Optical-resolution Microscopy Research Articles

Graphene synthesized using filtered cathodic vacuum arc technique and its applications

Amorphous carbon (a-C) films of varying thicknesses (0.5–18 nm) were deposited by the filtered cathodic vacuum arc technique on Ni/SiO2/Si substrate and then annealed in vacuum at 800 °C for 15 min and cooled down to room temperature to obtain graphene. The samples were characterized by Raman spectroscopy, transmittance, high resolution transmission electron microscope (HRTEM), optical microscopy and atomic force microscopy (AFM). Raman spectra showed that the values of I2D/IG, ID/IG and full width at half maximum of D and 2D peaks were in the range 0.18–0.51, 0.82–1.02, 104-208.1 cm−1 and 101.2–128.0 cm−1, respectively, for varying thicknesses of graphene. The transmittance decreases from 97 to 63.7% with the increase in thickness of prepared graphene film. The mobility of ∼725.4 cm2/V.s of field effect transistor using graphene synthesized from a-C film of 3 nm thickness has been obtained. HRTTEM and AFM image revealed monolayer graphene synthesized from a-C film of 1 nm thickness.

Supercontinuum-based Fourier transform infrared spectromicroscopy

Fourier-transform infrared (FTIR) spectromicroscopy combines the spatial resolution of optical microscopy with the spectral selectivity of vibrational spectroscopy. Synchrotron sources can provide diffraction-limited beams in the infrared, and therefore synchrotron-based FTIR spectromicroscopy is nowadays an indispensable tool for biology and materials science studies where high spatial resolution is required. However, the increasing need for accurate and highly spatially resolved characterization is calling for alternative laboratory-based sources to complement synchrotron radiation. To date, the low brightness of thermal emitters or high temporal coherence and narrow bandwidth or tunability of laser sources have hindered the progress of bench-top FTIR spectromicroscopy. Here, we demonstrate that fiber-based supercontinuum sources in the mid-infrared enable fast spectral mapping of localized material properties with close to diffraction-limited resolution (3 μm×3 μm) and pave the way to table-top, on-demand, fast, and highly spatially resolved studies. We illustrate these capabilities by imaging thin sections of human liver samples and compare the results and performance with those obtained using a synchrotron source.

Investigation on the InAs1–xSbx epilayers growth on GaAs (001) substrate by molecular beam epitaxy * * Project supported by the Polish National Science Centre (No. UMO-2015/17/B/ST5/01753).

Undoped and Be-doped InAs1–xSbx (0 ≤ x ≤ 0.71) epitaxial layers were successfully grown on lattice mismatched semi-insulating GaAs (001) substrate with 2° offcut towards <110>. The effect of the InAs buffer layer on the quality of the grown layers was investigated. Moreover, the influence of Sb/In flux ratio on the Sb fraction was examined. Furthermore, we have studied the defects distribution along the depth of the InAsSb epilayers. In addition, the p-type doping of the grown layers was explored. The InAsSb layers were assessed by X-ray diffraction, Nomarski microscopy, high resolution optical microscopy and Hall effect measurement. The InAs buffer layer was found to be beneficial for the growth of high quality InAsSb layers. The X-ray analysis revealed a full width at half maximum (FWHM) of 571 arcsec for InAs 0.87Sb0.13. It is worth noting here that the Hall concentration (mobility) as low (high) as 5 × 1016 cm−3 (25000 cm2V−1s−1) at room temperature, has been acquired.

Photopyroelectric Calorimetry Investigations of 8CB Liquid Crystal–Microemulsion System

In this work, the photopyroelectric technique has been used to investigate the phase transitions in a liquid crystal microemulsion by combining the simultaneous high temperature resolution thermal diffusivity measurements and optical polarization microscopy observations. It has been found that, during the conversion from the isotropic phase into the nematic one, the micelles are expelled from the nematic domains and remain confined in islands of isotropic material which survive down to the smectic temperature range. A hysteresis in the thermal diffusivity profiles between heating and cooling run over the isotropic–nematic transition temperature range has been observed which has been ascribed to the different micelles distribution into the sample volume during cooling and heating runs. Finally, the almost bulk-like behavior of the thermal diffusivity over the nematic–smectic phase transition confirms that a significant fraction of the micelles are expelled during the nucleation of the nematic phase.

