Application of optical microscopy in biochar characterization on selected examples

Presents several biological applications of near field optical microscopy, in combination with force microscopy. Aperture near field scanning optical microscopy (NSOM) with fluorescence detection gives (bio)chemical specificity and orientational information, in addition to the simultaneously acquired force image. This technique has large potential for DNA sequencing, molecular organization in monolayers, and study of the role of the cytoskeleton in cellular mobility in cell growth, cell migration, formation of protrusions, etc. Fluorescence NSOM gives high resolution on flat, not too deep surfaces. Fluorescence NSOM induces virtually no bleaching, as opposed to confocal fluorescence microscopy. Bright field NSOM in transmission generally yields a complicated contrast, caused by a mixture of dielectric and topographic contributions. Shear force feedback is essential in aperture NSOM operation with fibers, and operates on soft surfaces of cells and chromosomes. Ultimately, aperture NSOM is limited by low efficiency with a source brightness of typically 100 pW to 10 nW. Thus, in spectroscopic applications (fluorescence, Raman, etc.) photon noise will be a fundamental limit in the speed of imaging. Photon tunneling in combination with force microscopy allows routine scanning with a high optical lateral resolution. However, interference effects can be dominant on surfaces which display extensive scattering. As such, the application potential of PSTM to biological surfaces is rather limited. Clearly, the virtues of optics, non-invasiveness, high spectral resolution, and high time resolution all apply to the near field optical domain with its high spatial resolution, which adds extensively to the potential of scanning probe microscopy

9] Cytomechanics applications of optical sectioning microscopy

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

Image formation in focusing mirrors for surface plasmon polaritons (SPPs) is studied numerically. The transition from diffractive to geometrical optics is traced as a function of mirror size and surface polariton wavelength. The role of the mirror shape and the SPP propagation length in the image formation is also investigated. The small losses are shown to be helpful for imaging with surface waves, resulting in the increased contrast and reduced background of the images. Surface polariton focusing mirrors have recently found important applications in high-resolution optical microscopy based on short-wavelength SPP.

Photonic-chip-based light illumination has recently found applications in optical microscopy and nanoscopy methodologies. The photonic chip removes the dependency on imaging objective lenses to generate the required illumination patterns for different microscopy methods. Until now, all the reported chip-based optical microscopy methods exploit the evanescent field present on top of a waveguide surface and are thus inherently limited to two-dimensional microscopy. Here, we perform systematic simulation studies to investigate different chip-based waveguide designs for static and dynamic shaping of light beams in the free-space. The simulation studies have been carefully designed considering the photo-lithography limitations and wavelength spectrum (405 nm to 660 nm) that is of interest in fluorescence based optical microscopy and nanoscopy. We first report the generation of a quasi-Bessel beam (QBB) using an on-chip axicon made at the end facet of a planar waveguide to mimic light sheet illumination. This is extended to the implementation of a counter propagating QBB for lattice light-sheet applications. The double axicon, a derivative of the axicon generates superimposed Bessel beams (SBB). Its waveguide-based implementation is proposed and analyzed. Finally, we investigate an optical phased array (OPA) approach to allow dynamic steering of the output light in the free-space. The aim of this study is to find suitable waveguide design parameters for free-space beam shaping operating in the visible spectrum opening possibilities for three-dimensional chip-based optical microscopy.

This article presents an application of the digital imaging in the reading of a slide in optical microscopy, by applying a photometric correction of images issued out of the microscope, an interpolation of images captured by a CDD sensor (decoding) and the implementation of a mosaicking in order to obtain a virtual slide of big size. This study is particularly decisive in a context of telediagnosis applications in optical microscopy, using this type of sensor and manipulating images of large dimensions. We also propose some solutions which improve the efficiency of the telediagnosis chain, and the quality of the visualized images.

In this work, we present the special non-diffracting beams analysis by digital holography. The experimental realizations of non-diffracting beams (Bessel, Mathieus and superposition of co-propagating Bessel beams - Frozen Waves) are made through computer generated holograms reproduced in spatial light modulators (SLMs). And, the phase and intensity analysis of these special non-diffracting beams by digital holography. The results are in agreement with the theoretical predictions and are presenting excellent prospects for this beam type analysis with potential applications in optical micromanipulation, optical microscopy and optics communications.

