ISO 23124:2024

International
Standard
ISO 23124
First edition
Surface chemical analysis —
2024-04
Measurement of lateral and axial
resolutions of a Raman microscope
Analyse chimique des surfaces — Mesurage des résolutions
latérale et axiale d'un microscope Raman
Reference number
© ISO 2024
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General information . 1
4.1 Outline of the method . .1
4.2 Lateral resolution . .2
4.3 Axial resolution .2
5 Sample requirements . 3
5.1 Selection of the sample and sample requirement to measure the lateral resolution .3
5.2 Selection of the sample and sample requirement to measure the axial resolution .3
6 Experimental parameters to be specified . 3
6.1 Overview .3
6.2 Numerical aperture of objective lens .3
6.3 Size of confocal pinhole or the optics which works with similar function .4
6.4 Setting the parameters before the operation of the instrument .4
7 Data acquisition . 5
7.1 Data collection and analysis for lateral resolution .5
7.2 Data collection and analysis for axial resolution .5
7.3 Recording of the data . .5
Annex A (informative) Case study using dispersed carbon nanotubes and suspended graphene
as samples . 7
Bibliography .11

iii
Foreword
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iv
Introduction
The Raman microscope is usually built on an optical micro-spectroscopy platform and integrated with laser
input, laser line filter and spectrometer output. The laser focus is scanned on the sample and the Raman-
scattered photons are collected from each pixel to record the full spectrum. Raman spectral images contain
a variety of spectral information, such as the intensity, peak position, or peak width of certain Raman bands.
Spatial resolution is one of the main specifications of the Raman microscope. However, the definition and
the measurement procedures largely vary depending on the manufacturers of the Raman microscope,
therefore the general assessment of the spatial resolution has been limited. In this document, we provide a
standardized protocol that describes the measurement of the spatial resolution of a Raman microscope by
performing simple measurements using specific standard specimens.
In the Raman microscope, spatial resolution includes the lateral resolution and axial resolution. For this
evaluation, there are several methods, such as the straight edge method, narrow line method and grating
method. This document describes only the narrow line method for evaluation of the spatial resolution for
Raman measurement. A case study of the measurement is provided in Annex A.

v
International Standard ISO 23124:2024(en)
Surface chemical analysis — Measurement of lateral and axial
resolutions of a Raman microscope
1 Scope
This document describes a method for measuring the spatial resolutions, lateral and axial, of the Raman
microscope.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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/
3.1
numerical aperture
NA
number originally defined by Abbe for objectives and condensers, which is given by the expression nsin(u),
where n is the refractive index of the medium between the lens and the object and u is half the angular
aperture of the lens
[SOURCE: ISO 10934:2020, 3.1.10.4, modified — Note 1 to entry removed]
3.2
full width at half maximum
FWHM
region of the axial response symmetrical to the maximum peak where the signal falls to one-half of the
maximum peak signal
[SOURCE: ISO 25178-607:2019, 3.8]
3.3
signal-to-noise ratio
ratio of the signal intensity to a measure of the total noise in determining that signal
[SOURCE: ISO 18115-1:2023, 3.23, modified — Notes to entry have been removed]
4 General information
4.1 Outline of the method
Spatial resolution is the capacity for imaging fine detail, which is determined by the minimum spatial
resolution of two-point objects required for their observation as distinct objects. Various methods have

[1]
been proposed to determine the resolution, such as the straight edge, narrow line, and grating methods .
This protocol describes a measurement based on the narrow line method.
In confocal microscopy, the resolution is described by the full width at half maximum (FWHM) of the
[2][3]
confocal point spread function (PSF) using small objects . Besides FWHM of PSF, the FWHM of the
line spread function (LSF) with line-shaped objects for lateral resolution and spread functions of ultra-
thin or low reflective materials as well as LSF for axial resolutions are also applicable to determine spatial
resolutions of confocal microscopy. In the narrow line method, these features are available to evaluate the
lateral and axial resolution.
4.2 Lateral resolution
For measurement of lateral resolution, small objects, or line-shaped objects such as carbon nanotubes
(CNTs) or semiconductor nanowires with widths of tens of nm that provide a strong Raman signal are
recommended. The apparent size of small or diameter of line-shaped objects in a Raman map is a legitimate
measure of the lateral resolution if the actual diameters of line-shaped objects are less than one-fourth of
[2]
the lateral resolution . See Figure 1.
Key
1 objective lens
2 laser light
3 sample
4 scan direction
5 FWHM
Figure 1 — Scanning of laser focus over a line-shaped object for measurement of lateral resolution
4.3 Axial resolution
For measurement of axial resolution, small, line-shaped, (air-suspended) ultra-thin or low reflective
materials are required as standard specimens. For small, line-shaped, ultra-thin or low reflective materials,
the size, diameter, thickness, or penetration depth, respectively, shall be smaller than one-fourth of the axial
[1]
resolution, respectively . See Figure 2.

Key
1 objective lens
2 laser light
3 sample
4 scan direction
5 FWHM
Figure 2 — Scanning of laser focus through suspended ultra-thin materials for the measurement of
axial resolution.
5 Sample requirements
5.1 Selection of the sample and sample requirement to measure the lateral resolution
It is known that an object size less than one-fourth of the PSF or LSF contributes to the size shown in the Raman
[2]
image by less than 1 % . Considering the uncertainty of the whole measurement, this discrepancy may be
regarded to be negligible. Therefore, the size of small objects or the diameter of the line-shaped objects for
the measurements of lateral resolution shall be chosen to be less than one-fourth of the expected resolution
value. The size or diameter of the reference sample should preferably be verified by independent traceable
measur
...