Unique insight into protein-DNA interactions from single molecule atomic force microscopy

Protein-DNA interactions are pivotal for many essential biological processes. Atomic force microscopy (AFM) imaging of protein-DNA systems involved in DNA target site search, identification, and processing by proteins has contributed invaluable information to our understanding of the underlying mechanisms. The single molecule 3D resolution of AFM enables us to uncover stoichiometries and conformational properties of protein-DNA complexes. Its molecular resolution places AFM at the interface between the atomic resolution achievable by crystallography and the comparably poor (typically &gt; hundred nanometers) spatial resolution of optical microscopy. Furthermore, the transient character of protein interactions with nonspecific DNA sites, for example during their target site search renders these complexes difficult to resolve by standard ensemble methods. Here, we review current applications and capabilities of as well as novel advances in AFM imaging in protein-DNA interaction studies.

Достоверность выявления пронизывающих дислокаций в эпитаксиальных пленках с помощью структурно-чувствительного травления

AbstractCorrespondence between threading dislocations (TDs) in epitaxial films and the etch pits observed upon selective chemical etching of the samples was studied in Ge/Si(001) heterostructures. It is established that the density of TDs revealed in epitaxial films with thicknesses h ≤ 1 μm can be significantly understated because of insufficient resolution of optical microscopy. Recommendations are given that increase the reliability of PD density estimation by means of structure-sensitive etching.

The past, present and future of palynology in China&amp;mdash;Concentrate on Quaternary palynology

Palynology is a branch of botany with an increasingly important role in the development of modern nature science. It was originally established in Europe during the early twentieth century and has flourished in China since the 1950s. This paper discusses the development of palynology in China from the historical, contemporaneous, and future perspectives. Specifically, we focus on the development of Quaternary palynology since 1979. Quaternary palynology involves six research fields: plants and environment, Loess Plateau, ocean, the uplift of the Tibetan Plateau, global climate change, and environmental archaeology in this paper. Palynologists who study environmental changes mainly focus on the relationships among pollen, vegetation, and environment. The high-resolution analyses of pollen records in the surface soil and sediment profiles of Loess Plateaus have promoted the development of information about paleovegetation and paleoenvironment and enabled the comparison of past climate changes and effects of human activities with global changes. By analyzing pollen data from surface water samples and marine drilling cores, palynologists have made significant progress in elucidating the high-resolution evolutionary history of paleovegetation, paleoclimate, and paleomonsoon in South China Sea and its surrounding regions on the time scales of several million years. Such progress has benefitted research on global sea level changes. The uplift of the Tibetan Plateau has been a hot area of research for several years. Palynologists have explored the vegetational, climatic, and environmental features of different periods, such as the Late Glacial, the Last Glacial Maximum, the Younger Dryas, and the Holocene Megathermal, to reveal the mechanism and process of the uplift of the Tibetan Plateau. The application of Quaternary palynology to environmental archeology not only facilitates understanding of the needs of the economic life and food structure of ancestral humans, the evolution of paleoenvironment and the rise and fall of culture, but also promotes current research on the origin of human, agricultural and civilization. Although Quaternary palynology has made remarkable achievements in many research fields, three problems require urgent solutions. First, the low magnification and resolution of optical microscopy complicate palynological identification on the species level and introduce a degree of uncertainty to the reconstruction of paleovegetation, paleoenvironment and paleoclimate. Second, pollen does not show a simple linear relationship with vegetation. Thus, a reliable pollen-vegetation relationship model remains to be established. Third, research on modern pollen processes, which includes pollen production, transportation, source, deposition, and preservation, has a crucial role in the correct explanation of pollen data and quantitative reconstruction of the paleovegetation and paleoenvironment. Global changes and the impact of environmental evolution have provided excellent opportunities and new challenges for the development of Quaternary palynology in the new century. These opportunities and challenges are as follows: First, the accuracy of pollen identification must be improved to establish a reliable pollen-vegetation relationship model. Second, systemic theoretical studies on modern pollen processes, such as pollen production, transportation, source, deposition, and preservation, should be conducted using surfaces, atmosphere, water, and rock as media. Third, the reconstruction of paleovegetation and paleoclimate from the qualitative and quantitative analyses of high-resolution pollen assemblages should receive additional efforts. Pollen records should be analyzed using various mathematical methods. Finally, to distinguish the role of climate change and human activities in vegetation and climate dynamics, human land use data, surface and fossil pollen data, and meteorological data can be combined to quantify paleovegetation and land use patterns using appropriate mathematical models. Therefore, only by relying on the integration of multidisciplinary data and academic cooperation with colleagues at home and abroad can we further improve the Quaternary palynological research in China and better serve the needs of our society and national strategies.