Optical microscopy is widely used to visualize fine structures. When applied to bioimaging, its performance is often degraded by sample-induced aberrations. In recent years, adaptive optics (AO), originally developed to correct for atmosphere-associated aberrations, has been applied to a wide range of microscopy modalities, enabling high- or super-resolution imaging of biological structure and function in complex tissues. Here, we review classic and recently developed AO techniques and their applications in optical microscopy.

The real-time detection of objects in optical microscopy allows their direct manipulation, which has recently become a new tool for the control, e.g., of active particles. For larger heterogeneous ensembles of particles, detection techniques are required that can localize and classify different objects with strongly inhomogeneous optical contrast at video rate, which is often difficult to achieve with conventional algorithmic approaches. We present a convolutional neural network single-shot detector which is suitable for real-time applications in optical microscopy. The network is capable of localizing and classifying multiple microscopic objects at up to 100 frames per second in images as large as 416 times 416 pixels, even at very low signal-to-noise ratios. The detection scheme can be easily adapted and extended, e.g., to new particle classes and additional parameters as demonstrated for particle orientation. The developed framework is shown to control self-thermophoretic active particles in a heterogeneous ensemble selectively. Our approach will pave the way for new studies of collective behavior in active matter based on artificial interaction rules.

Based on the vector Debye theory, the tight focusing properties of a high-order polarized anomalous vortex (HPAV) beam are studied. The corresponding mathematical expressions of the HPAV beam are derived theoretically. We accomplish the inner and outer gear shapes of the focusing intensity where the number of the gear tooth can be modulated by polarization order. The results show that the focusing gear intensity can be flexibly modulated by initial polarization azimuth which may determine the trapping effects. Various charming focusing field patterns can be used to capture two kinds of different refractive indices particles simultaneously. The compactness of the intensity distribution can be freely adjusted by the HPAV beam topological charges and polarization order. The focal spot size, which is far beyond the Rayleigh diffraction limitation can be achieved. It may be expected to have potential applications in optical microscopy, imaging, optical telecommunication and other fields.

Recent advances in microchannel plate technology and their application to image intensification have made real-time, quantum-limited imaging a practical reality for applications in optical microscopy. Detectors employing both optical and electrical outputs allow systems to be configured for video rate or real-time data acquisition. Properly implemented in conjunction with digital image processing systems, these detectors are capable of accurately imaging single photons and, through temporal accumulation, producing quantitative, high-quality images over an exceptionally wide dynamic range.

Rapid and facile screening techniques to determine the effectiveness of solvents for cellulose or biomass dissolution can advance biomass processing research. Here, we report the use of a simple optical microscopy method to screen potential cellulose and lignin solvents. The described methodology was used to screen the dissolution of cellulose and lignin in two imidazolium-based ionic liquids (ILs), two phosphonium-based ILs, as well as a N,N-dimethylacetamide/lithium chloride (DMAc/LiCl) solution in less time than other techniques. The imidazolium-based ILs and the DMAc/LiCl were found to dissolve both cellulose and lignin. Also, it was observed that one of the phosphonium-based ILs dissolved lignin and not cellulose, demonstrating a potential for biomass fractionation applications. © 2011 Canadian Society for Chemical Engineering

The simultaneous recording of fluorescence intensities at two different wavelengths has a variety of applications in optical microscopy, particularly for ratiometric measurements to sense environmental conditions or for measuring fluorescence resonance energy transfer to probe protein conformation. We demonstrate that a Czerny–Turner monochromator can serve as a platform for dual-color imaging by inserting into the monochromator turret a glass wedge with different dichroic coatings on each side. The presence of a grating, a mirror, and the wedge on a single turret allows rapid switching between spectroscopy and different imaging modes, and the reflective optics in the monochromator minimizes chromatic aberrations.

Shaping perfect optical vortex with amplitude modulated using a digital micro-mirror device

The need for high-resolution metrology to verify lithography compliance with shrinking CD and overlay specifications has resulted in a variety of roadmap sanctioned approaches; notably, SEM, AFM and scatterometry. The application of optical microscopy has been relegated to overlay metrology. While high-resolution metrology is essential to the setup and qualification of lithographic processes, it is often ill suited to the demands of dynamic lithography control. The path to improved CD compliance is through improved on- produce dose and focus control. In control applications, enhanced sensitivity to what we are trying to control becomes a new handle on precision-to-tolerance. Sensitivity must be accompanied by ease of setup, speed and sampling. Modeling for correctable components of variation becomes an integral part of the metrology function. We show that the old dog optical microscopy can learn new tricks to encompass the control of dose and focus. In doing so it becomes the lead candidate for an integrated metrology solution.