Tomographic diffractive microscopy allows for imaging unlabeled specimens, with a better resolution than conventional microscopes, giving access to the index of refraction distribution within the specimen, and possibly at high speed. Principles of image formation and reconstruction are presented, and progresses towards realtime, three-dimensional acquisition, image reconstruction and final display, are discussed, as well as towards three-dimensional isotropic-resolution imaging.
The development of new nanomaterials, devices and systems is very much dependent on the availability of new techniques for nanometrology. There now exists many advanced optical imaging techniques capable of subwavelength resolution and detection, recently brought to the forefront through the 2014 Nobel Prize for chemistry for fluorescent STED and single molecule microscopy. Label-free nanoscopy techniques are particularly interesting for nanometrology since they have the advantages of being less intrusive and open to a wider number of structures that can be observed compared with fluorescent techniques. In view of the existence of many nanoscopy techniques, we present a practical classification scheme to help in their understanding. An important distinction is made between superresolution techniques that provide resolutions better than the classical λ/2 limit of diffraction and nanodetection techniques that are used to detect or characterize unresolved nanostructures or as nanoprobes to image sub-diffraction nanostructures. We then highlight some of the more important label-free techniques that can be used for nanometrology. Superresolution techniques displaying sub-100 nm resolution are demonstrated with tomographic diffractive microscopy (TDM) and submerged microsphere optical nanoscopy (SMON). Nanodetection techniques are separated into three categories depending on whether they use contrast, phase or deconvolution. The use of increased contrast is illustrated with ellipsometric contrast microscopy (SEEC) for measuring nanostructures. Very high sensitivity phase measurement using interference microscopy is then shown for characterizing nanometric surface roughness or internal structures. Finally, the use of through-focus scanning optical microscopy (TSOM) demonstrates the measurement and characterization of 60 nm linewidths in microelectronic devices.
Phase microscopy techniques regained interest in allowing for the observation of unprepared specimens
with excellent temporal resolution. Tomographic diffractive microscopy is an extension of
holographic microscopy which permits 3D observations with a finer resolution than incoherent light
microscopes. Specimens are imaged by a series of 2D holograms: their accumulation progressively
fills the range of frequencies of the specimen in Fourier space. A 3D inverse FFT eventually provides
a spatial image of the specimen.
Consequently, acquisition then reconstruction are mandatory to produce an image that could prelude
real-time control of the observed specimen. The MIPS Laboratory has built a tomographic
diffractive microscope with an unsurpassed 130nm resolution but a low imaging speed - no less than
one minute. Afterwards, a high-end PC reconstructs the 3D image in 20 seconds. We now expect
an interactive system providing preview images during the acquisition for monitoring purposes.
We first present a prototype implementing this solution on CPU: acquisition and reconstruction are
tied in a producer-consumer scheme, sharing common data into CPU memory. Then we present
a prototype dispatching some reconstruction tasks to GPU in order to take advantage of SIMDparallelization
for FFT and higher bandwidth for filtering operations. The CPU scheme takes 6
seconds for a 3D image update while the GPU scheme can go down to 2 or > 1 seconds depending
on the GPU class. This opens opportunities for 4D imaging of living organisms or crystallization
processes. We also consider the relevance of GPU for 3D image interaction in our specific conditions.
Many biological researches require observation of the sample at a sub-micrometer scale. However, most biological
samples are transparent, and thus are barely visible in conventional transmission microscopy. Techniques like
interference contrast or oblique illumination permit to record an improved contrast, but are useful for morphological
studies only, because the interaction of incoherent, non-polarized and polychromatic illumination with matter is very
complex, so that the recorded contrast cannot be linked to local physical properties of the sample, as for example the
index of refraction. We have developed a diffractive tomographic microscope, which permits the observation of
unstained-, transparent samples in 3-D. This device is based on a combination of microholography, which records the
field diffracted by the specimen in both amplitude and phase using a high numerical aperture objective and a phase
stepping interferometer, with a variable illumination of the sample (tomography) using a high numerical aperture
condenser. The successive holograms are numerically recombined in the Fourier space, and the reconstruction of the
specimen index of refraction distribution is based on the first Born approximation for weakly diffractive samples.
Examples of biological specimens observed with this technique are given, and possible evolutions of the instrument are
3-D optical fluorescent microscopy becomes now an efficient tool for volume investigation of living biological samples.
Developments in instrumentation have permit to beat off the conventional Abbe limit, in any case the recorded image
can be described by the convolution equation between the original object and the Point Spread Function (PSF) of the
acquisition system. If the goal is 3-D quantitative analysis, whether you improve the instrument capabilities, or (and)
you restore the data. These last is until now the main task in our laboratory. Based on the knowledge of the optical
Transfer Function of the microscope, deconvolution algorithms were adapted to automatic determine the regularisation
threshold in order to give less subjective and more reproducible results. The PSF represents the properties of the image
acquisition system; we have proposed the use of statistical tools and Zernike moments to describe a 3-D system PSF and
to quantify the variation of the PSF. This first step toward standardization is helpful to define an acquisition protocol
optimizing exploitation of the microscope depending on the studied biological sample.
We have pointed out that automating the choice of the regularization level; if it facilitates the use, it also greatly
improves the reliability of the measurements. Furthermore, to increase the quality and the repeatability of quantitative
measurements a pre-filtering of images improves the stability of deconvolution process. In the same way, the PSF pre-filtering
stabilizes the deconvolution process. We have shown that Zernike polynomials can be used to reconstruct
experimental PSF, preserving system characteristics and removing the noise contained in the PSF.
Fluorescent microscopes suffer from limitations; photobleaching and phototoxicity effects, or influence of the sample
optical properties to 3-D observation. Amplitude and phase of the object can be reached with optical tomography based
on a combination of microholography with a tomographic illumination. So indices cartography of the specimen can be
obtained, and combined with fluorescence information it will open new possibilities in 3-D optical microscopy.
The optical microscope has proven to be an invaluable tool in biology, because of its unique capabilities of 3-D imaging of living specimens. However, compared to other techniques, the achievable resolution is limited. Several techniques have been proposed to improve the resolution of the fluorescence microscope. The confocal set-up is the first of them. Interference effects can also be used to sharpen up the point spread function (PSF) in 4Pi microscopy. Another approach is the so-called STimulated Emission Depletion microscopy, which has permitted to decrease the resolution down to about 100 nm in three dimensions, and below 50 nm in either the x-y plane, or along the z-axis.