Synaptic phosphorylated α-synuclein in dementia with Lewy bodies.

Dementia with Lewy bodies is characterized by the accumulation of Lewy bodies and Lewy neurites in the CNS, both of which are composed mainly of aggregated α-synuclein phosphorylated at Ser129. Although phosphorylated α-synuclein is believed to exert toxic effects at the synapse in dementia with Lewy bodies and other α-synucleinopathies, direct evidence for the precise synaptic localization has been difficult to achieve due to the lack of adequate optical microscopic resolution to study human synapses. In the present study we applied array tomography, a microscopy technique that combines ultrathin sectioning of tissue with immunofluorescence allowing precise identification of small structures, to quantitatively investigate the synaptic phosphorylated α-synuclein pathology in dementia with Lewy bodies. We performed array tomography on human brain samples from five patients with dementia with Lewy bodies, five patients with Alzheimer's disease and five healthy control subjects to analyse the presence of phosphorylated α-synuclein immunoreactivity at the synapse and their relationship with synapse size. Main analyses were performed in blocks from cingulate cortex and confirmed in blocks from the striatum of cases with dementia with Lewy bodies. A total of 1 318 700 single pre- or postsynaptic terminals were analysed. We found that phosphorylated α-synuclein is present exclusively in dementia with Lewy bodies cases, where it can be identified in the form of Lewy bodies, Lewy neurites and small aggregates (<0.16 µm3). Between 19% and 25% of phosphorylated α-synuclein deposits were found in presynaptic terminals mainly in the form of small aggregates. Synaptic terminals that co-localized with small aggregates of phosphorylated α-synuclein were significantly larger than those that did not. Finally, a gradient of phosphorylated α-synuclein aggregation in synapses (pre > pre + post > postsynaptic) was observed. These results indicate that phosphorylated α-synuclein is found at the presynaptic terminals of dementia with Lewy bodies cases mainly in the form of small phosphorylated α-synuclein aggregates that are associated with changes in synaptic morphology. Overall, our data support the notion that pathological phosphorylated α-synuclein may disrupt the structure and function of the synapse in dementia with Lewy bodies.

Toward an Axial Nanoscale Ruler for Fluorescence Microscopy.

In the discussion of resolution in optical microscopy, axial precision has often come second to its lateral counterpart. However, biological systems make no special arrangements for our preferred direction of imaging. The ability to measure axial distances, that is, the heights of fluorophores relative to a plane of reference, is thus of paramount importance and has been the subject of several recent advances. A novel method is to modify the fluorescence emission based on the height of the individual fluorophore, such that its z-position is encoded somehow in the detected signal. One such approach is metal-enhanced energy transfer, recently extended to multicolor distance measurements and applied to study the topography of the nuclear membrane. Here, the fluorescence lifetime is shortened due to the proximity of the fluorophores to a thin metallic surface. Fluorescence lifetime imaging can therefore be used as an axial ruler with nanometer precision.

Polyamide Nanofibers Coating by Electrospinning Technique for Anti Corrosion Behavior

Poly amide (PA-6) nanofiber coatings on aluminum surface using electrospinning technique under two different voltage (24 kV & 34 Kv) were prepared. The coating morphology , roughness and 3D structural properties tested by using Atomic force microscopes (AFM), surface characterized by (SEM) microscopy, and high resolution optical microscopy (HROM) , Contact angle for hydrophobic behavior was tested by shape drop analyzer , and FTIR analysis for changing in crystalline structure was performed .The AFM images showed that the PA coating on the aluminum surface have a tight and twisted nanofiber structure with some beads through its morphology. SEM images shows the morphology beads of surface nanofibers . The electrochemical corrosion of aluminum without and with PA coating studied by subjected it to aerobic solutions of 3.5 wt.% sodium chloride (NaCl) . It has been found that the PA coatings decrease the corrosion currents and corrosion rate as well as increase the corrosion resistance for aluminum in the NaCl solution .

