Two‐dimensional structured illumination microscopy

In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms ‘quasi‐confocal images’ can be derived from a given number of such phase‐images. Here, we present an alternative structured illumination microscopy approach, which employs two‐dimensional patterns instead of a one‐dimensional one. While in one‐dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two‐dimensional approach it is shifted at a single, pattern‐dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern‐shift and ‐rotation. Moreover, our two‐dimensional approach also yields a better signal‐to‐noise ratio in the evaluated image.     

Comparison of single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.     

Curtailed light sheet microscopy for rapid imaging of macroscopic biological specimens

We study the feasibility of volume imaging from a few angular views/scans in a light sheet fluorescence microscopy. Two‐dimensional (2D) images (angular views) were acquired at an angular separation of 10° and volume images were constructed with merely 18, 9, and 6 views. We study the structural changes in a 5‐day old Zebrafish embryo labeled with Phalloidin TRITC that binds to F‐Actin of embryo cell. To collect the data, the specimen is rotated (for varying sampling angles Δθ) with respect to a fixed vertical axis passing through the volume‐of‐interest (yolk sac). In the proposed realization of selective plane illumination microscopy (SPIM) technique, the translation is completely avoided. Analysis shows rich structural information with marginal reduction in contrast. Comparison with the state‐of‐the‐art SPIM shows appreciable volume reconstruction (from an order less 2D scans) that may be good enough for rapid monitoring of macroscopic specimens. Microsc. Res. Tech. 79:455–458, 2016. © 2016 Wiley Periodicals, Inc.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

Optical coherence tomography as a tool for characterization of complex biological surfaces

The advent of scanning electron microscopy has facilitated our understanding of the biology in relation to surface microstructure of many invertebrates. In recent years, interest in biomimetics and bio‐inspired materials has further propelled the search for novel microstructures from natural surfaces. As this search widens in diversity to nurture deeper understanding of form and function, the need often arises to examine rare specimens. Unfortunately, most methods for characterization of the microtopography of natural surfaces are sacrificial, and as such, place limiting constraints on research progress in situations where only a few rare specimens are known, such as the rich resources lodged in natural history museum collections. In this paper, we introduce the use of optical coherence tomography (OCT) as a noninvasive tool for bioimaging surface microtopography of crab shells. The technique enables the capture of microstructures down to micron level using low coherence near‐infrared light source. OCT has allowed surface microtopography imaging on crab shells to be carried out rapidly and in a nondestructive manner, compared to the scanning electron microscope technique. The microtopography of four preserved crab specimens from Acanthodromia margarita, Ranina ranina, Conchoecetes intermedius and Dromia dormia imaged using OCT were similar to images obtained from scanning electron microscope, showing that OCT imaging retains the overall morphological form during the scanning process. By comparing the physical lengths of the spinal structures from images obtained from OCT and scanning electron microscope, the results showed that dimensional integrity of the images captured from OCT was also maintained.     

Depth enhancement of 3D microscopic living‐cell image using incoherent fluorescent digital holography

Multilayer images of living cells are typically obtained using confocal or multiphoton microscopy. However, limitations on the distance between consecutive scan layers hinder high‐resolution three‐dimensional reconstruction, and scattering strongly degrades images of living cell components. Consequently, when overlapping information from different layers is focused on a specific point in the camera, this causes uncertainty in the depiction of the cell components. We propose a method that combines the Fresnel incoherent correlation holography and a depth‐of‐focus reduction algorithm to enhance the depth information of three‐dimensional cell images. The proposed method eliminates overlap between light elements in the different layers inside living cells and limitations on the interlayer distance, and also enhances the contrast of the reconstructed holograms of living cells.     

MRT letter: Extended depth from focus reconstruction method for stretch zone measurement in 15-5PH steel

The stretch zone width (SZW) data for 15‐5PH steel CTOD specimens fractured at ?150°C to + 23°C temperature were measured based on focused images and 3D maps obtained by extended depth‐of‐field reconstruction from light microscopy (LM) image stacks. This LM‐based method, with a larger lateral resolution, seems to be as effective for quantitative analysis of SZW as scanning electron microscopy (SEM) or confocal scanning laser microscopy (CSLM), permitting to clearly identify stretch zone boundaries. Despite the worst sharpness of focused images, a robust linear correlation was established to fracture toughness (K_C) and SZW data for the 15‐5PH steel tested specimens, measured at their center region. The method is an alternative to evaluate the boundaries of stretched zones, at a lower cost of implementation and training, since topographic data from elevation maps can be associated with reconstructed image, which summarizes the original contrast and brightness information. Finally, the extended depth‐of‐field method is presented here as a valuable tool for failure analysis, as a cheaper alternative to investigate rough surfaces or fracture, compared to scanning electron or confocal light microscopes. Microsc. Res. Tech. 75:1155–1158, 2012. © 2012 Wiley Periodicals, Inc.     

Improved longitudinal resolution in tomographic diffractive microscopy with an ellipsoidal mirror

Tomographic diffractive microscopy is a technique, which is able to image transparent unstained samples with high resolution. The three‐dimensional distribution of the complex refractive index can be reconstructed quantitatively from the measured scattered fields under various illumination and detection angles, according to the diffraction tomography theorem. We propose a tomographic diffractive microscopy setup with an ellipsoidal mirror as the light collector. We demonstrate analytically and with numerical simulation that this approach permits to obtain images with drastically improved resolution.     

