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Applied Surface Science 281 (2013) 89– 95

Contents lists available at SciVerse ScienceDirect

Applied Surface Science

j ourna l ho me page: www.elsev ier .com/ locate /apsusc

ar field optical nanoscopy: How far can you go in nanometricharacterization without resolving all the details?

aul C. Montgomery ∗, Bruno Serio, Freddy Anstotz, Denis Montaneraboratoire des Sciences de l’Ingénieur, de l’Informatique et de l’Imagerie (ICube), UDS-CNRS, UMR 7367, 23 rue du Loess, 67037 Strasbourg, France

a r t i c l e i n f o

rticle history:vailable online 22 February 2013

eywords:anoscopyar field imagingiffraction limitedanomaterialsiomaterials

a b s t r a c t

In the development of nanomaterials and biomaterials, new characterization techniques are required thatovercome the challenges presented by the increasing dimensional ratio between the different entities tobe studied and the growing complexity introduced by the use of heterogeneous materials and technolo-gies. Diffraction limited far field optical nanoscopy techniques are receiving growing interest becauseof their ability to detect nanometer structures over very large fields and at high speed. We present aclassification scheme of the different types of optical nanoscopy techniques. In particular, we highlightfour categories of far field diffraction limited techniques based on increasing the contrast, measuring thephase, using deconvolution and using nano-markers. We demonstrate that by increasing the power of

ano-characterization detectability, observability or measurability, a wealth of information concerning nanometric structuresbecomes available even though all the lateral details may not be resolved. For example, it is possible todetermine the presence, the structure and orientation of nanostructures, to measure their density, posi-tion and 2D and 3D distribution and to measure nanometric surface roughness in bulk materials, surfaces,nano-layers, soft matter and cells. These techniques conserve all the advantages associated with classical

imag

imaging such as real time

. Introduction

Two of the challenges facing characterization in the devel-pment of new nano- and bio-materials and structures are theimensional ratio between the basic nano-structures and the fin-

shed system and the growing complexity due to the use ofnhomogeneous materials and different technologies. In the casef biomaterials, for example in layers of hydroxyapatite, the min-ral part of bones and teeth, morphological, chemical, optical andechanical information is necessary at several scales, from the nm

evel of the basic crystals to the millimetre level for the functionalaterial [1]. Such materials may also be incorporated with poly-ers or metal alloys for implants or on microelectronic circuits for

iochips.For high resolution imaging of the basic elements at the

anometre level, electron microscopy and near field microscopyre the techniques of choice. But as with all techniques, whileeing well adapted to certain applications, their limitations makehem unsuitable for others. For example, in electron microscopy,

he electron beam and the vacuum or near-vacuum conditions cane destructive for many types of samples. In near field microscopy,oint by point scanning limits the measurement bandwidth and the

∗ Corresponding author. Tel.: +33 388106231; fax: +33 388106230.E-mail address: paul.montgomery@unistra.fr (P.C. Montgomery).

169-4332/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsusc.2013.02.029

ing, non-invasiveness, non-destructiveness and ease of use.© 2013 Elsevier B.V. All rights reserved.

field of measurement. The physical presence of the tip also leads tomeasurement biases and a restriction to surface or near surfacecharacterization.

Far field optical microscopy has received renewed interest inrecent years for several reasons, not the least being because ofthe availability of high resolution and high speed cameras andimage processing that allow quantitative and real time analysis.In addition, the development of new super-resolution techniquesmakes it possible to go well beyond the limits of diffraction. Finally,there is the realization that the measurements made with far fieldimaging can contribute significantly to the gaining of a deeperunderstanding of the structural, physical and chemical propertiesof nano-materials, sensors and systems without having to resolveall the details.