Abstract 1866: Ultrahigh resolution optical coherence microscopy for label-free imaging of human cervical lesions ex vivo

Abstract Cervical cancer remains the fourth most common cause of cancer and the fourth leading cause of cancer death in women worldwide. In contrast to the decreasing prevalence in developed countries, incidence of cervical cancer remains high in developing countries, such as China, India and Chile. Effective screening of cervical cancer is crucial for early diagnosis of the disease. Ultrahigh resolution optical coherence microscopy (UHR OCM) is an emerging label free imaging technique that can be used to obtain three dimensional (3D) “optical biopsy” of biological samples with cellular resolution up to 2 mm beneath the tissue surface. The goal of this study is to assess the diagnostic value of UHR OCM for human cervical precancer and cancer screening and identify characteristic features in UHR OCM images that correlate with the gold standard histopathology. Six hundred and fifty eight (658) fresh cervical tissues from cervical biopsy, conization and panhysterectomy were obtained from 254 consenting volunteers. Multiple 3D UHR OCM images were obtained from each specimen before the sample was processed for standard histopathology evaluation. Of the 658 samples imaged, 474 had normal cervix or benign lesions, 110 had low grade squamous intraepithelial lesion (LSIL), 57 had high grade squamous intraepithelial lesions (HSIL) and 17 had invasive cancer. UHR OCM images were compared to histopathology slides obtained from the same specimens in order to identify diagnostic image features. Distinctive patterns for squamous epithelium, columnar epithelium, basement membrane, cervical inflammation and erosion, koilocytic cells, low grade and high grade squamous intraepithelial lesions and cervical cancer were clearly observed. Our results lay the foundation for future non-invasive optical evaluation of cervical tissue in vivo. This could lead to a less invasive and more effective screening strategy for cervical cancer based on OCM guided biopsies. Citation Format: Xianxu Zeng, Canyu Li, Xiaofang Wang, Jason Jerwick, Tao Xu, Yuan Ning, Linlin Zhang, Yutao Ma, Xiaoan Zhang, Chao Zhou. Ultrahigh resolution optical coherence microscopy for label-free imaging of human cervical lesions ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2017-1866

Hematuria en la niñez: revisión sistemática cualitativa

Introducción: la hematuria aislada en niños es un hallazgo frecuente en la práctica clínica diaria del médico general y el pediatra, y un reto diagnóstico para averiguar su etiología y manejo. Objetivo: describir las pautas básicas para el diagnóstico y manejo de la hematuria aislada en menores de 18 años por el médico general y el pediatra. Materiales y métodos: se realizó una búsqueda bibliográfica sistemática en las bases de datos PubMed, ScienceDirect, LILACS y Embase, utilizando palabras claves del DeCS (español) y MeSH (inglés), mediante las combinaciones con la conjunción “AND” o la disyunción “OR”, de manuscritos tipo estudios observacionales, ensayos clínicos, guías de práctica clínica y revisiones sistemáticas, publicados entre 1995 y 2017, que describieran el diagnóstico y el manejo básico de la hematuria. La calidad de los artículos fue evaluada por medio de los instrumentos PRISMA, CONSORT y STROBE, según correspondiera. Resultados: se identificaron 402 publicaciones, de las cuales 34 cumplieron con los criterios y 28 fueron seleccionadas para realizar esta revisión. Conclusiones: la hematuria aislada se define por el hallazgo de cinco eritrocitos por campo de alto poder en orina fresca centrifugada o más de cinco eritrocitos por microlitro en orina fresca no centrifugada. El diagnóstico y tratamiento se realiza por pasos: a) historia clínica y b) confirmación por microscopía óptica de alta resolución. Diferenciar la hematuria benigna de las que requieren paraclínicos de extensión es primordial para confirmarla más tempranamente, evitar procedimientos invasivos, disminuir el gasto en salud y hacer un seguimiento predictivo.

Nanoscale simultaneous chemical and mechanical imaging via peak force infrared microscopy.

Nondestructive chemical and mechanical measurements of materials with ~10-nm spatial resolution together with topography provide rich information on the compositions and organizations of heterogeneous materials and nanoscale objects. However, multimodal nanoscale correlations are difficult to achieve because of the limitation on spatial resolution of optical microscopy and constraints from instrumental complexities. We report a novel noninvasive spectroscopic scanning probe microscopy method-peak force infrared (PFIR) microscopy-that allows chemical imaging, collection of broadband infrared spectra, and mechanical mapping at a spatial resolution of 10 nm. In our technique, chemical absorption information is directly encoded in the withdraw curve of the peak force tapping cycle after illumination with synchronized infrared laser pulses in a simple apparatus. Nanoscale phase separation in block copolymers and inhomogeneity in CH3NH3PbBr3 perovskite crystals are studied with correlative infrared/mechanical nanoimaging. Furthermore, we show that the PFIR method is sensitive to the presence of surface phonon polaritons in boron nitride nanotubes. PFIR microscopy will provide a powerful analytical tool for explorations at the nanoscale across wide disciplines.

