MetVBadBugs project , one target was to probe the biocide uptake and quantification
inside the biomatrix by FTIR. This is challenging because FTIR spectroscopy requires reference materials or
a set of calibration samples in order to quantify antibiotic-uptake by using linear regression models. In this
project, different biocides in model biological surroundings and the characterisation of different biocides
accumulated in real a biological matrix were studied.
Infrared spectral fingerprints were successfully obtained from a wide range of biocides that act against
Gram-positive/negative bacteria and were compared with the biofilms' spectral characteristics, in order
to study spectral separation capability to the cellular environment itself (see Figure 3). It turned out that
most of the selected biocides can be distinguished from biological marker bonds. For calibration purposes,
two biocides accumulated in a model biomatrix were used to determine concentration-dependent mode
integrals.
ISO/DTR 4550:2025(en)
a) Scheme of a FTIR micro-spectroscopic setup for hyperspectral data acquisition

ISO/DTR 4550:2025(en)
b) Vibrational fingerprints of the biocides triclosan and linezolid and an E. coli biofilm
Key
-1
X wavenumber / cm
y v
1 IR microscope
2 hyperspectral imaging
3 triclosan
4 linezolid
5 biocides
6 E. coli biofilm
Figure 3 — FTIR micro-spectroscopic as a tool for measuring the uptake of biocides
The results from calibration samples clearly showed that the FTIR methodology is selective enough to gather
concentration-dependent calibration profiles for quantitative analysis for typical clinical concentrations.
7 X-ray fluorescence spectroscopy and related X-ray spectroscopy
7.1 General
In x-ray fluorescence spectroscopy (XRF), an inner-shell electron is excited by an incident x-ray photon,
with the ionized atom subsequently emitting a fluorescent photon. XRF analysis is a well-established, non-
destructive and multi-elemental analytical technique used in various fields of applications ranging from
materials science and semiconductor characterisation to geology and environmental issues as well as the
investigation of biological specimens. The emitted photon’s energy is specific to each outer-shell electron’s