The different approaches mentioned concerning near field, farfield and super-resolution optical techniques generally fall underthe term of nanoscopy. One thing that can be remarked whenapproaching this new field of nanoscopy is that the large num-ber of different techniques that exist can make it quite confusingwhen considering their performance and limitations. There are sev-eral different ways of classifying nanoscopy techniques. In Fig. 1we propose one particular classification scheme that although not

exhaustive, can help in better distinguishing between the differentfamilies of techniques that exist. A first level of classification con-cerns the distance at which the optical information is obtained, inthe far field, with an imaging objective, or in the near field with a

90 P.C. Montgomery et al. / Applied Surface Science 281 (2013) 89– 95

Fig. 1. The four sub-divisions of diffraction limited far field techniques (marked in blue) in the context of a global classification scheme for optical nanoscopy techniques.( rred to

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hysical probe placed in the nanometric vicinity of the surface suchs SNOM (scanning near-field optical microscopy).

Amongst the far field techniques, a second level of classifica-ion can be made concerning the lateral resolution attained, eitherimited by diffraction or by the super resolution technique used too beyond such limits. Resolving lateral details below the wave-ength of the light used while remaining in far field conditions, cane achieved by diffraction tomography (or DHM, digital holographicicroscopy), SIM (structured illumination microscopy), I5M, 4pi or

TED (stimulated emission depletion).The latter 3 techniques give the highest resolutions, from

00 nm down to 30 nm. In I5M and 4Pi fluorescence microscopy [2],uper-resolution is attained using the coherent addition of spheri-al wavefronts from two high numerical aperture objectives placedn opposite sides of the sample. In both cases, the central spot ofhe point spread function (PSF) is sharpened, leading to a 5–7-foldmprovement in the axial resolution of ∼100 nm. The characteris-ics of I5M [3] are the use of incoherent illumination, wide fieldetection and a stronger signal leading to faster image acquisi-ion but with the presence of more artefacts due to the strongeridelobes in the PSF.

In 4Pi microscopy on the other hand, the use of coherent illu-ination and pin-hole detection leads to less artefacts due to the

maller sidelobes along the optical axis of the PSF, but is slower dueo the need for point scanning. A further improvement in resolutionan be obtained in 4Pi microscopy by means of stimulated emissionepletion (STED) [4] using a saturated depletion of the fluorescenttate of marker molecules. One wavelength is used to create fluo-escence over a small diffraction spot and then a doughnut shapeding at another wavelength is used to deplete specific regions of theample so as to leave a much smaller central focal spot, effectivelyeducing in width the central spot of the PSF and resulting in a lat-ral resolution of 30 nm. It should be noted nonetheless, that theest resolutions are only attainable in fluorescence microscopy.

The second category of far field techniques concerns those thatre diffraction limited and that can be sub-divided into four cat-gories according to the method used to give the nanometricensitivity:

. Increasing the contrast by means of the illumination, the phase

or the polarization.

. Measuring the phase by interferometry.

. Using deconvolution in sub-pixel metrology techniques.

. Using nano-markers such as fluorochromes or gold nano-particles.

the web version of the article.)

It is this particular category of far field, diffraction limited tech-niques (marked in blue/grey boxes in Fig. 1) that is the subject ofthis paper. We provide some of the answers to the question of justhow far it is possible to go in nano-characterization without resolv-ing all the details. While being diffraction limited, these techniquescan nonetheless be used to obtain important information fromnano-structures while conserving all the advantages of classicalimaging [5]. For example, characterization can be performed overwide fields of hundreds of micrometres and even millimetres andat a very high rate that often allows real time measurement. The useof an optical probe is also non-invasive, non-destructive and non-toxic for living organisms. We pointed out the advantages of suchfar field nanoscopy techniques in several papers at the beginning ofthe 1990s [5–7] but it is only in more recent years that the idea hasbecome more popular. Such a review and identification of the basicprinciples involved in far field nanoscopy in the context of today’snano- and biomaterials could be useful to stimulate the develop-ment of new, powerful instrumentation. In the four sections thatfollow, we therefore describe a selection of far field, diffractionlimited nanoscopy techniques using contrast, phase measurement,deconvolution and nano-markers to illustrate some of the princi-ples behind them for performing nano-characterization.

