Optical far-field microscopy of single molecules with 3.4 nm lateral resolution

Optical far‐field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low‐temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of ~10 µm³, are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.     

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross‐talk between adjacent imaging channels. We demonstrate that a time‐multiplexed non‐linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single‐photon confocal system. The background becomes irrelevant for thin (< 15 µm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.     

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 µm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water–polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.     

Microinfrared reflection spectroscopic mapping: application to the detection of hydrogen-related species in natural quartz

A new method of microinfrared reflection spectroscopy and mapping analysis is briefly introduced. It was used to detect distributions and structures of hydrogen‐related species (e.g. H₂O, SiOH and SiH) in plastically deformed natural quartz. We used a Fourier transform‐infrared spectrometer with a microscopic imaging system fully automated for all microscope functions (e.g. focusing, aperture, stage motion and measurements). Mapping can be made in thin sections with a thickness of 50 µm at room temperature and low temperatures (77 K) using a liquid N₂ cooling system. Infrared reflection spectra were obtained by five scans for each point with a range from 4000 to 400 cm^?1. The spectra were measured five times within about 2.5 s at each position. The scanning interval was 100–150 µm using a 100 × 100 µm² aperture. All obtained spectral data were stored in computer memory to construct two‐dimensional mappings of infrared absorption. From the comparisons between infrared mapping images and deformation microstructures, in addition to the molecular H₂O around 3600–3400 cm^?1, the hydrogen‐related point defects (i.e. SiOH and SiH) around 970–900 cm^?1 within quartz grains and between grain boundaries increased with decreasing grain sizes (increasing plastic strain). The method can detect the SiOH and SiH along grain boundaries that enhance the hydrolytic weakening of natural quartz.     

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.     

Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies

Stereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three-dimensional (3D) data non-destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty-nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm³, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.     

A new confocal scanning beam laser MACROscope using a telecentric,f-theta laser scan lens

A new confocal scanning beam system (MACROscope) that images very large-area specimens is described. The MACROscope uses a telecentric, f-theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small-area, high-speed, high-resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large-area, high-speed, good-resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected-light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.     

An X-ray microscopy perspective on the effect of glutaraldehyde fixation on cells

X-ray microscopy (XRM) is the only microscopy technique that can provide high-resolution (30 nm) imaging of biological specimens without the need to fix, stain or section them. We aim to determine the effect, if any, of glutaraldehyde fixation on algae cells from the XRM perspective and thus provide beneficial information for both X-ray and electron microscopists on artefacts induced by glutaraldehyde fixation. Three species of microalgae, Microcystis aeruginosa, Anabaena spiroides and Chlorella vulgaris, were used in this study. XRM images were obtained from unfixed and glutaraldehyde-fixed cells and cell diameter and percentage X-ray absorbency were measured. The mean diameter of cells from fixed preparations was smaller than from unfixed preparations; the mean diameter of M. aeruginosa cells was significantly reduced from 3.92 µm in unfixed cells to 3.43 µm in fixed cells (P < 0.05); in C. vulgaris the diameter of cells was also significantly reduced from 3.50 µm in unfixed to 2.98 µm in fixed samples (P < 0.05); whereas there was no significant reduction in the diameter of A. spiroides cells (4.04–3.90 µm). The protein crosslinking mechanism of glutaraldehyde probably generated free water molecules, which play an important role in radiation damage induced by X-rays. This was seen as mass loss and cell shrinkage, which in the present study occurred more frequently in fixed cells than in unfixed cells. In addition, we demonstrated that the uptake of glutaraldehyde by cells makes all protein constituents in the cell organize into a closely packed configuration, thus causing a rise in the percentage of X-ray absorbency. In fixed cells, this rise was approximately by a factor of two compared with unfixed samples in which protein constituents inside the cell are arranged in their native form.     

Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope

A small diameter (600 µm) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non‐focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM–probe combination is an improvement over conventional fluorescence–probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

Confocal microscopy of the in-situ crystalline lens

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.     

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.     

