Laser Scanning Optical Microscopy Research Articles

Far-Field Super-Resolution Optical Microscopy for Nanostructures in a Reflective Substrate

The resolution of an optical microscope is determined by the overall point spread function of the system. When examining structures significantly smaller than the wavelength of light, the contribution of the background or surrounding environment can profoundly affect the point spread function. This research delves into the impact of reflective planar substrate structures on the system’s resolution. We establish a comprehensive forward imaging model for a reflection-type confocal laser scanning optical microscope, incorporating vector field manipulation to image densely packed nanoparticle clusters. Both theoretical and experimental findings indicate that the substrate causes an interference effect between the background field and the scattered field from the nanoparticles, markedly enhancing the overall spatial resolution. The integration of vector field manipulation with an interferometric scattering approach results in superior spatial resolution for imaging isolated particles and densely distributed nanoscale particle clusters even with deep subwavelength gaps as small as 20 nm between them. However, the method still struggles to resolve nanoparticles positioned directly next to each other without any gap, necessitating further work to enhance the resolving ability. This may involve techniques like deconvolution or machine learning-based post-processing methods.

Ultrahigh-resolution nonlinear optical imaging of the armchair orientation in 2D transition metal dichalcogenides

We used nonlinear laser scanning optical microscopy to study atomically thin transition metal dichalcogenides (TMDs) and revealed, with unprecedented resolution, the orientational distribution of armchair directions and their degree of organization in the two-dimensional (2D) crystal lattice. In particular, we carried out polarization-resolved second-harmonic generation (PSHG) imaging for monolayer WS2 and obtained, with high-precision, the orientation of the main crystallographic axis (armchair orientation) for each individual 120 × 120 nm2 pixel of the 2D crystal area. Such nanoscale resolution was realized by fitting the experimental PSHG images, obtained with sub-micron precision, to a new generalized theoretical model that accounts for the nonlinear optical properties of TMDs. This enabled us to distinguish between different crystallographic domains, locate boundaries and reveal fine structure. As a consequence, we can calculate the mean orientational average of armchair angle distributions in specific regions of interest and define the corresponding standard deviation as a figure-of-merit for the 2D crystal quality.

Non-Invasive Recognition of Poorly Resolved Integrated Circuit Elements

We present a non-invasive method for recognition of components in a digital CMOS integrated circuit (IC). We use a confocal infrared laser scanning optical microscope to collect multimodal images through the backside of the IC. Individual modes correspond to passive reflectivity measurements or active measurements, such as light-induced voltage alteration. The modes are registered and stored in a multidimensional data cube. We apply a machine learning algorithm using a binary representation to identify a variety of data structures from transistors to entire logic cells. Because of the compact representation, objects can be detected rapidly. We show that by increasing the number of imaging modes used to develop the descriptor, we can significantly increase recognition accuracy. The approach allows recognition of poorly resolved components, whose primary distinguishing features are below traditional optical resolution limits, and is general enough to be applied to multiple design processes. We believe this represents a significant step toward a fully non-invasive IC reverse engineering system.

The Materials Challenge in Diffraction‐Unlimited Direct‐Laser‐Writing Optical Lithography

two-photon excitation fluoscence a) The concept of stimulated emission depletion (STED) fl uorescence microscopy introduced by Stefan W. Hell has recently revolutionized the fi eld of optical microscopy [ 1–4 ] and has led to important applications in biology. [ 5 , 6 ] Spectacular lateral spatial resolutions down to less than 10 nm have been reported. [ 7 ] Conceptually, in sharp contrast to what was commonly believed for decades, the “diffraction limit” of far-fi eld optical microscopy is no longer a limit. Obviously, it would be highly desirable to translate this tremendous resolution enhancement in laser-scanning optical microscopy into a corresponding advance in optical lithography , especially in two-photon direct-laser-writing (DLW). [ 8–10 ] DLW can be viewed as the three-dimensional analogue of two-dimensional (planar) electron-beam lithography. If arbitrary nanostructures could be made by STED-DLW with feature sizes of 10 nm in all three dimensions, a nanotechnologists’ dream would come true. However, the step from microscopy to lithography is not simple at all and particularly requires developing suitable photoresist material systems. [ 3 ] Early work on DLW inspired by STED has used one-photon absorption [ 11 ] or one-color schemes. [ 12 ] Both results have been highly encouraging, but they do not yet allow for complete fl exibility in three-dimensional lithography. In contrast to this work, [ 11 , 12 ] our approach is to directly beat the inter-system crossing rate of the photoinitiator molecules by effi cient stimulated emission. As we will argue and show by experiments below, commercially available photoinitiators and photoresists containing them are far from ideal for the purpose of STED-DLW. Thus, in this communication, we present a novel class of photoresist systems, an example of which is successfully applied to DLW. In regular DLW, femtosecond laser pulses are very tightly focused into the volume of a photoresist. [ 8–10 ] By means of twophoton absorption, only a tiny volume is suffi ciently exposed by the light. By computer-controlled scanning of the relative positions of focus and resist via piezoelectric actuators, almost