Ultrastructure of cocoa fruits ( Theobroma cacao ) of cultivars with contrasting susceptibility to Moniliophthora roreri

Moniliasis of cocoa is an important disease caused by the fungus Moniliophthora roreri (Cif & Park) (Evans et al 1978). A study was conducted on the external features of fruits of two cocoa cultivars with different degrees of susceptibility to M. roreri also a comparative study of the exocarp tissues of healthy fruits was conducted. Scanning Electron Microscopy (SEM) and High Resolution Optical Microscopy (HROM) were used respectively. For SEM, sixty-day-old healthy fruits of cvs Pound-7 (susceptible) and UF-296 (moderately resistant) were collected from CATIE's germplasm collection in Turrialba and La Lola, Costa Rica. Apparently, no external anatomic differences between the fruits of the Pound 7 and the UF-296 (moderately resistant) cultivars exist, except for the star-like trichomes in the latter. Both show an alveolar epidemis on which there are noticeable anomocitic-type stomata and a large number of glandular trichomes. For HROM, healthy Pound-7 and UF-296 fruits from La Lola were used, as well as cv. UF-273 resistant to the pathogen. The major histological feature of cv UF-273 and UF-296 was a cellular arrangement of the subepidermic parenchyma which was more compact, probably containing larger amounts of phenolic substances in the vacuoles, as compared to the Pound 7.

Review of interferometric spectroscopy of scattered light for the quantification of subdiffractional structure of biomaterials.

Optical microscopy is the staple technique in the examination of microscale material structure in basic science and applied research. Of particular importance to biology and medical research is the visualization and analysis of the weakly scattering biological cells and tissues. However, the resolution of optical microscopy is limited to ? 200 ?? nm due to the fundamental diffraction limit of light. We review one distinct form of the spectroscopic microscopy (SM) method, which is founded in the analysis of the second-order spectral statistic of a wavelength-dependent bright-field far-zone reflected-light microscope image. This technique offers clear advantages for biomedical research by alleviating two notorious challenges of the optical evaluation of biomaterials: the diffraction limit of light and the lack of sensitivity to biological, optically transparent structures. Addressing the first issue, it has been shown that the spectroscopic content of a bright-field microscope image quantifies structural composition of samples at arbitrarily small length scales, limited by the signal-to-noise ratio of the detector, without necessarily resolving them. Addressing the second issue, SM utilizes a reference arm, sample arm interference scheme, which allows us to elevate the weak scattering signal from biomaterials above the instrument noise floor.

Influences of matrix viscosity on alignment of multi-walled carbon nanotubes in one-dimensional confined space

The effects of matrix viscosity on alignment of multi-walled carbon nanotubes (MWCNTs) and physical properties of high density polyethylene (HDPE)/MWCNTs composite nanofibers via nanolayer coextrusion are studied. The alignment states are altered depending upon the matrix viscosity of the composite nanofibers. The morphology and nano-sized diameter of the nanofibers are observed on scanning electron microscope. To characterize the prioritized alignment and dispersion of MWCNTs in the matrix, transmission electron microscopy, polarized Raman spectra, and high resolution optical microscopy are used. It is found that the alignment of MWCNTs in higher viscosity matrix of the nanofibers is better than that in lower viscosity. The aligned MWCNTs composite nanofibers have smaller slope variations of rheological curves with increasing content of MWCNTs. In addition, the higher aligned HDPE/MWCNTs composite nanofibers exhibit higher decomposition temperature and enhanced mechanical properties. However, the poorly aligned nanofibers have higher electrical and thermal conductivities due to the easily formed three-dimensional network.

Image scanning microscopy: an overview.

