Label-free quantitative phase imaging and analysis of airborne pollen

DUV lithography has successfully adopted both bright and dark mask tonalities. This gives the freedom to chip manufacturers to choose the optimum combination of mask and resist tonality for their product [1]. In EUV lithography, however, there has been a clear preference for dark field masks, driven by the prevalence of positive tone resist processes, and their relative insensitivity to multilayer defects. Future customer nodes, however, may require negative tone (metal-oxide) resist processes [2][3], resulting in a requirement to use bright field masks. Therefore, a deeper understanding of bright and dark field imaging is needed in order to provide guidance to ASML customers in choosing the optimal approach. In this work we consider the fundamentals of bright and dark field imaging based on the diffraction theory of aerial image formation [4]. We will show that bright field imaging has an intrinsic potential for higher optical NILS (normalized image log-slope), especially for isolated features, but with a lower depth of focus. The theoretical results are compared to rigorous simulations. Experimental bright vs dark-field results is also presented for comparison. Wafer based data has been obtained on an NXE:3400 scanner, whilst aerial image measurements have been obtained using the Aerial Image Measurement System for EUV (AIMS® EUV) at Zeiss. These experimental results confirm the theoretical expectations. The main goal of the paper is to draw attention to bright versus dark field comparison for EUV and to kick off more studies in this direction.

We report on a single capture approach for simultaneous incoherent bright field (BF) and laser-based quantitative phase imaging (QPI). Common-path digital holographic microscopy (DHM) is implemented in parallel with BF imaging within the optical path of a commercial optical microscope to achieve spatially multiplexed recording of white light images and digital off-axis holograms, which are subsequently numerically demultiplexed. The performance of the proposed multimodal concept is firstly determined by investigations on microspheres. Then, the application for label-free dual-mode QPI and BF imaging of living pancreatic tumor cells is demonstrated.

1. The optical transparency of unstained live cell specimens limits the extent to which information can be recovered from bright-field microscopic images because these specimens generally lack visible amplitude-modulating components. However, visualization of the phase modulation that occurs when light traverses these specimens can provide additional information. 2. Optical phase microscopy and derivatives of this technique, such as differential interference contrast (DIC) and Hoffman modulation contrast (HMC), have been used widely in the study of cellular materials. With these techniques, enhanced contrast is achieved, which is useful in viewing specimens, but does not allow quantitative information to be extracted from the phase content available in the images. 3. An innovative computational approach to phase microscopy, which provides mathematically derived information about specimen phase-modulating characteristics, has been described recently. Known as quantitative phase microscopy (QPM), this method derives quantitative phase measurements from images captured using a bright-field microscope without phase- or interference-contrast optics. 4. The phase map generated from the bright-field images by the QPM method can be used to emulate other contrast image modes (including DIC and HMC) for qualitative viewing. Quantitative phase microscopy achieves improved discrimination of cellular detail, which permits more rigorous image analysis procedures to be undertaken compared with conventional optical methods. 5. The phase map contains information about cell thickness and refractive index and can allow quantification of cellular morphology under experimental conditions. As an example, the proliferative properties of smooth muscle cells have been evaluated using QPM to track growth and confluency of cell cultures. Quantitative phase microscopy has also been used to investigate erythrocyte cell volume and morphology in different osmotic environments. 6. Quantitative phase microscopy is a valuable, new, non-destructive, non-interventional experimental tool for structural and functional cellular investigations.

The objective of this study was to investigate pollen morphology and ranges of intraspecific variability of Abies alba Mill. Pollen grains were collected from nine clonal seed orchards of A. alba in the Sudety Mountains, (South-Western Poland). At each seed orchard, 4–6 grafts were selected. Each individual (graft) was represented by 30 pollen grains and 1440 pollen grains were measured totally. Eight quantitative and four qualitative features of pollen grains were analysed. The diagnostic features of pollen grains for the studied species were: Exine surface of pollen corpus (cappa and leptoma) and sacci, the length of the polar axis (P), pollen shape (P/E ratio), and a new trait—saccus shape (A/B ratio — saccus width (A) to his length (B)). Pollen features made possible to differentiate seven individual genotypes (samples). To our knowledge, this is the first time that the intraspecific and interindividual variability of pollen grains of A. alba were investigated. The most different were the pollen grains from samples—genotypes 13 (Bystrzyca Kłodzka) and 18 (Jugów), and also (although to a lesser extent) genotypes—11 (Kamienna Góra), 30, 31 (Jugów), and 44 (Szklarska Poręba). No significant relationships were observed between the pollen grain traits and the geographical location of the collection sites.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells invitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression invivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

