Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin

Multi-photon fluorescence microscopy (MPFM) is a powerful technique for imaging scattering, biological specimens in depth. In addition to the sectioning effect generated by the point-like excitation volume, the near-infrared wavelengths used for multi-photon excitation allow deeper penetration into optically turbid specimens. In physiological specimens, the optical properties such as the scattering coefficients and refractive indices are often heterogeneous. In these specimens, it is not clear which type of immersion objective can provide optimized images in-depth. In particular, in-depth dermatological imaging applications using MPFM requires such optimization to obtain qualitative and quantitative information from the skin specimens. In this work, we address this issue by comparing the performances of two common types of high numerical aperture (NA) objectives: water-immersion and oil-immersion. A high-quality water-immersion objective (Zeiss, 40 x C-Apochromat, NA 1.2) and a comparable oil-immersion objective (Zeiss, 40 x Fluar, NA 1.25) were used for in-depth imaging of autofuorescent excised human skin and sulforhodamine B treated human skin specimens. Our results show that in the epidermal layers, the two types of immersion objectives perform comparably. However, in the dermis, multi-photon imaging using the oil immersion objective results in stronger fluorescence detection. These observations are most likely due to the degraded point-spread-function (PSF) caused by refractive index mismatch between the epidermis and the dermis.     

A procedure for increasing the contrast of biological specimens in edge-projection TEM

Rotary shadowing has been used to increase the image contrast of biological specimens during edge-projection imaging in the transmission electron microscope (TEM). In this imaging mode, biological specimens are adsorbed from aqueous solution onto a highly curved substrate and observed in a direction parallel to its surface. High contrast TEM images are obtained at 200 kV when a 1–3 nm layer of tungsten is thermally evaporated onto the substrate at an angle of about 9°. Individual adsorbates are clearly delineated by the smooth, continuous, and fine-grained tungsten layer that surrounds them. TEM images obtained with this technique can provide a unique view of biological adsorbates on metal, insulator or semiconductor substrates.     

A universal fluid cell for the imaging of biological specimens in the atomic force microscope

Recently, atomic force microscope (AFM) manufacturers have begun producing instruments specifically designed to image biological specimens. In most instances, they are integrated with an inverted optical microscope, which permits concurrent optical and AFM imaging. An important component of the set‐up is the imaging chamber, whose design determines the nature of the experiments that can be conducted. Many different imaging chamber designs are available, usually designed to optimize a single parameter, such as the dimensions of the substrate or the volume of fluid that can be used throughout the experiment. In this report, we present a universal fluid cell, which simultaneously optimizes all of the parameters that are important for the imaging of biological specimens in the AFM. This novel imaging chamber has been successfully tested using mammalian, plant, and microbial cells. Microsc. Res. Tech. 76:357–363, 2013. © 2013 Wiley Periodicals, Inc.     

Digital holographic microscopy and focusing methods based on image sharpness

Digital holographic microscope allows imaging of opaque and transparent specimens without staining. A digitally recorded hologram must be reconstructed numerically at the actual depth of the object to obtain a focused image. We have developed a high‐resolution digital holographic microscope for imaging amplitude and phase objects with autofocusing capability. If the actual depth of an object is not known a priori, it is estimated by comparing the sharpness of several reconstructions at different distances, which is very demanding in means of computational power when the recorded hologram is large. In this paper, we present 11 different sharpness metrics for estimating the actual focus depths of objects. The speed performance of focusing is discussed, and a scaling technique is introduced where the speed of autofocusing increases on the order of square of the scale ratio. We measured the performance of scaling on computer‐generated holograms and on recorded holograms of a biological sample. We show that simulations are in good agreement with the experimental results.     

Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron–specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy-loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy-filtered microscope and to assess its utility for thick-section microscopy. We found that for imaging thick biological specimens (> 0.5 μm) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy-filtering at the most probable energy loss (in this case at the first plasmon energy-loss peak). Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.     

Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0·3–1·0 μm). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than ～10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the ‘restored’ images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.     

生物体内部结构三维显微成像技术研究

概述了生物体内部结构的几种传统的成像技术 ,介绍了一种新的生物体内部结构三维显微成像方法。该方法对生物体作连续切片 ,由 CCD显微摄像系统获取切片的序列图像 ,然后由计算机进行三维图像重构 ,最终得到生物体内部结构的三维显微图像     

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.     

Specimen-induced distortions in light microscopy

Specimen‐induced aberrations affect the imaging properties in optical 3D microscopy, especially when high numerical aperture lenses are used. Studies on aberrations are often properly concerned with the degradation of image quality such as compromised resolution or reduced signal intensity. Apart from these, aberration effects can also introduce geometric image distortions. The effects, discussed here are particularly strong when thick biological specimens are investigated. Using a high numerical aperture interferometer, we measured wavefront aberrations in transmission mode and quantified geometric distortions associated with specimen‐induced aberrations. This assessment for a range of biological specimens allows estimation of the accuracy of spatial measurements. The results show that high‐resolution spatial measurements can be significantly compromised by specimen‐induced aberrations.     