For almost a century, the resolution of optical microscopy was thought to be limited by Abbé's law describing the diffraction limit of light. At the turn of the millennium, aided by new technologies and fluorophores, the field of optical microscopy finally surpassed the diffraction barrier: a milestone achievement that has been recognized by the 2014 Nobel Prize in Chemistry. Many super-resolution methods rely on the unique photophysical properties of the fluorophores to improve resolution, posing significant limitations on biological imaging, such as multicoloured staining, live-cell imaging and imaging thick specimens. Structured Illumination Microscopy (SIM) is one branch of super-resolution microscopy that requires no such special properties of the applied fluorophores, making it more versatile than other techniques. Since its introduction in biological imaging, SIM has proven to be a popular tool in the biologist's arsenal for following biological interaction and probing structures of nanometre scale. SIM continues to see much advancement in design and implementation, including the development of Image Scanning Microscopy (ISM), which uses patterned excitation via either predefined arrays or raster-scanned single point-spread functions (PSF). This review aims to give a brief overview of the SIM and ISM processes and subsequent developments in the image reconstruction process. Drawing from this, and incorporating more recent achievements in light shaping (i.e. pattern scanning and super-resolution beam shaping), this study also intends to suggest potential future directions for this ever-expanding field.

A processing method with high efficiency for low density polyethylene nanofibers reinforced by aligned carbon nanotubes via nanolayer coextrusion

A method for large-scale processing of low density polyethylene (LDPE) nanofibers reinforced by aligned multi-walled carbon nanotubes (MWCNTs) is achieved via nanolayer coextrusion. The morphology and nano-sized diameter of the nanofibers are observed on scanning electron microscope. The prioritized alignment and dispersion of MWCNTs along the fiber axis are determined by transmission electron microscopy, polarized Raman spectra, and high resolution optical microscopy. The dispersion of MWCNTs in nanofibers is better than that in homologous micron-fibers. In addition, MWCNTs can promote the crystallization of LDPE in varying degrees which depend on the fiber diameter and MWCNTs content. As a result of the alignment of MWCNTs, the nanofibers exhibit enhanced mechanical properties. The tensile strength of LDPE(1 wt% MWCNTs) nanofibers is more than twice of pure nanofibers (without MWCNTs). The volume resistivity of the LDPE(MWCNTs) nanofiber mats containing 9 wt% MWCNTs can be reduced to 100 Ω cm.

Recent progress on super-resolution imaging and correlative super-resolution microscopy

The diffraction of the finite aperture in the optical imaging system restricts further improvement of the resolution of optical microscopy, which is called the diffraction limit. Since raised by Ernst Abbe in 1873, the problem of diffraction limit has been one of the foci of academic research. In recent years, with the rapid development of related fields such as the development of optoelectronic devices including high energy lasers and high sensitivity detectors and the development of new fluorescent probes, the problem of diffraction limit in optical microscopy ushered in a new opportunity, and super-resolution microscopy (SRM) has made remarkable achievements in the past decade. The basic principles of diffraction limited resolution in both space and frequency domains are reviewed, and on this basis, the mechanisms for the various SRM technologies to circumvent the diffraction limit and improve the resolution are explained in detail. The development trends and research directions of various SRM techniques are also introduced. As a new and important development trend of SRM, correlative super-resolution microscopy and its recent progress are reviewed, including correlative studies on SRM and time-lapse live cell fluorescence microscopy, fluorescence lifetime imaging microscopy, spectrometry and spectroscopy, electron microscopy, atomic force microscopy, etc. The role and significance of various correlative super-resolution microscopy are discussed. The future development of super-resolution microscopy and correlative super-resolution microscopy is also prospected.

Generalized vector wave theory for ultrahigh resolution confocal optical microscopy.

Polarization modulation of a tightly focused beam in a confocal imaging scheme is considered for incident and collected light fields. Rigorous vector wave theory of a confocal optical microscopy is developed, which provides clear physical pictures without the requirement for fragmentary calculations. Multiple spatial modulations on polarization, phase, or amplitude of the illuminating and the detected beams can be mathematically described by a uniform expression. Linear and nonlinear excitation schemes are derived with tailored excitation and detection fields within this generalized theory, whose results show that the ultimate resolution achieved with the linear excitation can reach one-fifth of the excitation wavelength (or λ/5), while the nonlinear excitation scheme gives rise to a resolution better than λ/12 for two-photon fluorescence excitation and λ/20 for three-photon fluorescence excitation. Hence the resolution of optical microscopy with a near-infrared excitation can routinely reach sub-60 nm. In addition, simulations for confocal laser scanning microscopy are carried out with the linear excitation scheme and the fluorescent one, respectively.