Differential phase contrast in scanning optical microscopy

High-quality high-resolution transmission and reflection images produced using a scanning optical microscope and the split-detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.     

Super‐resolution optical microscopy based on scannable cantilever‐combined microsphere

We report an ingenious method of super‐resolution optical microscopy utilizing scannable cantilever‐combined microsphere. By scanning the microsphere over the sample surface in a cantilever‐combined microsphere‐sample contact state, super‐resolution images can be acquired at arbitrary sample regions through near‐field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub‐diffraction‐limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy. Microsc. Res. Tech. 78:1128–1132, 2015. © 2015 Wiley Periodicals, Inc.     

Polarization contrast in reflection near-field optical microscopy with uncoated fibre tips

Using cross-hatched, patterned semiconductor surfaces and round 20-nm-thick gold pads on semiconductor wafers, we investigate the imaging characteristics of a reflection near-field optical microscope with an uncoated fibre tip for different polarization configurations and light wavelengths. It is shown that cross-polarized detection allows one to effectively suppress far-field components in the detected signal and to realize imaging of optical contrast on the sub-wavelength scale. The sensitivity window of our microscope, i.e. the scale on which near-field optical images represent mainly optical contrast, is found to be ≈100 nm for light wavelengths in the visible region. We demonstrate imaging of near-field components of a dipole field and purely dielectric contrast (related to well-width fluctuations in a semiconductor quantum well) with a spatial resolution of ≈100 nm. The results obtained show that such a near-field technique can be used for polarization-sensitive imaging with reasonably high spatial resolution and suggest a number of applications for this technique.     

Image contrast reduction mechanism in full‐field optical coherence tomography

Correct interpretation of image contrast obtained with full‐field optical coherence tomography (FFOCT) technique is required for accurate medical diagnosis applications. In this work, first, the characteristics of microscopic structures of tissue that generate the contrast in en‐face tomographic image obtained with FFOCT are discussed. Then an overview is given of the parameters that affect image contrast. Finally, the contrast correction factor for correct image interpretation and the contrast limits to practical FFOCT systems are outlined.     

High‐speed optical imaging of liquid film flow and liquid macro‐slip over free surfaces with different surface energies

High‐speed optical imaging is introduced as a visualization method to investigate the film‐flow properties of liquids with different surface tensions and viscosities (water, poly‐alpha‐olefin oil, and glycerol) over free rotating surfaces with different surface energies and polarities (steel, and two different diamond‐like‐carbon ‐ DLC coatings, i.e. DLC, and F‐DLC). It was found that the polar surface energy strongly influences the structural dynamics of the liquid film's flow and the film's slip. Namely, a decrease in the polar surface energy results in a less stable film with de‐wetting areas and breakups into streams, as well as in a larger amount of film slip, which was most clearly expressed by the F‐DLC. It was also found that the combination of a high surface tension and a low viscosity provides the largest amount of liquid slip, with the most obvious breakup of the liquid film being observed with water, which clearly exhibits these properties.     

Harmonic demodulation and minimum enhancement factors in field‐enhanced near‐field optical microscopy

Field‐enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near‐field information recorded in a field‐enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe–sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near‐field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near‐field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe–sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field‐enhanced scanning optical microscopy.     

Fibre-optic nonlinear optical microscopy and endoscopy

Nonlinear optical microscopy has been an indispensable laboratory tool of high‐resolution imaging in thick tissue and live animals. Rapid developments of fibre‐optic components in terms of growing functionality and decreasing size provide enormous opportunities for innovations in nonlinear optical microscopy. Fibre‐based nonlinear optical endoscopy is the sole instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible to a conventional optical microscope. This article reviews the current development of fibre‐optic nonlinear optical microscopy and endoscopy, which includes crucial technologies for miniaturized nonlinear optical microscopy and their embodiments of endoscopic systems. A particular attention is given to several classes of photonic crystal fibres that have been applied to nonlinear optical microscopy due to their unique properties for ultrashort pulse delivery and signal collection. Furthermore, fibre‐optic nonlinear optical imaging systems can be classified into portable microscopes suitable for imaging behaving animals, rigid endoscopes that allow for deep tissue imaging with minimally invasive manners, and flexible endoscopes enabling imaging of internal organs. Fibre‐optic nonlinear optical endoscopy is coming of age and a paradigm shift leading to optical microscope tools for early cancer detection and minimally invasive surgery.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

Near‐field optical microscopy based on microfabricated probes

We demonstrate high resolution imaging with microfabricated, cantilevered probes, consisting of solid quartz tips on silicon levers. The tips are covered by a 60‐nm thick layer of aluminium, which appears to be closed at the apex when investigated by transmission electron microscopy. An instrument specifically built for cantilever probes was used to record images of latex bead projection patterns in transmission as well as single molecule fluorescence. All images were recorded in constant height mode and show optical resolutions down to 32 nm.     

Dimensional metrology of lab‐on‐a‐chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in‐house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser‐scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three‐dimensional metrology of critical internal structures in lab‐on‐a‐chip devices because scans can be performed rapidly and noninvasively prior to their use.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, Inc.     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.     

Fibre‐optical microendoscopy

Microendoscopy has been an essential tool in exploring micro/nano mechanisms in vivo due to high‐quality imaging performance, compact size and flexible movement. The investigations into optical fibres, micro‐scanners and miniature lens have boosted efficiencies of remote light delivery to sample site and signal collection. Given the light interaction with materials in the fluorescence imaging regime, this paper reviews two classes of compact microendoscopy based on a single fibre: linear optical microendoscopy and nonlinear optical microendoscopy. Due to the fact that fluorescence occurs only in the focal volume, nonlinear optical microendoscopy can provide stronger optical sectioning ability than linear optical microendoscopy, and is a good candidate for deep tissue imaging. Moreover, one‐photon excited fluorescence microendoscopy as the linear optical microendoscopy suffers from severe photobleaching owing to the linear dependence of photobleaching rate on excitation laser power. On the contrary, nonlinear optical microendoscopy, including two‐photon excited fluorescence microendoscopy and second harmonic generation microendoscopy, has the capability to minimize or avoid the photobleaching effect at a high excitation power and generate high image contrast. The combination of various nonlinear signals gained by the nonlinear optical microendoscopy provides a comprehensive insight into biophenomena in internal organs. Fibre‐optical microendoscopy overcomes physical limitations of traditional microscopy and opens up a new path to achieve early cancer diagnosis and microsurgery in a minimally invasive and localized manner.     

Three‐dimensional optical transfer function in differential confocal microscopy

To reveal the fundamental characteristics of differential confocal microscopy (DCM), its imaging properties were analysed by studying the 3D optical transfer function (OTF). The zero transfer at zero frequency along the axial direction in DCM, which has not been well understood and is considerably different from the transfer behaviour in conventional confocal microscopy (CM), was elucidated. The integral expressions of the OTFs for CM and DCM and the subsequent simulation results showed that DCMs have higher transfer capability than CM in the axial direction at medium and high frequencies. Conventionally, the relative optimal defocusing amount in DCMs are determined through calculations of the gradient of the point spread functions in the spatial domain. In contrast, in this study, the OTF performances were compared and the optimal defocusing amount was found to be between 5 and 7.     

Pure optical contrast in scattering-type scanning near‐field microscopy

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO₄–UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunner's glands, the Golgi cisternae were well defined with the KMnO₄–UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO₄–UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO₄ oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.     

In vivo three-dimensional imaging of plants with optical coherence microscopy

Achieving the ability to non‐destructively, non‐invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics‐based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three‐dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non‐invasive, non‐destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.