2. Nanoscopy using high contrast for detection of nmstructures

When the size of an object under a conventional optical micro-scope is smaller than the Rayleigh limit, the intensity decreasesrapidly to the point of the object becoming no longer visible. Thiscan be described in terms of Rayleigh scattering; when the objectsize d � �, the intensity of the scattered light decreases as a func-tion of d6. But since scattering is uniform in all directions, one wayof making nano-particles visible in the far field is simply to increasethe contrast. For images containing small features that are presenton a large uniform background, the contrast is given by the Webercontrast, CW:

CW = IMax − IMin

IMin(1)

where IMax is the maximum intensity (intensity of the features)and IMin is the minimum intensity (background intensity). For an8 bit image depth, CW therefore varies from 1 to 255. In imageshaving structures containing equivalent bright and dark features,

P.C. Montgomery et al. / Applied Surface Science 281 (2013) 89– 95 91

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ig. 2. The observation of nano-structures in crystals of GaAs using near IR microscoeld microscopy (P. Montgomery 1990). (b) Cloud of 10 nm nano-particles (indicate

I, 1990). (c) Extended atomic sized dislocation path made visible by phase contrast

he contrast is given by the Michelson contrast, CM, which variesetween 0 and 1 and is defined by:

M = IMax − IMin

IMiax + IMin(2)

.1. Dark field microscopy

In classical dark field microscopy [5,6] an increase in contrasts obtained by illuminating the sample with highly inclined lightays that do not enter the objective lens, leaving only the lightcattered by the sample details. The image in Fig. 2(a) shows theddition of three images taken at different depths in a sample ofaAs using near IR dark field microscopy. The contrast of the parti-les is fairly low (CW = 1–2) since they are near to the resolution limitf about 1 �m in size and the background intensity is far from zeroIMin = 20–80 grey levels) due to light diffused from scatterers abovend below the observation plane. But after image processing andolour coding, the result gives a 3D impression of the distribution oftrings of microprecipitates that in fact decorate a series of loopedislocations that are atomic in size. While nano-structures are notirectly visible in these conditions, this result illustrates how it isossible nonetheless to observe them indirectly in the presencef the microprecipitates that act as “native-markers” and whenhey are extended beyond the lateral resolution limit of the micro-cope. This example could therefore have been used in Section 5 tollustrate the use of nano-markers.

A novel way of considerably increasing the contrast in dark fieldicroscopy is by using a thin beam of laser illumination at 90◦ to the

irection of observation with laser scanning tomography (LST) tovoid illuminating the scatterers above and below the observationlane. The results in Fig. 2(b) show just what can be achieved with

his technique. A near IR (� = 1 �m) laser beam has been focusedn the cleaved face of a crystal of GaAs which is then scanned touild up an image of the distribution of defects. While most of thecatterers are micrometre sized microprecipitates, the fainter ones

) Strings of 1 �m sized microprecipitates decorating looped dislocations using darkrow) using LST (1 mm × 1 mm) (M. Castagné & P. Gall-Borrut, IES, Univ. Montpellierscopy (P.C. Montgomery, IES, Univ. Montpellier II, 1989).

(indicated by an arrow) consist of 10 nm sized microprecipitates (asverified by TEM) which appear after annealing [6,7]. The nanopar-ticles act like point sources that are visible individually if theirdensity is less than 109–1010 cm−3 or as a continuous backgroundlevel if their density is greater. While LST is very useful for measur-ing defect densities in bulk crystals, it is not suitable for analysingdefects in thin layers since the observation plane is limited to adistance of more than 5 �m from the surface. The use of severalillumination wavelengths [8] and the effects of polarization in morerecent improvements of the LST technique allow the determinationof the shape and nature of the different nanoparticles present.

2.2. Preferential chemical etching and Nomarski (DIC)

A powerful although destructive technique for revealing nano-defects in the sub-surface of crystals is by the use of preferentialchemical etching, which produces pits and hillocks according to thetype of defects present. This surface structure, which is often nano-metric in height, can then be observed using Nomarski microscopy(or DIC, differential interference microscopy) [9] or quantified usingphase stepping microscopy (see Section 3.2).

