IPP team: Photonics Instrumentation and Processes

Multimodal microscopy

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P. Montgomery, M. Flury, F. Anstotz, S. Lecler, V. Maioli, D. Montaner, A. Nahas, F. Salzenstein.

What is multimodal nanoscopy?

The IPP team has been at the forefront of research in the field of interference microscopy for many years [P. Montgomery et al., ACS Omega, 2023]. The aims are several. More recently, they concern improving the lateral resolution to beyond the diffraction limit without using markers. The addition of different imaging modalities allows using the same imaging system to access different types of information such as microscopic surface topography (static and moving), the high-resolution structure of transparent layers and the local spectroscopic response. The use of dedicated environmental chambers allows the study of specific parameters of samples in a controlled environment. The study of specific algorithms allows optimisation of fringe processing and the extraction of the required information.

The team is also extending its field of investigation of samples from the historical application areas of materials and micro- nanotechnologies to those of the biological field. It does this through strong links with local actors in the field (ICS, IPCMS, IGBMC, IPHC, etc.), starting from the fundamental understanding of the basic principles involved, through the development of prototype instrumentation, right through to the technology transfer of the techniques developed.

Interference microscopy, often called WLSI (White Light Scanning Interferometry) is typically used to measure nanometric surface roughness using PSM (Phase Shifting Microscopy) and deeper structures using CSI (Coherence Scanning Interferometry).

Scanning interference microscopy is a powerful technique based on far field optical reflection microscopy and interferometry that can be used for extracting information on micro- and nanostructures embedded in complex materials, devices and microsystems. While the technique is classically used for measuring static microscopic surface roughness and topography, we have developed other measurement modes and various techniques for improving the measurements (see review in [P. Montgomery et al., ACS Omega, 2023]).

The following topics are covered:
Tomography: high-resolution characterization of thick (>500 nm) transparent, semi-transparent or translucent layers of materials such as glass layers, polymer films, colloidal layers [E. Halter et al., Appl. Surf. Sci., 2010] or layers of hydroxyapatite (biomaterials) [E. Pecheva et al., Langmuir, 2007]. Instead of just detecting a single fringe signal corresponding to the surface at each point to measure surface roughness, several fringe signals along the optical axis can be detected at each point that correspond to buried surfaces or structures. To do this, an image stack is built up by scanning the whole depth of the film to be measured followed by image processing to reveal the XZ image, or "B-scan" that shows a cross sectional image of the film and buried structures and is known as OCT (Optical Coherence Tomography). Image processing is necessary to increase the signal to noise ratio, such as with the High Dynamic Range (HDR) technique [A. Leong-HoÏ et al., Opt. Letts., 2016].

These techniques are also useful for characterising nanostructured photonic devices developed in the theme Photonics Modeling and Simulation.

4D microscopy (3D+time): 3D topographic measurement in real time of surfaces that evolve aperiodically, such as moving samples and those found in soft materials, microsystems (MEMS and MOEMS) and chemical reactions. The fringes are scanned continuously over the depth to be measured and a high speed camera is used in combination with fast FPGA (Field Programmable Gate Array) cabled logic processing to give real time measurements of surface roughness and structure [A. Dubois et al., Eur. Phys. J.: Appl. Phys., 2002], [P. Montgomery et al., Ch. 11 in Full-Field Optical Coherence Microscopy: technology and applications, Ed. A. Dubois, 2016].
The following results show 3D measurements in real time (18 3D images per second) of a metallic contact on silicon moving sideways at a speed of 200 µm/s (x40 Mirau objective, axial range of 5 µm).

Fringe processing algorithms: development of fringe processing techniques for specific imaging modalities. Peak Fringe Scanning Microscopy (PFSM) [P. Mon tgomery et al., Proc. SPIE, 1993] is a very compact algorithm, particularly suited for implementation in FPGA processing in real time measurement [P. Montgomery et al., Ch. 11 in Full-Field Optical Coherence Microscopy: technology and applications, Ed. A. Dubois, 2016]. Teager-Kaiser Energy Operators (TKEO) [F. Salzenstein et al., Eurasip J. Appl. Signal Process., 2005] are also compact but more robust, having the advantages of efficiency and simplicity for tracking the instantaneous amplitude and frequency of AM-FM signals and particularly useful for detecting the weak signals in tomography [F. Salzenstein et al., Opt. Expr., 2014]. TKEO has been improved further using pre-filtering and demodulation processing to remove the noise and offset component, and to retrieve the fringe envelope to determine the surface height and internal structural information [G. Gianto et al., Appl. Opt., 2016].

Fourier Transform (FT) algorithms are particularly suitable for local spectroscopy measurements [R. Claveau et al., Phys. Stat. Sol. (c), 2017].

Optical nanoscopy: study of basic principles of optical nanoscopy techniques for imaging and characterizing microscopic structures with one or more dimensions at the nanometer scale [P. Montgomery, Nanotechnology, 1990] either optically resolved using super-resolution or unresolved but detectable using nanodetection [A. Leong-Hoï et al., Nanotech., Sci. & App., 2015]. The two following classification schemes developed give a better perspective of the main techniques available, the second one taking into account microsphere imaging.

