Paul Montgomery is a senior research scientist with the CNRS at the Engineering science, computer science and imaging laboratory (ICube) in Strasbourg, France. He has over 30 years' experience in developing optical instrumentation for the characterization of materials and biomaterials. He is currently interested in label-free far field nanoscopy, microsphere-assisted interference microscopy, local spectroscopy and the use of environmental chambers for measuring specific parameters. He is a senior member and a member of the Board of Directors of SPIE as well as being a member of IOP and SFO.
Structural imaging using light microscopy is a cornerstone of histology and cytology. However, the utility of the optical microscope for diagnostic imaging is limited by the fundamental tradeoff between the field of view and spatial resolution and a reliance on exogenous dyes to generate sufficient image contrast. Fourier Ptychographic Microscopy (FPM) is a complex imaging modality with the potential to overcome these limitations by recovering high-resolution images of sample amplitude and phase from a set of low-resolution raw images captured under inclined illumination. In this article we explore the application of FPM to clinical imaging using a simple, low-cost FPM system and simulated and experimental data to explore the influence of both image acquisition parameters and hardware configuration on image quality and imaging throughput. The practical performance of the method is investigated by imaging peripheral blood films and histological tissue sections. We find that, at the cost of increased computational complexity, FPM increases the information capture capacity of the optical microscope significantly, allowing label-free examination and quantification of features such as tissue and cell morphology over large sample areas.
KEYWORDS: Spectroscopy, Interferometry, Reflectivity, Spectrometers, Calibration, Refractive index, Near field scanning optical microscopy, Near field optics, Microscopy, Microscopes
The large spot size of a few mm2 with spectrometers and a few thousand µm2 with ellipsometers means that classical spectroscopic characterization is limited to that of bulk materials. In the development of more recent heterogeneous materials in which there exists local variations between materials on a microscopic scale, a much smaller spot size is required for optical characterization. Several new techniques have been developed for performing local spectroscopy, such as by color camera microscopy, hyperspectral imaging microscopy, scattering type scanning near field optical microscopy (s-SNOM) or spectroscopic optical coherence tomography (s-OCT). Concerning the latter, the related technique of coherence scanning interferometry (CSI) also allows local spectroscopy by applying Fourier Transform processing to the local polychromatic interference fringe signal. This technique offers the advantages of not requiring an external spectrometer since an interferometer is incorporated in the microscope objective, but challenges remain in order to correctly adjust and calibrate the system.
In this paper we present some of our latest results of using CSI to accurately measure the local spectra at a microscopic scale with a spot size a little larger than that defined by the diffraction limit, of around 1 µm. Results demonstrate measurements of local reflectance spectra at the surface of a heterogeneous sample and on small structures buried within or under a transparent layer. Other theory has been developed to allow the measurement of local transparent layer thickness and refractive index. As well as performing local point measurements, we show how with a single scan over the optical axis, 2D cartographic maps can be made of reflectance spectra together with the topographic height map of the same area. Any nanometric height errors present due to phase on reflection errors linked to the presence of complex refractive indices can then be corrected using the spectroscopic information.
Interference microscopy is a non-destructive full-field imaging method, mainly used to measure the surface topography of different samples. In this work, two designs for improving the signal quality are described. The first consists of an original vertically orientated breadboard interferometer, in a Linnik configuration. The mechanical design of the arms allows the independent control and alignment of the coherence and the focal plane positions for optimizing fringe contrast. A low noise 16-bit camera is used to improve the sensitivity. The second interferometer is based on a Thorlabs tube system, with a Nikon Mirau Objective and a white LED, all controlled with IGOR Pro software or Labview, with the aim of being more compact, flexible and mobile. For both systems, an evaluation of the interferometric signal quality is performed, whereas the difference in lateral resolution by considering the 3D nature of the interferometric system, or only its 2D imaging abilities, is explored.
In the domain of optical metrology, white light interference microscopy is mainly known for performing micro and nano surface profilometry. This is achieved by identifying the envelope peak of the fringe signal. However, the polychromatic signal is rich in information and spectral characterization may be performed through the Fourier analysis of the signal, which gives local spectroscopic information about the sample surface. The use of CSI for studying transparent layers has also been well-developed since the analysis of the reflected light provides both structural and spectral information on the layer. Through the spectral analysis of the reflected light, it has been shown that the morphological properties of a thin film structure, namely the thickness and the refractive index, can be precisely measured. In this case, either the amplitude or the phase of the thin film total reflectance spectrum are used to recover the thickness. The technique is based on the best fit between the experimentally measured spectrum with that of the theoretical model using a non-linear least-squares algorithm. Usually, this spectral method is used to investigate thin films having a thickness that does not exceed a few hundred nanometers. In this work, we apply a similar technique, based on the magnitude of the total reflectance spectrum, to study thicker transparent layers. In this case, we show that precautions regarding the effective numerical aperture of the system need to be considered to obtain consistent values of both the refractive index and the thickness. In addition, we demonstrate the possibility of extracting the depth-resolved reflectance spectra of a buried interface independently from the spectral response of the surface. The consistency of these different spectra is demonstrated by comparing the results with those obtained using a program based on electromagnetic matrix methods for stratified media. The lateral spatial resolution of the measurements attained is a spot size of around 0.84 μm for spectrally characterizing small structures.
