Phase calibration of a basic bright-field microscope for 3D metrology of transparent samples at the nanoscale

Optical retrieval of the structure of transparent objects at the nano-scale requires adapted methods capable of probing their interaction with suitable light. Scattering events, for instance, depend on the content and the arrangement of the medium encountered by the light along its propagation path. Physical models of scattering were used in the past to understand image formation and phase retrieval in transparent objects. Here, we considered one of them, which is based on the acquisition of defocused images obtained with partially coherent illumination, and explored its phase retrieval capability over a wide range of illumination angles. We used a basic transmission bright-field microscope with a bandpass filter and an adjustable illumination aperture. The bandpass filter was used to select a specific wavelength range to avoid light dispersion in the sample and maximize illumination capabilities. The adjustable illumination aperture was used to probe and assess the calibration over a wide range of illumination angles, which give access to different parts of the spatial frequency spectrum of the sample. We subsequently employed a computational algorithm to retrieve the local 3-dimensional phase-shift induced on the light field by the scattering through the sample. We imaged several types of samples to explore the calibration and the results in different experimental configurations. We employed: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis, (2) custom-made nanosteps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis. We first verified the model prediction in the spatial frequency domain for the scattering induced by our samples, and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to 0.5. This enabled us to calibrate a very simple optical system in a wide range of illumination angles to obtain quantitative 3D metrology of transparent samples at the nanoscale.

3D nanometrology of transparent objects by phase calibration of a basic bright-field microscope for multiple illumination apertures

Martinus Gijs, Daniel Migliozzi, Bingying Zhao

Optical retrieval of the structure of transparent objects at the nanoscale requires adapted techniques capable of probing their interaction with light. Here, we considered a method based on calibration of the defocusing with partially coherent illumination and explored its phase retrieval capability over a wide range of illumination angles. We imaged: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis,(2) custom-made nano-steps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis, and (3) human cancer cells deposited on a glass substrate to assess the results of the calibration on complex transparent 3-dimensional samples. We first verified the model prediction in the spatial frequency domain and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to0.5. Finally, we studied the dependence of the phase retrieval of a complex nanostructured object on the illumination aperture.

2020

On the Characteristics of Highly Confined Light Fields

Myun Sik Kim

The Investigation of highly confined light fields created by different mechanisms, such as refraction, diffraction, and scattering, are the main subject of this thesis. We distinguish a specific scattering phenomenon of dielectric microspheres from general definition of light scattering that includes refraction and diffraction. Light confinement has been studied by numerous researchers in theoretical ways. However, the experimental approaches for microstructures were always limited due to the lack of resolution and functionalities of the measurement system. To understand the characteristics of optical fields propagating in free space or emerging form microstructures, intensity is not always sufficient. It is therefore necessary to measure the phase of light. The main goal of this work was to set up an optical instrument that is able to measure the amplitude and phase of the light field in the real 3D object space. By combining an optical interferometer (Mach-Zehnder type) with an optical microscope, we have developed a multi-functional high-resolution interference microscope (HRIM). Phase shifting interferometry (PSI) and the five frame (Schwider-Hariharan) algorithm have been introduced in the HRIM in order to directly measure the phase. Auxiliary techniques have been applied for the first time to the experimental setup of the HRIM. For example, multiple-wavelengths light sources for the study of the spectral dependency, immersion of object space for an enhanced resolution and a phase telescope (Bertrand lens) to image and manipulating the Fourier plane (the back focal plane of the objective) have been demonstrated. In addition to a plane-wave illumination, the engineering of the illumination beams allows to broaden the range of experimental investigations for a given sample, such as wavefront management by focused Gaussian beams and focused Bessel beams, and polarization-induced beam shaping for particular shapes of localized illuminations to a small sample. Our main subject is the confinement of light. Three physical optical mechanisms are discussed in detail: refraction, diffraction and scattering. Discussion is done by examples. We have chosen effects that represent the different effects of physical optics at a microscale: highly focused Gaussian beam for refraction, the spot of Arago and Talbot effect for diffraction and photonic nanojets for scattering. Note that even though scattering in general includes refraction and diffraction, here we use the term "scattering" only for scattered hotspots, i.e., photonic nanojets, by dielectric microspheres. Our study contributes to the understanding of strong confinement in submicron or sub-wavelength dimensions by such effects and show for the first time experimental data on phase and amplitude fields. Basic physical principles, like the Gouy phase shift for focused light were demonstrated for the photonic nanojet, for a complex optical phenomenon where focusing is based on scattering rather than refraction. We present the capabilities of the HRIM system through a series of measurements of aforementioned problems for highly confined light fields. To meet the proper experimental conditions for each problem of interest, the HRIM employs several auxiliary techniques of conventional microscopy, e.g., multiple-wavelengths light sources, an immersion technique, and a manipulation of the Fourier plane (the back focal plane of the objective). Engineering of the illumination including polarization-induced beam shaping broadens the range of applications of the instrument and we illustrate this by examples. Furthermore, the HRIM can be operated in two distinguished modes for measurements of the axial phase evolution, "longitudinal-differential mode" or "propagation mode". The longitudinal differential mode directly visualizes peculiar phase evolutions along the optical axis used to prove the phase anomalies. The HRIM with auxiliary techniques therefore serves as a powerful tool for the investigation of highly confined light fields in small volumes.