Micro‐CT measurements of tumoral vessels supplied by portal circulation in hepatic colorectal metastasis mouse model

The purpose of this study was to elucidate the micro CT findings of tumoral vessels supplied by portal circulation during establishment of hepatic metastasis of colorectal cancer in a mouse model. Hepatic metastases were induced in 15 BALB/c mice through the injection of murine colonic adenocarcinoma tumor cells into the mesenteric vein. Micro‐CT imaging of the tumoral vessels was obtained to clarify the microvascular architecture. We evaluated the sinusoidal structure, diameter of the tumoral vessels (DTV) and blood vessel density (BVD) according to tumor sizes ranging from 201 to 3,000 µm in diameter. A total of 116 tumors were observed on day 15 after cell injection. The mean diameter of a normal hepatic sinusoid was 11.7 ± 2.0 µm on micro CT. The DTV supplied by the portal vein of tumors measuring 1,001–1,500 µm in diameter was greater than that of tumors 200–1,000 µm in diameter. The mean BVD from the portal vein gradually decrease according to size of tumor from 201 to 3,000 µm in diameter (r² = ?0.584, P < 0.01). The characteristics of tumoral vessels supplied by portal circulation during establishment of hepatic colorectal metastases were well visualized with micro‐CT imaging. Microsc. Res. Tech. 77:415–421, 2014. © 2014 Wiley Periodicals, Inc.     

Accurate measure of laser irradiance threshold for near-infrared photo-oxidation with a modified confocal microscope

Femtosecond mode‐locked lasers are now being used routinely in multiphoton fluorescence and autofluorescence spectroscopy, are just beginning to be used in refractive surgery, and may be used in the future diagnosis of skin cancer. Pulses from these lasers induce non‐linear effects in resultant tissue interactions. Using a modified confocal microscope with dispersion compensation and accurate measurements of beam diameter, a very low threshold was measured for photochemical oxidation in cultured cells. The measured threshold showed non‐linear photo‐oxidation at a peak irradiance and photon‐flux density of 8.4 × 10⁸ W cm^?2 and 3.4 × 10²⁷ photons cm^?2 s^?1, respectively (90‐fs pulse). The impact of these findings is significant to those using ultrashort lasers because they provide a tangible reference point (microscope‐independent) for the generation of photo‐oxidative stress in laser‐exposed tissues, and because they highlight the importance of dispersion compensation in minimizing collateral tissue damage.     

Near-field scanning optical microscopy and near-field induced photocurrent investigations of buried heterostructure multiquantum well lasers

Buried heterostructure multiquantum well laser devices are investigated utilizing a near-field scanning optical microscope to characterize and correlate the surface topography, optical output and electronic properties of the device. Near-field photocurrent imaging has been used to accurately measure the unbiased buried heterostructure multiquantum well device in cross-section, successfully revealing the distribution of pn-junctions and their associated fields. Moreover, this has been accurately correlated with the physical structure of the device determined by simultaneous shear-force imaging of the surface. Topographic structure is manifested as a result of strain relaxation (∼10⁻¹⁰ m) of the cleaved cross-section. These imaging modes are similarly correlated with the optical output of the operational device mapped with 50 nm lateral resolution. The collection-mode measurements detected electroluminescence external to the active region, highlighting the existence of carrier recombination away from the multiquantum well device region. The combination and correlation of different near-field scanning optical microscope imaging modes proved powerful in the analysis of the buried heterostructure multiquantum well device, and was shown to assist in the identification of current leakage pathways within the structure.     

Magneto-optical near-field microscopy of ultrathin films in ultrahigh vacuum

In this study, guiding of surface plasmon polaritons excited at a gold film surface along corrugation‐free channels in regions that are covered with randomly located surface scatterers, is considered using near‐field microscopy for imaging of surface plasmon polariton intensity distributions at the surface. In the wavelength range 713–815 nm, we observed complete inhibition of the surface plasmon polariton propagation inside the random structures composed of individual (≈ 70 nm high) gold bumps (and their clusters) placed on a 55 nm thick gold film with a bump density of 75 µm^?2. We demonstrate well‐defined surface plasmon polariton guiding along corrugation‐free 2 µm wide channels in random structures and, in the wavelength range 738–774 nm, low‐loss guiding around 20° bends having a bend radius of ≈ 15 µm.     

