1、BSI Standards PublicationBS ISO 18337:2015Surface chemical analysis Surface characterization Measurement of the lateralresolution of a confocalfluorescence microscopeBS ISO 18337:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 18337:2015.The UK participati
2、on in its preparation was entrusted to TechnicalCommittee CII/60, Surface chemical analysis.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible f
3、or its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 81132 6ICS 71.040.40Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy a
4、nd Strategy Committee on 30 June 2015.Amendments issued since publicationDate Text affectedBS ISO 18337:2015 ISO 2015Surface chemical analysis Surface characterization Measurement of the lateral resolution of a confocal fluorescence microscopeAnalyse chimique des surfaces Caractrisation des surfaces
5、 Mesurage de la rsolution latrale dun microscope confocal fluorescenceINTERNATIONAL STANDARDISO18337First edition2015-06-15Reference numberISO 18337:2015(E)BS ISO 18337:2015ISO 18337:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015, Published in SwitzerlandAll rights reser
6、ved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
7、 at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 18337:2015ISO 18337:2015(E)Foreword ivIntroduction v1 Scope . 12 Term
8、s and definitions . 13 Symbols and abbreviated terms . 14 General 14.1 Background information 14.2 Types of CFM operation 24.2.1 General 24.2.2 Stage scanning CFM . 24.2.3 Laser scanning CFM 24.2.4 Spinning disk CFM 24.3 Parameters that affect the lateral resolution of a CFM . 34.3.1 General 34.3.2
9、Objective lens 34.3.3 Detection pinhole size and focal length of the tube lens . 34.3.4 Collimation and purity of the laser illumination beam 34.3.5 Polarization of the laser illumination 34.3.6 Excitation and emission wavelengths 34.3.7 Image contrast . 35 Measuring the lateral resolution by imagin
10、g a small object . 35.1 Background information 35.2 Selection of the sample and sample requirements 45.3 Setting the parameters prior to operating the instrument . 45.4 Data collection and analysis 55.4.1 Selecting a proper spot . 55.4.2 Extracting a line profile using band-average process 55.5 Reco
11、rding the data . 6Annex A (informative) Sample preparation and example of data and analysis . 7Bibliography .10 ISO 2015 All rights reserved iiiContents PageBS ISO 18337:2015ISO 18337:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standa
12、rds 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 organ
13、izations, 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 f
14、urther maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/direc
15、tives).Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the
16、Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related
17、to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 201 Surface chemical analysis.iv ISO 2015 All rights r
18、eservedBS ISO 18337:2015ISO 18337:2015(E)IntroductionConfocal fluorescence microscopes (CFMs) are laser scanning confocal microscopes (LSCMs) operated in a fluorescence imaging mode so as to obtain a fluorescence image of a sample. Fluorescence is the light emitted by a molecule or solid lattice dur
19、ing relaxation after undergoing photon absorption and electronic excitation. The fluorescence wavelength, intensity and spectral shape are specific to the electronic structure of the material; therefore, fluorescence spectroscopy and imaging techniques are useful for chemical characterization and an
20、alysis. Among the optical imaging and spectroscopy tools, CFM yields a high spatial resolution that is advantageous for analysing nanomaterials and thin films. The spatial resolution is one of the most important performance factors for a CFM.The spatial resolution of a technique refers to the maximu
21、m resolvability of two adjacent objects. This value is often characterized in different ways by the manufacturers. The spatial resolution of a CFM is characterized by both the lateral and axial resolution, which have different values and are not necessarily dependent on one another. In this Internat
22、ional Standard, one convenient and effective method for measuring the lateral resolution of a CFM is presented. This method is suitable for use by non-expert operators. ISO 2015 All rights reserved vBS ISO 18337:2015BS ISO 18337:2015Surface chemical analysis Surface characterization Measurement of t
23、he lateral resolution of a confocal fluorescence microscope1 ScopeThis International Standard describes a method for determining the lateral resolution of a confocal fluorescence microscope (CFM) by imaging an object with a size much smaller than the expected resolution.2 Terms and definitionsFor th
24、e purposes of this document, the following terms and definitions apply.2.1point spread functionresponse of an imaging system to a point source or point object2.2lateral resolutiondistance measured either in the plane of the sample surface or in a plane at right angles to the axis of the image formin
25、g optics over which changes in composition can be separately established with confidenceNote 1 to entry: See Reference1.3 Symbols and abbreviated termsOL objective lensAPD avalanche photodiodeFWHM full width at half-maximumCFM confocal fluorescence microscopeNA numerical aperturePSF point spread fun
26、ctionQD quantum dotPMT photomultiplier tube4 General4.1 Background informationLaser scanning confocal microscopes (LSCMs) scan a tightly focused laser beam over a sample and record the optical intensity at each pixel to form a two-dimensional image. LSCMs have a pinhole in front of the photo detecto
27、r or a spectrometer input slit at the conjugate focal plane of the laser focus at the sample. Light originating from the non-focal plane is largely prevented from reaching the photo detector. Confocal configurations significantly improve the contrast in an image, and the spatial resolution may be in
28、creased2.INTERNATIONAL STANDARD ISO 18337:2015(E) ISO 2015 All rights reserved 1BS ISO 18337:2015ISO 18337:2015(E)CFM is one of the most widely used LSCM operation modes because it provides a fluorescence image or spectrum. In the fluorescence mode, the incident laser light is blocked by a long pass
29、 filter, and only the Stokes-shifted fluorescence light is detected by the photo detector or spectrometer. An image is formed, depending on the fluorescence wavelength, and the image displays good contrast compared to images obtained through other optical imaging techniques. Imaging multiple colours