EPFL2011

Optical microscopy for imaging through scattering media

Xin Yang

Optical microscopy has been widely used in the life sciences and biological research for several decades. It possesses many advantages over x-ray radiography, computed tomography, ultrasound imaging and magnetic resonance imaging, in terms of being low cost, high resolution, harmless and capable of multiple contrast mechanism, etc. However, the weakness of optical microscopy is the inability to perform deep tissue imaging. Owing to the short wavelength of optical waves, it experiences much more scattering than longer waves in inhomogeneous materials such as biological tissues. Scattering scrambles the wavefront and limits the penetration depth of optical microscopy to only hundreds of microns. We explore in this thesis, novel optical approaches to image through scattering media. There are two main categories of the state of art techniques: imaging with ballistic photons, which requires separation of ballistic photons from the sneak or multiple scattered ones; and imaging with scattered photons, which relies on manipulation of the scattered photons according to the specific scattering properties. We primarily focus on the second category in this thesis. Taking advantage of the reversibility of light, phase conjugation has a long history in aberration compensation. A deterministic wave is incident on a scattering medium and results in a random scattered field. If we record the full information of the scattered field (amplitude and phase) and generate a phase conjugated replica of it, the phase conjugated wave can follow the same path and reconstruct the original deterministic wave after passing back through the scattering medium. In this work, phase conjugation is achieved using digital method, called digital phase conjugation. The scattered field is recorded through digital holography, and a spatial light modulator is applied to construct the phase conjugated wave. Second harmonic radiation imaging probes are used as the original light source, rendering the generation of a focus behind the scattering medium. The memory effect, which indicates the correlation between speckles with different incident angles, is exploited to move the focus around and obtain three-dimensional scanning microscopy behind scattering medium. Speckle scanning microscopy is the second method demonstrated in this thesis for imaging through turbid media. In contrast to digital phase conjugation, which requires access from both sides of the scatterers to measure the scattered field, in speckle scanning microscopy, excitation and collection can be achieved from the same side. The specific scattering events are not taken into account, instead, the statistic property of the scatterers plays a major role. This technique makes full use of two facts: 1. The statistically averaged autocorrelation of speckle fields approaches a delta function; 2. When a speckle pattern scans across a fluorescence object, the collected fluorescence intensity is proportional to the convolution of the speckle field and the object. Linking these two facts with delicate calculation, we can eventually minimize the influence of the speckle, and reconstruct two-dimensional images of objects behind turbid medium. Encouraged by the success of two-dimensional speckle scanning microscopy, we investigate more advanced possibilities by simulation, including three-dimensional speckle scanning and reference based speckle scanning techniques. [...]