Developments in molecular SIMS depth profiling and 3D imaging of biological systems using polyatomic primary ions

In principle mass spectral imaging has enormous potential for discovery applications in biology. The chemical specificity of mass spectrometry combined with spatial analysis capabilities of liquid metal cluster beams and the high yields of polyatomic ion beams should present unprecedented ability to spatially locate molecular chemistry in the 100 nm range. However, although metal cluster ion beams have greatly increased yields in the m/z range up to 1000, they still have to be operated under the static limit and even in most favorable cases maximum yields for molecular species from 1 µm pixels are frequently below 20 counts. However, some very impressive molecular imaging analysis has been accomplished under these conditions. Nevertheless although molecular ions of lipids have been detected and correlation with biology is obtained, signal levels are such that lateral resolution must be sacrificed to provide a sufficient signal to image. To obtain useful spatial resolution detection below 1 µm is almost impossible. Too few ions are generated! The review shows that the application of polyatomic primary ions with their low damage cross‐sections offers hope of a new approach to molecular SIMS imaging by accessing voxels rather than pixels to thereby increase the dynamic signal range in 2D imaging and to extend the analysis to depth profiling and 3D imaging. Recent data on cells and tissue analysis suggest that there is, in consequence, the prospect that a wider chemistry might be accessible within a sub‐micron area and as a function of depth. However, these advances are compromised by the pulsed nature of current ToF‐SIMS instruments. The duty cycle is very low and results in excessive analysis times, and maximum mass resolution is incompatible with maximum spatial resolution. New instrumental directions are described that enable a dc primary beam to be used that promises to be able to take full advantage of all the capabilities of the polyatomic ion beam. Some new data are presented that suggest that the aspirations for these new instruments will be realized. However, although prospects are good, the review highlights the continuing challenges presented by the low ionization efficiency and the complications that arise from matrix effects. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:142–174, 2011     

Silver deposition on freeze-dried cells allows subcellular localization of cholesterol with imaging TOF-SIMS

Imaging time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) was used for characterization and subcellular localization of organic ions in leucocytes adhering to glass surfaces. The cells were fixed by freeze drying in 0.15 m ammonium formate buffer at pH 7.2–7.4. The freeze‐dried cells were sputter‐coated with silver, and the silver surface was analysed with imaging TOF‐SIMS. TOF‐SIMS spectra were recorded by scanning the primary ion beam over the analysis area and acquiring positive mass spectra of the ions leaving the surface. The relative brightness of each pixel within the analysis area reflects the signal intensity of a selected ion in that pixel. Data were collected separately at high mass resolution m/Δm > 7000 and at high lateral resolution (= 0.5 µm). The images were analysed by principal component analysis (PCA). The glass‐adhering cells showed a well defined attachment area with a diameter of up to 20 µm, and an equally well defined cell body, containing the nucleus, with a diameter of 8–10 µm. On the raw data images, the obtained cholesterol distributions were consistent with a higher cholesterol content of the cell membrane in the attachment area than in the cell body. Using PCA analysis, silver‐cationized molecular cholesterol was found localized mainly in the attachment area of the cells. Cholesterol was also seen at higher concentration in circular spots of ≤ 1 µm in diameter, probably representing caveolae.     

Magnetic characterization of microscopic particles by MO-SNOM

This paper reports on the development of a magneto‐optical scanning near‐field optical microscope and the experimental near‐field study of the domain structure for a model magnetic particle of 16 × 16 µm² of a Co_70.4Fe_4.6Si₁₅B₁₀ amorphous thin film, deposited on a silicon substrate. We present the topographic, optical and magneto‐optical differential susceptibility (MODS) images of the particle. Imaging by using the local MODS reveals the domain structure. These images are also used for positioning the tip in order to acquire local hysteresis loops, with submicrometre spatial resolution.