2.3. Phase contrast microscopy

Another way of indirectly observing extended atomic defectsin crystals such as dislocations, is to increase the contrast of smallvariations in the refractive index associated with the strain fieldaround the dislocation, using phase contrast microscopy. The imagein Fig. 2(c) shows exceptional results of the “pinning” effect ofmicroprecipitates on the dislocation path in a crystal of tin dopedGaAs. The path is made visible by the extended Cottrel atmosphere

around the decorated dislocation using near IR phase contrastmicroscopy [10]. Image integration to reduce camera noise was alsoused, resulting in a very low but nonetheless sufficient contrast ofCM = 0.01 (Fig. 3).

92 P.C. Montgomery et al. / Applied Surface Science 281 (2013) 89– 95

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ig. 3. Study of fluctuations in shape of a giant vesicle using strobed phase contrast

b) t = 1 s.

A more recent application of phase contrast microscopy in nano-haracterization, is in the measurement of the elastic properties ofipid membranes through the study of thermal shape fluctuationsf giant vesicles [11]. Although the thickness of the membranes ofhe unilamellar giant vesicles (10 �m radius) is less than 5 nm, theifference in the refractive index compared with the aqueous sur-oundings is sufficient to reveal the edge of the sphere in the phaseontrast mode. To avoid blurring due to the rapid and complex ther-al fluctuations, stroboscopic illumination with a xenon flashlamp

s used to freeze the movement. The system has been used to suc-essfully estimate the friction between the monolayers comprisinghe bilayer of the membrane, as well as the bending elasticity mod-lus of the blocked exchange of molecules between the monolayers.uantitative measurement and mapping of the refractive index oficro- and nanostructures can now be performed with modified

ersions of phase contrast and DIC microscopy.

.4. Surface enhanced ellipsometric contrast microscopy (SEEC)

Polarized light can also be used to increase the contrast ofano-structures. The SEEC (surface enhanced ellipsometric con-rast, commercially known as Sarfus) technique, uses well designednti-reflection coatings on dedicated substrates for use in crossedolarized light to decrease the background intensity (IMin) and thus

ncrease the contrast by a factor of 10–100 [12]. The presence of nmayers or nanoparticles changes the state of the polarization, thusroducing contrast and becoming visible.

The results in Fig. 4(a) show an image from a video of the crystal-ization of a 9 nm thick layer of polystyrene-b-polyethylene oxidePS-b-PEO) diblock copolymers. In Fig. 4(b), reflection interferenceontrast microscopy (RICM) has been used with an immersion

urf to observe the spreading of a COS cell onto a fibronectinoated surface, revealing the pseudopods and cytoskeleton directlyithout labelling. Phenomena that drive the adhesion of cells on

urface structures can also be studied by this means. The SEEC

ig. 4. The observation of nano-layers and cell structures using the SEEC technique (© NanG. Reiter, ICSI, Mulhouse, France). (b) COS cell spreading onto a fibronectin coated surfac

scopy (V. Vitkova, Laboratory of Liquid Crystals, ISSP, BAS, Sofia, Bulgaria). (a) t = 0 s.

technique is finding use in a growing number of applicationsin nano-characterization, such as for example in the study ofnano-dots, nanowires, graphene, crystallites, thin films, polymers,membranes, polyelectrolytes, soft lithography, biochips and livecells.

3. Nanoscopy using phase for nm roughness measurement

The second family of far field diffraction limited nanoscopy tech-niques is that based on the measurement of the phase of the lightreflected from a surface. Comparison of the wavefront of the lightfrom the sample with that from a flat reference surface results ininterference fringes and the conversion of the phase into intensitythat allows the measurement of nm surface roughness and shape.