Enhanced resolution: different techniques are being developed to enhance the lateral resolution in optical microscopy. Since 2016 we have been studying the use of glass microspheres to improve the resolving power in 2D reflection microscopy for high detail imaging [S. Perrin et al., Opt. & Las. Tech., 2018] and in interference microscopy for nanometrology [I. Kassamakov et al., Sci. Rep., 2017][S. Perrin et al., Appl. Opt., 2017]. Studies are performed from both theoretical and experimental points of view. The theoretical study of the imaging properties of microspheres and resolution enhancement is carried out using rigorous 2D FEM simulation under COMSOL. A rigorous model is also being developed for surface topography measurement using interference microscopy and in particular, using Phase Shifting Microscopy (PSM).

Experimental results of improved resolution imaging and surface topography measurements using microspheres are demonstrated.

Another method to enhance the lateral resolution is by using Fourier Ptychography Microscopy. In this technique, a larger numerical aperture in an optical microscope is synthesized from a set of images acquired with a series of illumination angles using coherent light. Image processing is then carried out in Fourier space to produce a single image with higher resolution than the initial images captured at low resolution. A particularity of this technique is being able to produce images sensitive to the phase of the light passing through the sample, revealing changes in refractive index and details that are different to the images produced in a standard microscope, particularly useful for the high resolution imaging of biological samples.

Local spectroscopy: local characterization of the optical properties of materials, layers and buried interfaces at a local scale of 3.5x3.5 µm and hyperspectral mapping over large areas.

A Linnik interferometer is used to first scan the white light interference patterns along the optical axis to give an interferometric stack. The interferograms at each pixel are then processed using Fourier Transform processing and the modulus of the result gives the "effective spectrum" at each pixel. To remove the optical effects of the instrumentation, the "spectral transfer function" is obtained by measuring a calibrated sample. Dividing the first by the second gives the spectral reflectivity at that point [R. Claveau et al., Phys. Stat. Sol. (c), 2017]. Performing the processing at each pixel, a complete spectral cube can be obtained, allowing spectral mapping [S. Marbach et al., Opt. Letts., 2021]. By the use of windowing of the fringe signal, depth resolved spectra can be obtained in transparent layers [R. Claveau et al., Opt. Expr., 2017].

Results can then be taken from the spectral cube to give the reflectance map at a given wavelength or the reflectance spectra from specific pixels to show the optical properties of different materials. Using colorimetry, the reflectance results can then be used to determine the local colour, or the colour as observed by human eye under typical midday light. This is achieved by first determining the trichromatic components XYZ at each pixel of the spectral cube and then the trichromatic coordinates on the colour locus (CIE 1931 with D65 illuminating spectra). The following results show some experimental measurements compared with the theoretical values of the previous copper layer on glass [S. Marbach et al., Opt. & Las. in Eng., 2023]:

While the colour for copper has been correctly determined, the glass has a slightly yellowish tinge due to the limited illumination spectral range in the blue-UV (460-780 nm).

Environmental chambers: measurement of specific material parameters using dedicated environmental chambers to control parameters such as temperature, pressure, humidity or the immersion medium.

The following example shows the use of environmental chambers under a Linnik microscope to measure the mechanical properties of ultra-thin (30-60 nm) polymer films (collaboration with ICS, Strasbourg). The "nanobubble" inflation method is used, consisting of inflating an array of "nanobubbles" formed by the polymer film on a grid of 5 µm diameter holes. Two prototype chambers were developed, the first to control the temperature and pressure [P. Chapuis et al., Rev. Sci. Instr. 2017] and the second to control the temperature, pressure and humidity [P. Montgomery et al., ACS Omega, 2023].

Using the very sensitive technique of PSM, the shape of the "nanobubbles" can be monitored over several hours in order to measure the relaxation and then determine the creep compliance. Measuring at the nanometer scale over such a long period is challenging, requiring the development of a careful measurement protocol in order to obtain significant results.

Other types of microscopy are also being developed, particularly for biomedical applications:

Full field OCT (Optical Coherence Tomography): reconstruction of volumetric information inside turbid media such as organic tissue, by making use of the zero path difference in coherence scanning interferometry (CSI) to detect only ballistic photons that have been backscattered by buried structures and sample interfaces.

Light sheet microscopy: 3D imaging inside tissue and small animals (e.g. zebra fish, C. elegans worm…) by scanning an oblique sheet of light across the volume of the sample and stitching the images together.


  • PHC: Tassili
  • ANR: LatexDry
  • CNRS: NewMaps ("Time", collaboration with ICS)
  • Grand Est region: MIRAGE (FRCR)
  • University of Strasbourg: Idex conferences
  • SPIE: SPIE Photonics Europe
  • ICube internal projects: LocalSpec-PV


  • Indonesian government: 2 Scholarships
  • CNRS: PICS, Cooperation, ACO (collaboration with ESPCI, Paris), pRECISION ("Instrumentation at its limits challenge", collaboration with ICS)
  • Grand Est Region: SuperMic (post-doc), MIRAGE (FRCR)
  • SATT Conectus Alsace: CAM4D, Nano3D, SIBIC
  • Industrial: Eotech SA, Michelin
  • ICube internal projects: RTPPP, PiMeNtS, GLOBAL-INNOV