Quantitative comparison of bright field and annular bright field imaging modes for characterization of oxygen octahedral tilts

An experimental evaluation of bright and dark field imaging modes in high voltage electron microscopy

The aim of this study is verification of the taxonomic usefulness of the pollen grain features studied, based on pollen morphology of 32 wild species from all 4 subgenera and all 10 sections of the genus Rosa, mainly for delimitation of subgenera, sections, and species. The measurements and observations were carried out with both light microscopy and scanning electron microscopy. Only correctly formed pollen grains (30 per specimen) were measured, and 960 pollen grains were examined in total. They were analyzed for 14 quantitative features of pollen grains and exine sculpturing and the following qualitative ones: outline, shape, and operculum structure. Our study revealed that the diagnostic features of pollen grains studied were: exine sculpture, length of polar axis, and pollen shape (P/E ratio). On the basis of the above characters, five species were isolated and the remaining ones were included in several groups isolated on the basis of exine sculpture types. The following three exine sculpture types occurred in the species studied: granular-verrucate (in R. stellata), striate-psilate (in R. multibracteata and R. multiflora), and striate (the remaining species). R. banksiae is characterized by small pollen grains, while R. setigera has strongly elongated pollen with P/E ratio >1.5. Exine sculpture features considered to be diagnostic should be treated as auxiliary because they fail to differentiate individual species, although they can be helpful in distinguishing groups of species of similar exine sculpture. The arrangement of the species examined on a dendrogram only slightly corroborates division of the Rosa genus into subgenera and sections currently adopted in taxonomy (Rehder 1940). An interesting result was reported for the species studied from the Caninae (R. agrestis, R. canina, R. dumalis, R. jundzillii, and R. rubiginosa) section which, despite hybrid nature, with the exception of R. villosa, grouped in the same, most separated group of species.

Quantitative phase imaging (QPI) is an advanced label-free imaging technique that can quantify pathlength changes in biological samples at the nanometer scale without staining or tagging. White blood cells (WBCs) are the key components of the body’s immune system. However, automatic detection and segmentation of WBCs from QPI data can be challenging as QPI may lack the needed intrinsic specificity and contrast compared to a stained brightfield image. Morever, typical supervised learning methods require a large number of annotations for model training which involves time-consuming efforts and clinical expertise, and may not be available often. Motivated by the effectiveness of the recently developed contrastive learning-based semi-supervised segmentation methods and considering the intrinsic properties of QPI, the main contribution of this work is to implement an efficient semi-supervised segmentation method to segment WBCs from QPI data when only a small amount of ground-truth labels are available. A DeeplabV3+ and DeeplabV2 architecture were used as a backbone of the segmentation network with a modified convolution module, specifically to capture the subtle morphological details of WBCs in QPI. The preliminary results showed significant improvement in terms of quantitative metrics compared to the supervised training methods. An exploratory study was also performed to investigate the effect of the contrastive learning module on segmentation performance. To the best of our knowledge, this is the first application of the semi-supervised contrastive learning method in the WBC segmentation framework from QPI.

Multistage morphological segmentation of bright-field and fluorescent microscopy images

High resolution dark field (DF) images of the superstructures of the pyrrhotite (Fe1-xS) and bornite-digenite (Cu5FeS4-Cu9S5) series can be related to structure. Further, they provide more detail than bright field (BF) images. The same objective aperture size and stigmater settings were used for DF as for BF imaging; symmetrical arrangements of diffracted beams in the objective aperture were used. Images that can be related to structure were obtained at the defocus value giving the greatest image contrast, thereby enabling proper defocusing without requiring extensive through-focus series.For the minerals of interest, diffraction patterns consist of many superstructure reflections and a few subcell reflections. BF images contain primarily features of the superstructure, presumably because the subcell reflections fall far from the axis of the objective lens and thus are affected by spherical and chromatic aberrations and beam divergence. Likewise, DF images formed with a similar arrangement of beams as that in BF contain only features of superstructure, but with reverse contrast to BF.