30、 in one image is also possible.The spatial resolution is one of the most important features characterizing the performance of an LSCM or CFM. The lateral resolution and axial resolution must be determined separately and must be treated independently. The lateral resolution is important especially wh
31、en a CFM is used for the imaging and chemical analysis of thin films or nanoscale objects, in which the axial dimension is significantly less than the typical value of the axial resolution of a CFM.The spatial resolutions of instruments tend to be characterized in different ways by different manufac
32、turers. This work provides one convenient and effective method for measuring the lateral spatial resolution of a CFM instrument so as to be suitable for use by a non-expert operator. The terms and analysis procedure described here are according to ISO 185163.4.2 Types of CFM operation4.2.1 GeneralBe
33、low, we describe different modes of CFM operation according to the laser focus scanning technique with respect to the sample. This International Standard principally treats stage scanning-type and laser scanning-type CFMs.4.2.2 Stage scanning CFMStage scanning CFMs are characterized by a moving stag
34、e that implements a scanning function while the laser focus remains unmoved with respect to the microscope frame. Because the optics involved in the CFM are stationary, the beam path is simple and nearly maintenance-free. Aberrations or drift in the optical alignment are minimized. The scan area siz
35、e is limited only by the mechanical movement of the sample scanning stage, and allows for large-area scans.Scanning speeds in these CFMs are relatively slow, and the sample may be affected by any rapid movements of a scanning stage and, therefore, may not be suitable for imaging delicate cells.4.2.3
36、 Laser scanning CFMOff-axis beam scanning techniques can incur aberrations that degrade the image resolution. A fast oscillating mirror set may be used to scan a beam across a sample more rapidly than is possible in stage scanning-type CFM. The scanning rate can sometimes reach values of a few kHz l
37、ine scan speed. In this approach, the sample does not move, thereby preserving the condition of the sample.Because the optical ray is off-axis during laser focus scanning, some aberrations may be introduced into the image. The need for a scanning head makes this technique more complicated than techn
38、iques based on stage scanning. The size of a scan area is limited because an objective will admit off-axis laser light within only limited angle. The size of a scanning area also depends on the magnification of the objective. The use of low magnification objectives (resulting in a large scan area) w
39、hich at the same time have a high NA (which results in a good collection efficiency and an improved resolution) is preferential in this case.4.2.4 Spinning disk CFMHigh-speed spinning disks involve multiple laser foci and conjugated detector pinholes.2 ISO 2015 All rights reservedBS ISO 18337:2015IS
40、O 18337:2015(E)4.3 Parameters that affect the lateral resolution of a CFM4.3.1 GeneralLateral resolution assessments are made by measuring the lateral size of a PSF on a CFM. The fundamental limit in the spatial resolution of a light microscope is given by:Abbe Resolutionx,y= /2NA, Abbe Resolutionz=
41、 2/NA2Comprehensive reviews of the relevant parameters and an experimental protocol for determining the size of a PSF are available elsewhere2,5,6,7.4.3.2 Objective lensThe objective lens (OL) directly affects the lateral resolution of a CFM image because the size of the laser focus and the collecti
42、on volume are determined by the NA of the OL.4.3.3 Detection pinhole size and focal length of the tube lensThe confocal pinhole in front of a photo detector eliminates light originating outside of the focal volume in the sample. Smaller pinholes increase the resolution; however the amount of light t
43、hat contributes to the image is also reduced, which can reduce the contrast in the image. Therefore the optimum size of the detection pinhole for a particular sample and application must be selected with the discretion of the operator. The imaged size of the detection pinhole on the sample plane by
44、the tube lens and the OL is more important than the actual size of the detection pinhole. Therefore the magnification of the OL and the tube lens should also be specified.4.3.4 Collimation and purity of the laser illumination beamThe expected performance of an OL can be achieved provided that the il
45、lumination beam is collimated and fills the back aperture of the OL.4.3.5 Polarization of the laser illuminationThe polarization of the input laser affects the shape and size of a PSF. Linearly polarized illumination tends to elongate the PSF along the direction of polarization8.4.3.6 Excitation and
46、 emission wavelengthsThe wavelength of the light used in CFM has the direct effect on the size of PSF as described by Abbe resolution in 4.3.1. Furthermore, in the fluorescence imaging, the emission wavelength is different from (longer than) the excitation wavelength and this can lower the resolutio
47、n2.4.3.7 Image contrastGood contrast in a CFM image is required for the accurate measurement of the lateral resolution. The cleanness of the optics, signal strength, noise, sensitivity of the photo detector, throughput of the beam path, and beam polarization are empirical factors that affect the con
48、trast of a CFM image.5 Measuring the lateral resolution by imaging a small object5.1 Background informationA very small object may be imaged to estimate the lateral resolution of a CFM. This approach is advantageous because one can obtain a two-dimensional profile that directly shows the PSF of the
49、CFM from a single CFM image. The lateral resolution can be defined as the full width at half-maximum (FWHM) of the PSF. Because the finite size of a small object contributes to the observed size of the ISO 2015 All rights reserved 3BS ISO 18337:2015ISO 18337:2015(E)object in the CFM image, the observed image of the object represents the convolution of the PSF and the spatial distribution of the small object. A wide range of samples may be used in this method, including fluorescent nanoparticles, such as light-emitting pol