Design considerations for refractive solid immersion lens: Application to subsurface integrated circuit fault localization using laser induced techniques

With fast scaling and advancement of integrated circuit (IC) technology, circuitries have become smaller and denser. New materials and more sophisticated designs have evolved. These changes reduced the effectiveness of conventional laser induced fault localization techniques. Since IC fault localization is the most critical step in failure analysis, there are strong motivations to improve both spatial resolution and sensitivity of such systems to meet the new challenges from advanced technology. Refractive solid immersion lens (RSIL) is well known to enhance the laser spot size which directly affects resolution and sensitivity in back side fault localizations. In practice, it is difficult to operate RSIL at the ideal configurations to obtain the smallest spot resolution. It is necessary to understand the resolution performance at the other design focal planes. Besides resolution, there are also other factors that affect sensitivity in a RSIL enhanced system. This paper identifies and characterizes key RSIL design parameters to optimize RSIL performance on laser induced techniques. We report that the most efficient conditions are achieved close to aplanatic RSIL design to within 20-25 microm (for a 1 mm diameter lens), and the backing objective should be the minimum numerical aperture required for optimum resolution performance. The size of the mechanical clear aperture opening should be large enough (>80%) to exploit the advantage of aplanatic RSIL. RSIL is developed on a laser scanning optical microscope in this work, and a resolution of 0.3 microm (for a wavelength of 1340 nm) was achieved over a range of operating conditions. A quantitative resolution of 0.25 microm is achieved and a pitch structure of 0.4 microm is easily resolvable. Close to 15 times enhancement in laser induced signal is obtained.

Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation

In imaging anisotropic samples with optical microscopy, a controlled, polarized light source can be used to gain molecular information of fibrous materials such as muscles and collagen fibers. However, the delivery of the polarized excitation light source in a system such as a laser scanning optical microscope often encounters the problem of the polarization ellipticity altering effects of the optical components. Using a half-wave plate and a quarter-wave plate, we demonstrate that the polarization ellipticity altering effect of the dichroic mirror in an epi-illuminated multiphoton laser scanning microscope can be corrected, and that this approach can be used to obtain polarized second-harmonic generation (SHG) images of rat tail tendon and mouse leg muscle. The excitation polarization dependence of the SHG intensity is fitted to determine the ratio of the second-order susceptibility tensor elements associated with type I collagen in the rat tail tendon and myofibril in the mouse leg muscle. Our methodology can be applied to polarized SHG imaging without sample rotation. This approach has great potential for imaging noncentrosymmetric biological samples, providing structural information on the molecular scale in addition to morphological information of tissues.

Flexibility and bending loss of waveguiding molecular fibers self-assembled from thiacyanine dye

The flexibility and waveguiding properties of fiber-shaped aggregates self-assembled from thiacyanine dye molecules in solution were investigated. Sharply bent fibers were prepared by drop-casting a solution containing the fibers on a glass substrate and immediately soaking up the solution. It was found that the fibers could be smoothly bent down to a radius of curvature R ≈ 2 μm. Fluorescence microscopy revealed that the fibers guide their fluorescence along such sharp bends. The bending losses were quantitatively measured using laser scanning optical microscopy.

Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

Unique laser-scanning optical microscope for low-temperature imaging and spectroscopy

Low-temperature optical characterization of single quantum nanostructures can reveal detailed information on structure-dependent properties of these materials. We describe the development of a unique laser-scanning optical microscope capable of low-temperature single molecule/particle imaging and spectroscopy. Making use of the magnification of a microscope objective, the laser- scanning scheme of the present microscope allows for high-repeatability imaging over large sample areas. The microscope is utilized to measure the low-temperature Raman scattering spectra of individual single-walled carbon nanotubes and single molecule fluorescence spectra of conjugated polymers. The developed low-temperature microscope can be applied to study a wide array of nanomaterials at a single particle level.