Characterizing the shape of pollen is crucial for plant breeding, taxonomy, and conservation, and studies on pollen viability can help breeding initiatives. This study examined the male fertility and pollen morphology of six Cucurbitaceae species with potential for both economic and nutritional benefit. Pollen grains were acetolyzed for morphological study, and a light compound microscope and an LEICA digital microscope were used to determine the exine stratification as well as other characteristics of pollen grains. The Cucurbitaceae family is eurypalynous, as evidenced by the noticeable differences in pollen physical characteristics between species. The shapes of pollen might be oblate, sub-oblate, or oblate-spheroidal. Pollen viability percentage was determined by using different staining methods-Acetocarmine test, Lugol’s test and Aniline blue test respectively. M. dioica, or spiny gourd, had the highest pollen viability percentage (94.4%), while L. acutangula, or Chinese okra, had the lowest percentage (80.83%) when it was fresh. Using Lugol's test, six Cucurbitaceae species' pollen grains were found to be starchy by nature. In Lugol's test, M. dioica showed the highest percentage of pollen viability, whereas M. charantia showed the lowest percentage of pollen viability following the anthesis of the flowers (at fresh). Species identification relies heavily on pollen morphology, particularly in this diverse family where many species exist. High viability rates encourage seed production and fertilization, which are necessary for effective hybrid development and conservation.

Quantitative phase imaging (QPI) has emerged as a new digital histopathologic tool as it provides structural information of conventional slide without staining process. It is also capable of imaging biological tissue sections with sub-nanometer sensitivity and classifying them using light scattering properties. Here we extend its capability further by using optical scattering properties as imaging contrast in a wide-field QPI. In our first step towards validation, QPI images of 10 major organs of a wild-type mouse have been obtained followed by H&E-stained images of the corresponding tissue sections. Furthermore, we utilized deep learning model based on generative adversarial network (GAN) architecture for virtual staining of phase delay images to a H&E-equivalent brightfield (BF) image analogues. Using the structural similarity index, we demonstrate similarities between virtually stained and H&E histology images. Whereas the scattering-based maps look rather similar to QPI phase maps in the kidney, the brain images show significant improvement over QPI with clear demarcation of features across all regions. Since our technology provides not only structural information but also unique optical property maps, it could potentially become a fast and contrast-enriched histopathology technique.

Quantitative phase imaging (QPI) techniques is an emerging field aimed at studying weakly scattering and absorbing specimens. QPI techniques provide 3-d images with high contrast, compared to qualitative imaging techniques like bright field imaging, phase contrast imaging, differential interference contrast imaging. White light diffraction phase microscopy (wDPM) is one of the QPI techniques which provide both amplitude and phase images simultaneously with single shot. It also uses common path interferometric geometry which provide better temporal sensitivity compared to other interferometric techniques. Also wDPM uses the white light as the illumination source which makes this imaging technique unique in speckle free imaging. The rate of recording the cellular dynamics is only limited by the camera speed. In this work we developed the wDPM techniques and characterized the system in terms of resolution and sensitivity. We also used the Fast phase reconstruction algorithm to reconstruct the 3d phase form the recorded interference pattern. We recorded the interference pattern of E.coli and reconstructed the 3-d phase images with high sensitivity. We also demonstrated the cell division in E.coli using this technique.

We evaluated the printability of patterns relevant for Logic Metal at P28nm (L/S and T2T) on wafer using EUV single expose. We compare illumination sources with and without fading correction as well as Bright field / Dark field mask tonalities and NTD MOR / PTD CAR resist. In simulations, Bright field (BF) imaging gives better image quality than Dark field (DF) at small pitch/CD. It also enables smaller T2T. To avoid tone inversion (assuming dual damascene processing), BF imaging requires the use of a NTD resist. On wafer, exposure latitudes increase for a BF/NTD choice, concurrent with simulations, even after correcting out SEM shrinkage. Also, T2T CD is reduced. In terms of illumination, we compare dipole sources to fading corrected sources. As fading correction, we have both induced aberrations (Z6-corrected dipole) and monopoles. As expected, a fading correction significantly reduces best focus differences of L/S through pitch and T2T. Moreover, the Z6-corrected dipole is optimal to print small T2T with better uniformity. Finally, we observe that PTD and NTD MOR resist utilize the same aerial image differently. NTD resist can leverage pupil shapes with high exposure latitude, but low depth of focus, better than PTD resist. Fading correction via induced aberrations naturally produces such sources. In summary, the preferred option is a Z6-corrected dipole for best focus alignment and sharp T2T, together with BF imaging to allow higher L/S exposure latitudes and small T2T. Combining this choice with NTD MOR resist avoids tone inversion and leverages the illumination source optimally.