ISO/DTR 4550:2025(en)
transition to the inner-shell vacancy. Thereby, the emitted fluorescence radiation is characteristic of the
element and can be identified by X-ray detectors which can be either an energy dispersive detector (e.g.
silicon drift detectors) or a wavelength dispersive spectrometer using a grating or a crystal as dispersive
element. The concentration or mass deposition of the elements can then be determined by a quantification
[29]
model that evaluates the intensity of the characteristic fluorescence radiation detected. Therefore, the
procedure is expected to be calibrated using a reference material or a dedicated calibration sample which is
very similar to the sample of interest. With this, the chemical analysis is traceable. This procedure prevents
quantification error induced by non-linear matrix effects which are associated, for example, with (X-ray)
absorption effects or secondary enhancement effects by photo electrons. This can become a major issue,
especially when dealing with biological samples with light elements.
[30][31]
In contrast to calibrated XRF analysis, reference-free XRF is possible. Here, physical traceability is
established using radiometrically calibrated instrumentation, e.g. well-known X-ray sources and detectors,
[32]
as well as knowledge on atomic fundamental parameters for quantification. Reference-free XRF can
[33][34]
not only contribute to the analysis surface contamination, but also the assessment of thickness and
[35]
composition of micro- and nanolayers as well as the composition of bulk specimens. Additionally, if the
angle of incidence of the exciting radiation is varied with respect to the surface of a flat sample, the resulting
X-ray standing wave field can also probe sub-surface regions depending on the incident photon energy and
angle. Because photons generally have a higher information depth than electrons, elemental depth profiling
by grazing incident XRF (GIXRF) enables the analysis of buried nanolayers and interfaces from a few to
several hundreds of nanometres below the surface for very flat samples. If combined with near-edge X-ray
absorption fine structure spectroscopy (NEXAFS), GIXRF also facilitates the analysis of different chemical
[36]
states of buried nanolayers .
Generally, XRF is an elemental sensitive technique, sample preparation as well as preplanning the series
of experiments is key for a successfully measurement campaign. For example, if sample preparation adds
an element to a system in which the same element is present and to be studied, there will most likely be a
deteriorated contrast dependent on the concentration of the additive. Furthermore, analysing the uptake
of a biocide or antibiotic (e.g. triclosan, a chlorine-containing antibacterial agent found in many consumer
and personal health-care products and used for surgical cleaning treatments) in a bacterial biofilm can be
complicated, because chlorine is also present as an ion in biological systems (cellular content 10 mM to 20 mM).
7.2 Calibration of quantitative XRF measurements
To calibrate quantitative XRS for biofilm characterization reference samples such as ionic liquids (1-butyl-
3-methylimidazolium iodide), C-matrices model membranes with added iodo-benzoic-acid, PVD-iodine,
and triclosan with 5 % and 10 %, implantations of iodine or other ions of interest, e.g. Ag to track silver
sulfadiazine, into low Z crystal matrices have proven to be suitable. Planarly homogeneous samples can be
used as calibration samples using reference-free utilizing XRS analysis for the calibration procedure as a
[34],[37],[38]
primary method because it yields the absolute mass deposition . Using these reference samples
enables a traceable calibration of techniques like XPS, FTIR or Raman spectroscopy for absolute quantitative
analysis.
7.3 XRF analysis of bacteria and biofilms
This subclause presents a brief overview of recent XRF based studies. Most publications are based on (high-
resolution) X-ray (scanning) fluorescence spectroscopy used as a high-resolution imaging tool to extract
spatial as well as chemical information to monitor metals in biological systems (see Figure 4). As stated in
Reference [39], X-ray fluorescence analysis can provide high trace element sensitivity, image whole cells and
quantify elements on a per cell basis. It usually does not require sectioning. Cells or bacteria can be analysed
close to their natural, hydrated state by using cryogenic sample preparation approaches. In Reference [40],
Dynes et al. investigated the spatial distribution (at 50 nm resolution) as well as the chemical speciation of
metals in a microbial biofilm cultivated from river water. In a similar study by Hunter, Hitchcock, Dynes et
[41]
al. , the spatial distribution of iron species throughout Pseudomonas aeruginosa biofilms was analysed to
assess the influence of chemical heterogeneity on biomineralization. High-energy X-ray fluorescence and
X-ray absorption fine structure (XAFS) measurements on single pseudomonas fluorescens strain NCIMB
11764 cells were carried out in Reference [41]. Differences between planktonic and adhered cells in the
morphology, elemental composition and sensitivity to Cr(VI) of hydrated cells at spatial scales of 150 nm
were presented.
ISO/DTR 4550:2025(en)
Key
X energy/ keV
Y intensity / counts
1 (biological) sample
2 absolute mass deposition and/or elemental map of sample
3 (scanning) X-ray fluorescence analysis on (biological sample)
4 deconvolution and quantification of spectra
5 data
6 fit
7 background
SOURCE Reference [43], adapted with permission from the authors.
Figure 4 — Basic principle of (quantitative scanning) X-ray fluorescence analysis on biological tissue
If scanning X-ray fluorescence microscopy can be combined with confocal laser scanning microscopy,
transmission electron microscopy or others and exactly the same region of a sample can be aligned and
measured, it is possible to derive a detailed multi-microscopic (highest-resolution, elemental, chemical and
[44] [45]
functional information) correlative map of a biofilm structure and composition. Wolfe-Simon et al.
went even further and combined scanning electron microscopy, nanoSIMS and micro-X-ray absorption near-
edge spectroscopy to study a bacterium, strain GFAJ-1, isolated from Mono Lake, California, that is able to
substitute arsenic for phosphorus to sustain its growth. Other notable XAFS studies on bacterial cell walls
are References [46] and [47].
A different approach is taken in Reference [48], where Schneck et al. quantitatively determined ion
distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence.
Another established method to analyse bacteria is X-ray crystallography, a technique in which an X-ray beam
is diffracted by a crystalline sample, the corresponding electron densities are calculated from the diffraction
pattern, and finally the mean positions of atoms as well as their chemical state are derived from the electron
densities. This method allows for the investigation of attraction, attachment, insertion and orientation of
[49]
peptides, as well as the orientation of lipids and the thickness and integrity of the lipid bilayer of bacteria,
[50] [51] [52]
bacterial cell wall remodelling processes and in general research on (bacterial) enzymes , .

ISO/DTR 4550:2025(en)
8 Secondary ion mass spectrometry
8.1 General
Secondary ion mass spectrometry (SIMS) imaging is a label-free and matrix-free method that enables
multiplexed chemical imaging of sample surfaces. Through scanning a focused beam of ions on the sample
surface, SIMS imaging can be achieved by acquiring spatially resolved mass spectra and generating
ion images by any signals in mass spectra. In addition, by ablating layers of the sample with sputter ion
beams, 3D molecular images can be reconstructed. With the improvement of primary ion sources and mass
spectrometers, SIMS is increasingly used for biological samples, such as brain tissues, neurons, single cells
[53]
and bacteria . With small cluster or monatomic ion beams, SIMS offers a lateral resolution better than
[54]
500 nm, enabling imaging of single cells at sub-cellular level. The introduction of polyatomic and cluster
ion sources has significantly improved the signal
...