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy.

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 microm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 degrees C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.

Multi-photon microscopy: seeing more by imaging less.

Since the earliest microscopes of Leuwenhoek and Hooke in the 17th century, optical designers have strived to maximize a key performance characteristic of each new instrument model—that of contrast. Put simply, contrast is how well we can discern useful information about a microscopic specimen from unwanted background and noise. Resolution (the ability to accurately measure features) and sensitivity (the ability to detect features) are intimately related to contrast. The Rayleigh criterion for resolution describes how far apart two tiny objects must be so that are clearly contrasted from the darker space between them. Sensitivity defines the faintest feature that can be contrasted from the background. Why is this seemingly philosophical discussion important? The answer is clear; we can improve both sensitivity and effective resolution by improving contrast between what we want to see and what we don’t. Scanning optical microscopes in general (23) deliver improved contrast in a number of different ways. A regular film or digital camera, mounted on a conventional microscope, takes a snapshot or a complete picture of the desired scene at the press of a button. The laser-scanning (optical) microscope (LSM) is quite different. The picture is built up in stages, rather as an artist might construct a work on canvas. The scene or field-of-view is surveyed, and the color and brightness of different regions are assessed and then represented by appropriate amounts of paint applied in small strokes, dots, etc. Surveying in the LSM is achieved by scanning a focused beam of laser light through the sample. One or more photodetectors are used to measure the brightness, color, or other properties at various points, and the resulting values are used to “paint” each corresponding point (or pixel) on a display screen. The final part of this analogy is the most important. The painter does not make an exact copy of the scene on the canvas but makes an interpretation according to her artistic tendencies. While the LSM is somewhat more impartial, it too has a tendency to inexactitude. This is called the microscope’s “response”, and since we are building-up our picture pointby-point, we can easily engineer our microscope’s “point-response” to get the kind of pictures we want. More precisely, we can increase the contrast of selected features or parts of the specimen.

Fine pit pattern formation by EB-writing for a high density optical recording

Possibility of fine pattern formation for an optical recording with a high recording density is researched using a conventional electro–optical system. Then, we checked whether conventional optics or near-field optics can read them out for high density recording. The conventional optics in a laser scanning optical microscope with a blue laser (a wavelength of 405 nm) and a high NA (numerical aperture) of 0.95 or 1.4 was used. The EB drawing technique provides patterns with a minimum size of 30 nm×100 nm. However, the advanced optics reads out fine pattern with a minimum size of 160 nm×320 nm using an NA of 0.95 and a wave length of 405 nm. We conclude that we have to develop multilevel memory technology and near-field optics to achieve 100 Gb/in 2.

Second Harmonic Generation Imaging (SHG) in the Non-Linear Optical Microscopy of Living Cells

In recent years there has been considerable interest in two and three-photon excited fluorescence in laser scanning optical microscopy. Because absorption is confined tot he focal plane of the objective, these techniques provide intrinsic optical sectioning without the use of a confocal aperture. In addition, photobleaching and phototoxicity are greatly reduced above and below the focal plane. We have adapted a two-photon microscope to utilize surface second harmonic generation (SHG) as a new contrast mechanism for nonlinear optical biological imaging.Surface SHG was first described by Shen [1] and arises from the second order nonlinear susceptibility, χ(2). Signal will only arise from a non-centrosymmetric environment such as an interfacial region. Thus this technique has the potential to probe cellular membranes at high specificity. Further, since SHG results from an induced polarization and not absorption, photobleaching considerations are greatly reduced over fluorescence based methods.

Autofluorescence and Mineral Content of Carious Dentine: Scanning Optical and Backscattered Electron Microscopic Studies

The aim of this study was to investigate the extent of natural intrinsic fluorescence in carious human dentine and any correspondence of such autofluorescence (AF) to the mineral distribution within the lesion. Two investigative techniques were used, both employing the same sample set and fields in diamond-polished block surfaces of polymethylmethacrylate-embedded carious teeth. AF at emission wavelengths > 515 nm, excited by 488-nm laser light, was assessed using a confocal laser scanning optical microscope (CLSM), the recordings made under standard operating conditions. The relative mineral content was assessed using digital backscattered scanning electron microscopy (20 kV BSE-SEM). The AF intensity correlated with the level of demineralisation as seen by BSE, but the depth of the lesion as seen by CLSM was significantly greater implying that the AF is not directly related to the mineral component.