Visualization of biological specimens by cryoHVEM

This article describes the operation and the characteristics of cryoHVEM imaging of biological specimens using a top-entry cryostage. The procedure for inserting frozen specimens into the microscope column is also presented. Whole mounts were thus observed under optimal imaging conditions by combining: (i) fixation by fast freezing for structure preservation without exposure to chemicals, (ii) observation in the hydrated (frozen) state or in the dried state without exposure to the atmosphere after the initial fixation by freezing, and (iii) ultrastructural visualization with the key imaging factors of resolution, penetration and beam-induced damage at their best by high-voltage electron microscopy.     

Computed tomography of cryogenic biological specimens based on X-ray microscopic images

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment, rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state.     

Real imaging and size values of Saccharomyces cerevisiae cells with comparable contrast tuning to two environmental scanning electron microscopy modes

The combined application of electron microscopy (EM) is frequently used for the microstructural investigation of biological specimens and plays two important roles in the quantification and in gaining an improved understanding of biological phenomena by making use of the highest resolution capability provided by EM. The possibility of imaging wet specimens in their "native" states in the environmental scanning electron microscope (ESEM) at high resolution and large depth of focus in real time is discussed in this paper. It is demonstrated here that new features can be discovered by the elimination of even the least hazardous approaches in some preparation techniques, that destroy the samples. Since the analysis conditions may influence the morphology and the extreme surface sensitivity of living biological systems, the results obtained from the same cultured cell with two different ESEM modes (Lvac mode and wet mode) were compared. This offers new opportunities compared with ESEM-wet/Lvac-mode imaging, since wet-mode imaging involves a real contrast and gives an indication of the changes in cell morphology and structure required for cell viability. In this study, wet-mode imaging was optimized using the unique ability of cell quantities for microcharacterization in situ giving very fine features of topological effects. Accordingly, the progress is reported by comparing the results of these two modes, which demonstrate interesting application details. In general, the functional comparisons have revealed that the fresh unprocessed Saccharomyces cerevisiae cells (ESEM-wet mode) were essentially unaltered with improved and minimal specimen preparation timescales, and the optimal cell viability degree was visualized and also measured quantitatively while the cell size remained unchanged with continuous images.     

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.     

The effects of total internal reflection on the spread function along the axis in confocal microscopy

A broadening and splitting of the axial spread function is observed when high-numerical-aperture (NA) oil-immersion objectives are used on a confocal microscope to examine dielectric interfaces when the refractive index below the boundary is lower than the NA of the objective. The phenomena is due to total internal reflection probably as a consequence of the Goos-Hänchen shift. If total internal reflection occurs when undertaking confocal microscopy, this shift creates obvious problems when the optical sectioning capabilities must be optimal in reflectance mode and more subtle difficulties can arise when examining fluorescent emission. Alternatively, deliberately inducing total internal reflection can be used to estimate the refractive index in component parts within foams, emulsions and aerated specimens where such measurements can be relatively difficult to make by other means. Furthermore, the examination of total internal reflection with a confocal microscope permits the phenomena of total internal reflection itself to be probed with very high illumination intensities without disturbing the boundary conditions with an external probe. Finally, other changes in the apparent position of the focus were noted to occur when high-NA oil-immersion objectives are used to examine specimens such as metal mirrors.     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

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.     

Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review.

When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures.     

Super‐aperture metrology: overcoming a fundamental limit in imaging smooth highly curved surfaces

The imaging of smooth, highly curved or tilted surfaces is widely recognized as one of the most challenging and unsolved problems in optical imaging and metrology today. The reason is that even when such surfaces are imaged using high aperture microscope objectives the steepness of the features causes the light to be reflected in such a way that it is not captured by the lens. This is true even in the limiting case of unity numerical aperture since the illuminating light may also be reflected in the forward direction. In order to overcome this fundamental problem we have developed a method whereby such specimens are covered with a readily removable organic fluorescent film thereby creating an isotropic scattering surface. We show that we are readily able to detect slopes with angles close 90° using a 0.75 NA objective – an 82% improvement over the theoretical aperture limit. Issues of variation in film thickness deposition are shown to be readily accommodated. This approach may be used with other fluorophore materials, organic or inorganic, since there is no need for biocompatibility in this application.     

Aberration correction for confocal imaging in refractive-index-mismatched media

A major limitation to the use of confocal microscopes to image thick biological tissue lies in the dramatic reduction in both signal level and resolution when focusing deep into a refractive-index-mismatched specimen. This limitation may be overcome by measuring the wavefront aberration and pre-shaping the input beam so as to cancel the effects of aberration. We consider the images of planar and point objects in brightfield, single-photon fluorescence and two-photon fluorescence imaging. In all cases, the specimens are imaged using an oil-immersion objective through various thicknesses of water. The question of finite-sized pinhole is addressed and it is found, in general, that it is sufficient to correct only the first two or three orders of spherical aberration to restore adequate image signal level and optical resolution, at imaging depths of up to 50-100 wavelengths.     

Automated microscopy system for mosaic acquisition and processing

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide‐field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast‐scanning, multiphoton, confocal microscope fitted with a motorized, high‐precision stage and custom‐developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.