Light scattering microscopy with angular resolution and its possible application to apoptosis

An inverted microscope has been modified for light scattering experiments with high angular resolution in combination with transmission, wide‐field fluorescence or laser scanning microscopy. Supported by simulations of Mie scattering, this method permits detection of morphological changes of 3T3 fibroblasts on apoptosis and formation of spherically shaped cells of about 20 μm diameter, in agreement with visual observation. Smaller sub‐structures (e.g. cell nuclei) as well as cell clusters may possibly contribute to the scattering behaviour. Results of 2‐dimensional cell cultures are confirmed by 3‐dimensional multicellular spheroids of 3T3 fibroblasts and HeLa 2E8 cervix carcinoma cells, where in most cases no morphological changes are discernable. This offers some advantage of light scattering microscopy for label‐free detection of apoptosis and may represent a first step towards label‐free in vivo diagnostics.     

Comparative authentication of three “snow lotus” herbs by macroscopic and microscopic features

“Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). To differentiate three representative easily‐confused snow lotus herbs, namely, Saussurea involucrata (Kar. et Kir.) Sch.‐Bip, Saussurea laniceps Hand.‐Mazz., and Saussurea medusa Maxim., macroscopic features of the three herbs were systemically observed, and microscopic features were compared by using ordinary light microscopy, polarized light microscopy and scanning electron microscopy (SEM). The results indicate that, as for macroscopic identification, capitula situation and arrangement, and as for microscopic identification, pollen grains, nonglandular hairs, glandular hairs, and cells of inner surface of the microdiodange can be used to authenticate the three snow lotus herbs. Comprehensive table comparing the characteristics were presented in this study. SEM has been found to provide a number of unique characteristics of pollen grains. Based on the observation of pollen grains, evolution sequence of the three species was speculated. The present method was proven to be efficient, convenient, simple, and reliable, which was successfully applied to the authentication of three snow lotus herbs. Microsc. Res. Tech.1 77:631–641, 2014. © 2014 Wiley Periodicals, Inc.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans

The utility of cuticular autofluorescence for the visualization of copepod morphology by means of confocal laser scanning microscopy (CLSM) was examined. Resulting maximum intensity projections give very accurate information on morphology and show even diminutive structures such as small setae in detail. Furthermore, CLSM enables recognition of internal structures and differences in material composition. Optical sections in all layers and along all axes of the specimens can be obtained by CLSM. The facile and rapid preparation method bears no risk of artefacts or damage occurring to the preparations and the visualized specimens can be used for later analyses allowing for the investigation of irreplaceable type specimens or parts of them. These features make CLSM a very effective tool for both taxonomical and ecological studies in small crustaceans; however, the maximum thickness of the specimens is limited to a few hundred micrometers. Three‐dimensional models based on CLSM image stacks allow observation of the preparations from all angles and can permit, improve and speed up studies on functional morphology. The visualization method described has a strong potential to become a future standard technique in aquatic biology due to its advantages over conventional light microscopy and scanning electron microscopy.     

Correlation of two‐photon in vivo imaging and FIB/SEM microscopy

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two‐photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long‐term two‐photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three‐dimensional high‐resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.     

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.     

Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.     

Three-dimensional representation of curved nanowires

Nanostructures, such as nanowires, nanotubes and nanocoils, can be described in many cases as quasi one‐dimensional curved objects projecting in three‐dimensional space. A parallax method to construct the correct three‐dimensional geometry of such one‐dimensional nanostructures is presented. A series of scanning electron microscope images was acquired at different view angles, thus providing a set of image pairs that were used to generate three‐dimensional representations using a matlab program. An error analysis as a function of the view angle between the two images is presented and discussed. As an example application, the importance of knowing the true three‐dimensional shape of boron nanowires is demonstrated; without the nanowire's correct length and diameter, mechanical resonance data cannot provide an accurate estimate of Young's modulus.     

A novel approach for correlative light electron microscopy analysis

Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three‐dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Statistical analysis of reduced pair correlation functions of capillaries in the prostate gland

Blood capillaries are thread‐like structures that may be considered as an example of a spatial fibre process in three dimensions. At light microscopy, the capillary profiles appear as a planar point process on sections. It has recently been shown that the observed pair correlation function g(r) of the centres of the fibre profiles on two‐dimensional sections may be used to estimate the reduced pair correlation function of stationary and isotropic fibre processes in three dimensions. In the present study, we explored how this approach may be extended to statistical analysis of reduced g‐functions of capillaries from multiple specimens of different groups and with replicated observations. The methods were applied to normal prostatic tissue compared with prostate cancer. Confidence intervals for the mean reduced g‐functions of groups were estimated for fixed r‐values parametrically using the t‐distribution, and by bootstrap methods. Each estimated reduced g‐function was furthermore characterized in terms of its first maximum and minimum. The mean length of capillaries per unit tissue volume was significantly higher in prostate cancer tissue than in normal prostate tissue. Significant differences between the mean reduced g‐functions of malignant and benign lesions could be demonstrated for two domains of r‐values. In general, bootstrap‐based confidence intervals were slightly wider than parametrically estimated confidence intervals. Falsely negative lower bounds of the intervals, which sometimes arose using the parametric approach, could be avoided by the bootstrap method. Testing of group mean values for significant differences by the bootstrap method yielded more conservative results than multiple t‐tests. The functional value of the first maximum of the reduced g‐function and a global statistical parameter of short‐range ordering was significantly reduced in the carcinoma group. Prostate cancer tissue is more densely supplied with capillaries than normal prostate tissue and the three‐dimensional arrangement of the vessels differs with respect to interaction at various distance ranges. In the local approach used here, bootstrap methods can be used as a robust statistical tool for the computation of confidence intervals and group comparisons of mean reduced g‐functions at specific ranges of interaction.