Disorganization of dystrophin costameric lattice in Becker muscular dystrophy.

We studied by high-resolution immunofluorescence (HRI) and by confocal laser scanning optical microscopy (CLSOM) the costameric organization of dystrophin and vinculin at the surface membrane of muscle fibers from 4 young boys with Becker muscular dystrophy (BMD). By HRI, the surface membrane of normal fibers showed regular parallel bands encircling the fiber at the level of I and M band. In BMD fibers, the dystrophin bands were stretched apart, interrupted, or did not show the intermediate band encircling the M band. By CLSOM, computer reconstruction of muscle surface membrane showed disorganization of the costameric dystrophin lattice at the membrane level in BMD muscle, in contrast with the preservation of the costameric lattice organization of vinculin.

Fluorescence photon antibunching from single molecules on a surface

Fluorescence correlation spectroscopy of individual Rhodamine 6G (R6G) molecules immobilized on a silica surface is performed in air at room temperature using confocal laser scanning optical microscopy (CLSM). The high excitation irradiance in CLSM is used to saturate single R6G molecules, and to observe fluorescence photon antibunching. An experimental arrangement is described that improves the overall coincidence counting efficiency at high irradiance. Photophysical parameters obtained from the saturation data are used to model the antibunching behavior. This is the first reported example of photon antibunching from individual quantum systems on a surface.

Studies on the structure of sperm heads of Eledone cirrhosa by means of CLSM linked to bioimage-oriented devices.

We have used a confocal laser scanning optical microscope imaging device and a bioimage-oriented workstation equipped for augmented reality to study the helical sperm head of the octopus Eledone cirrhosa. This approach allows us to study different complex organisational motifs due to the spatial arrangement of linear helical structures. We consider this helical specimen an enlarged copy of one of the most important biostructures governing cell functioning such as chromatin-DNA. Moreover, this very same sample is made of highly compacted chromatin that can be studied at higher resolution, i.e., by means of scanning force microscopy. Fluorescence optical sectioning has been used to enter the spatial organisation. Three-dimensional images of single, twisted, and folded fibers are shown.

Time-variant analysis of organelle and vesicle movement during phagocytosis in Paramecium primaurelia by means of fluorescence confocal laser scanning microscopy.

Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical microscope (CLSM) to display and analyze formation, movement, and fusion of vesicles during the phagocytosis of Paramecium primaurelia, in the x-y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intracellular movement of food vacuoles from their formation at the cytostome to their egestion at the cytoproct was visualized, and food vacuoles were selected in a specific digestion stage. Small pinocytic vesicles are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enlarge the membrane of the nascent food vacuoles or fuse with stage II food vacuoles, when the vacuoles of stage II increase their size, changing from an acidic to an alkaline status. A multimodal analysis of confocal fluorescence images and the false-color technique were used to visualize vesicle movement vs. time. Starting from three images of the same cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color corresponding to each sampling point in time. The composite image shows that vesicles move away from the food vacuole in a scattered manner exhibiting changes in direction.

Time‐variant analysis of organelle and vesicle movement during phagocytosis in Paramecium primaurelia by means of fluorescence confocal laser scanning microscopy

Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical microscope (CLSM) to display and analyze formation, movement, and fusion of vesicles during the phagocytosis of Paramecium primaurelia, in the x-y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intracellular movement of food vacuoles from their formation at the cytostome to their egestion at the cytoproct was visualized, and food vacuoles were selected in a specific digestion stage. Small pinocytic vesicles are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enlarge the membrane of the nascent food vacuoles or fuse with stage II food vacuoles, when the vacuoles of stage II increase their size, changing from an acidic to an alkaline status. A multimodal analysis of confocal fluorescence images and the false-color technique were used to visualize vesicle movement vs. time. Starting from three images of the same cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color corresponding to each sampling point in time. The composite image shows that vesicles move away from the food vacuole in a scattered manner exhibiting changes in direction. © 1996 Wiley-Liss, Inc.

Laser Reviews. Near-field Laser-Scanning Microscope.

As a new version of laser-scanning optical microscope, near-field scanning optical microscope or NSOM is getting very popular in microscope-research society. In this review, why and how the NSOM is important in the basic research and the application fields of microscopy. The principle and the new analysis of image formation by NSOM is discussed with some experimental results.