Interference microscopy is a widely used technique in optical metrology for the characterization of materials and in particular for measuring the micro and nanotopography of surfaces. Depending on the processing applied to the interference signal, either topographic analysis of the sample can be carried out by identifying the envelope peak of the fringe signal, which leads to 3D surface imaging, or spectral analysis may be performed which gives spectroscopic measurements. By applying a Fourier transform to the interference fringes, information about the source spectrum, the spectral response of the optical system, and the reflectance spectrum of the surface at the origin of the interferogram can be obtained. By using a sample of known reflectivity for calibration, it is possible to extract the spectral signature of the entire system and therefore to deduce that of the surface of interest. In this paper, we first explain theoretically how to retrieve the reflectance information of a surface from the interferometric signal. Then, we present some results obtained by this means with a white light scanning Linnik interferometer on different kinds of samples (silicon, tin oxide (SnO2), indium tin oxide (ITO)). The initial results were slightly different from those obtained with a conventional optical spectrometer until averaged temporally and were improved even further when averaged spatially. We show that the reflectance of the surface can be calculated over the given wavelength range of the effective spectrum, which is defined as the source spectrum multiplied by the spectral response of the camera and the spectral transmissivity of the optical system. We thus demonstrate that local spectroscopic measurements can be carried out with an interference microscope and that they match well with those measured with an optical spectrometer model Lambda19 UV-VIS-NIR from Perkin Elmer. A simulation study is also presented in order to validate the method and to help identify the potential sources of errors in the spectroscopic analysis.
Full-field optical coherence tomography (FF-OCT) based on white-light interference microscopy, is an emerging noninvasive imaging technique for characterizing biological tissue or optical scattering media with micrometer resolution. Tomographic images can be obtained by analyzing a sequence of interferograms acquired with a camera. This is achieved by scanning an interferometric microscope objectives along the optical axis and performing appropriate signal processing for fringe envelope extraction, leading to three-dimensional imaging over depth. However, noise contained in the images can hide some important details or induce errors in the size of these details. To firstly reduce temporal and spatial noise from the camera, it is possible to apply basic image post processing methods such as image averaging, dark frame subtraction or flat field division. It has been demonstrate that this can improve the quality of microscopy images by enhancing the signal to noise ratio. In addition, the dynamic range of images can be enhanced to improve the contrast by combining images acquired with different exposure times or light intensity. This can be made possible by applying a hybrid high dynamic range (HDR) technique, which is proposed in this paper. High resolution tomographic analysis is thus performed using a combination of the above-mentioned image processing techniques. As a result, the lateral resolution of the system can be improved so as to approach the diffraction limit of the microscope as well as to increase the power of detection, thus enabling new sub-diffraction sized structures contained in a transparent layer, initially hidden by the noise, to be detected.
Transparent layers such as polymers are complex and can contain defects which are not detectable with classical optical inspection techniques. With an interference microscope, tomographic analysis can be used to obtain initial structural information over the depth of the sample by scanning the fringes along the Z axis and performing appropriate signal processing to extract the fringe envelope. By observing the resulting XZ section, low contrast, sub-μm sized defects can be lost in the noise which is present in images acquired with a CCD camera. It is possible to reduce temporal and spatial noise from the camera by applying image processing methods such as image averaging, dark frame subtraction or flat field division. In this paper, we present some first results obtained by this means with a white light scanning interferometer on a Mylar polymer, used currently as an insulator in electronics and micro-electronics. We show that sub-μm sized structures contained in the layer, initially lost in noise and barely observable, can be detected by applying a combination of image processing methods to each of the scanned XY images along the Z-axis. In addition, errors from optical imperfections such as dust particles on the lenses or components of the system can be compensated for with this method. We thus demonstrate that XZ section images of a transparent sample can be denoised by improving each of the XY acquisition images. A quantitative study of the noise reduction is presented in order to validate the performance of this technique.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.