3.1. Interference reflection microscopy (IRM)

A simple interferometric setup exists for studying the behaviourof live cells on microscope slides using RICM (also known as IRM,interference reflection microscopy). Viewing through the glass sub-strate, interference is produced between the substrate surface andthe cell membrane, producing fringes that can be quantified so asto measure the distance between the membrane and the substratesurface in adhesion studies for example [13].

3.2. Interference microscopy

By optimizing the optical configuration of the interferometer ina dedicated objective, the fringe contrast, CM, which is also knownas the fringe modulation or visibility in interferometry, can havea high value between neighbouring bright and dark fringes, cor-

responding to a height difference of �/4, or roughly 170 nm invisible light. Thus even with an 8 bit image depth, measurement ofintermediate intensities of fringes in combination with optimizedphase stepping algorithms allows the measurement of nanometric

olane). (a) The crystallization of a 9 nm thick layer of PS-PEO in the reflection modee using RICM (M.P. Valignat, INSERM, Marseille).

P.C. Montgomery et al. / Applied Surface Science 281 (2013) 89– 95 93

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ig. 5. Deep surface structure measured with coherence scanning interferometry

ideo of chip moving sideways at 200 �m/s using 4D microscopy.

r better surface roughness with phase stepping microscopy (PSM)5,7,14]. The high axial sensitivity is also well adapted for measur-ng and cataloguing the defects after preferential chemical etchingsee Section 2.2). PSM has even been employed in a type of com-inatorial technique for optimizing the laser fluence for annealinghin layers of amorphous Si for flat panel displays [15]. To achievehis, a single measurement of the 3D roughness is made of a sam-le placed at the edge of the laser impact which has a “top hat”

ntensity profile and therefore a linear variation of the laser fluenceith distance along one of the axes. Although the exact shape of the

rystals of c-Si so formed are undersampled since their size is nearo the resolution limit of the microscope, the variation in rough-ess with laser fluence is quite sufficient to be able to identify the

ptimal value of the laser fluence for a large and constant grain size.

A more recent variation of the PSM technique, QLSI (quadriwaveateral shearing interferometry), allows the measurement of phase

ig. 6. Position referenced microscopy (P. Sandoz, J.A. Galéano, FEMTO-ST, Besanc on, Fraodies (green). (b) The pseudo-periodic reference structure. (c) Alignment of two successigure legend, the reader is referred to the web version of the article.)

, Strasbourg). (a) Study of dehydration of living cell. (b) Single image taken from

variations in a single image and therefore higher speed analysis. Ahigh resolution wavefront sensor based on a 2D hole grating (mod-ified Hartman mask) in front of the CCD is used in combinationwith FT analysis. The technique is particularly useful in the studyof cell membrane dynamics and intracellular activities due to thehigh sensitivity [16].

For measuring deeper surface roughness, white light interfer-ometry is used in coherence scanning interferometry (CSI), alsoknown as WLSI (white light scanning interferometry) over manymicrometres or tens of micrometres. The technique is widely usedin materials analysis [1,4] and can also be used for studying celldynamics, such as the effects of dehydration in living cells (Fig. 4(a)).For measuring deep structures in real time, our own 4D microscopy

technique, based on a high speed camera and FPGA processing iscapable of measuring moving surfaces up to a 3D image rate of 25images per second (Fig. 4(b)) [17].

nce). (a) Confocal image of fibroblasts (red) in the process of absorbing apoptoticve images following correction. (For interpretation of the references to color in this

9 d Surface Science 281 (2013) 89– 95

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. Nanoscopy by deconvolution for nanometre positioning

In the case of simple, regular structures such as small particlesr straight edges, a third family of far field nanoscopy techniquesxists using deconvolution, based on the knowledge of the opticalransfer function of the optical system for sub-pixel or sub-voxel

etrology.

.1. Edge and particle positioning

White light interferometry was used in early sub-pixel tech-iques for helping to better measure the positions of structuraldges in integrated circuits, with a lateral measurement uncer-ainty in position of 10–20 nm [18]. In LST, analysing the 3Distribution of microprecipitates inside semiconductor crystals at

sub-micron scale can be carried out by measuring the intensityesponse of the particles as a function of either the Gaussian beamrofile of the laser beam or the sinc2 longitudinal PSF of the opti-al system, giving a measurement uncertainty of 0.1 �m along theptical axis [7].

.2. Position referenced microscopy

A more recent version of sub-pixel metrology has been devel-ped for the precise repositioning of live cells using a carefullyesigned reference mask consisting of a pseudo-periodic patternlaced just below the sample (Fig. 5(b)) [19]. With a lateral mea-urement uncertainty of 20 nm, this technique allows the absoluteeference positioning of live specimens such as that shown inig. 5(a) for studying the absorption of apoptotic bodies by fibro-lasts. The evolution of the cell activity can be monitored over longeriods of tens of hours by removing the petri dish from a controllednvironment for imaging on the microscope at regular intervals andsing the mask to correct for positioning errors (Figs. 5(c) and 6).

. Nanoscopy using nano-markers for nanometreeasurement of position or structure

The fourth and last group of techniques considered is bysing nano-markers to study nano-structures. Apart from “native-arkers” such as microprecipitates decorating dislocations in

rystals, mentioned in Section 2.1, the use of “foreign-markers”uch as fluorochromes and gold nano-particles are finding exten-ive use in the study of nano-structures.

.1. Total internal reflection microscopy (TIRF)

While different fluorochromes are used to target many differentypes of nano-structures in fluorescence imaging, the contrast cane enhanced even further by reducing the background intensityIMin) using total internal reflection at an interface (Fig. 7(a)), orIRF (total internal reflection microscopy) [20].

The evanescent field excites only the fluorophores that arettached to structures within a depth of about 100 nm. The image inig. 7(b) shows the adhesion of a cell on a substrate. TIRF can alsoe used for following the movement of nanometric vesicles andor studying the dynamics of ensembles of single molecules nearell membranes [20]. Quantified analysis of individual moleculesan be performed with stochastic techniques such as PALM (photo-ctivated localization microscopy) and STORM (stochastic opticaleconstruction microscopy).

.2. Gold nanoparticles (AuNP)

Finally, in place of fluorophores, nano-particles of gold (AuNP)an also be used as useful markers, becoming point sources that

Fig. 7. Total internal reflection microscopy. (a) Layout for illumination in TIRF. (b)Study of cell adhesion on substrate (© Leica Microsystems).

are not resolved but are observable. One example is the use of twophoton microscopy to map out the nanometric structure of collagenfibres using a single 5 nm AuNP that is moved along the fibre bymeans of a photo-acoustic effect from a pulsed laser [21].

6. Conclusion

In this paper we have provided some of the answers to the ques-tion of just how far it is possible to go in nano-characterizationusing diffraction limited far field nanoscopy techniques withoutresolving all the details. We have underlined the growing interestin this new field of nanoscopy techniques that although limited bydiffraction, nonetheless allow the detection, observation or mea-surement of many different types of nanostructures right down tothose that are atomic in size, although extended in space. A varietyof different techniques exist based on increasing the contrast, mea-suring the phase, using deconvolution techniques, or by employingnano-markers such as fluorophores or gold nano-particles. By thesemeans it then becomes possible to not only detect the presence ofnano-structures but also to perform quantified 2D and 3D mapping,density measurements, structural and orientation identification,nanometric surface and interface roughness measurements andnanometric positional measurement concerning bulk materials,surfaces, nano-layers, soft matter and biological cells. Real timeanalysis can be performed for tracking movement, for followinga transformation or for studying dynamic functions such as thosein living cells.

Acknowledgements

Thanks are extended to P. Gall-Borrut (IES, UniversitéMontpellier II, France), V. Vitkova (BAS, Sofia, Bulgaria), N. Médard(Nanolane, France), R. Serra (Phasics, Palaiseau, France), P. Sandoz(FEMTO-ST, Besanc on, France), and J.M. Ellens (Leica Microsystems,Nanterre, France) for permission to use their material in this paper.

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