Label‐free imaging characteristics of colonic mucinous adenocarcinoma using multiphoton microscopy

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity‐like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin‐eosin (HE) stained images. Second harmonic generation and two‐photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini‐endoscopy in real‐time early diagnosis of CRC. SCANNING 35: 277‐282, 2013. © 2012 Wiley Periodicals, Inc.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Label‐free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy

Glioblastoma multiforme (GBM‐WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10–12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.     

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.     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.     

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two‐photon excitation microscopy including 2‐photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2‐photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in‐tandem combination of 2‐photon fluorescence and second harmonic generated signal microscopy as two‐modality microscopy allows for in situ co‐localization imaging of various microstructural components in the whole‐mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2‐photon‐controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two‐modal 2‐photon microscopy/tomography, acting as an efficient and sensitive non‐injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non‐linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.     

Application of Fourier transform‐second‐harmonic generation imaging to the rat cervix

We present the application of Fourier transform‐second‐harmonic generation (FT‐SHG) imaging to evaluate the arrangement of collagen fibers in five nonpregnant rat cervices. Tissue slices from the mid‐cervix and near the external orifice of the cervix were analyzed in both two‐dimensions (2D) and three‐dimensions (3D). We validate that the cervical microstructure can be quantitatively assessed in three dimensions using FT‐SHG imaging and observe collagen fibers oriented both in and out‐of‐plane in the outermost and the innermost layers, which cannot be observed using 2D FT‐SHG analysis alone. This approach has the potential to be a clinically applicable method for measuring progressive changes in collagen organization during cervical remodeling in humans.     

Strain mapping accuracy improvement using super‐resolution techniques

Super‐resolution (SR) software‐based techniques aim at generating a final image by combining several noisy frames with lower resolution from the same scene. A comparative study on high‐resolution high‐angle annular dark field images of InAs/GaAs QDs has been carried out in order to evaluate the performance of the SR technique. The obtained SR images present enhanced resolution and higher signal‐to‐noise (SNR) ratio and sharpness regarding the experimental images. In addition, SR is also applied in the field of strain analysis using digital image processing applications such as geometrical phase analysis and peak pairs analysis. The precision of the strain mappings can be improved when SR methodologies are applied to experimental images.     

Blind deconvolution of 3D fluorescence microscopy using depth‐variant asymmetric PSF

The 3D wide‐field fluorescence microscopy suffers from depth‐variant asymmetric blur. The depth‐variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth‐variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth‐variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480–494, 2016. © 2016 Wiley Periodicals, Inc.     

Automatic fluorescent tag detection in 3D with super-resolution: application to the analysis of chromosome movement

In this paper, we describe an algorithmic framework for the automatic detection of diffraction‐limited fluorescent spots in 3D optical images at a separation below the Rayleigh limit, i.e. with super‐resolution. We demonstrate the potential of super‐resolution detection by tracking fluorescently tagged chromosomes during mitosis in budding yeast. Our biological objective is to identify and analyse the proteins responsible for the generation of tensile force during chromosome segregation. Dynamic measurements in living cells are made possible by green fluroescent protein (GFP)‐tagging chromosomes and spindle pole bodies to generate cells carrying four fluorescent spots, and observe the motion of the spots over time using 3D‐fluorescence microscopy. The central problem in spot detection arises with the partial or complete overlap of spots when tagged objects are separated by distances below the resolution of the optics. To detect multiple spots under these conditions, a set of candidate mixture models is built, and the best candidate is selected from the set based on χ²‐statistics of the residuals in least‐square fits of the models to the image data. Even with images having a signal‐to‐noise ratio (SNR) as low as 5–10, we are able to increase the resolution two‐fold below the Rayleigh limit. In images with a SNR of 5–10, the accuracy with which isolated tags can be localized is less than 5 nm. For two tags separated by less than the Rayleigh limit, the localization accuracy is found to be between 10 and 20 nm, depending on the effective point‐to‐point distance. This indicates the intimate relationship between resolution and localization accuracy.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

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.     

Increasing microscopy resolution with photobleaching and intensity cumulant analysis

Super‐resolution fluorescence microscopy and its applications for analysis of biological structures are evolving rapidly field. A number of approaches aimed at overcoming the fundamental limit imposed by diffraction have been proposed in recent years. Here we present a modification of super‐resolution optical fluctuation imaging (SOFI), a technique based on spatio‐temporal evaluation of the optical signal from independently fluctuating emitters. Instead of rapid, reversible photoswitching, photobleaching is used to produce irreversible transitions between emitting and nonemitting states of the fluorochrome molecules. Simulated images are used to demonstrate that, in the absence of noise, the proposed SOFI modification increases the efficiency of transfer of high spatial frequencies in a fluorescence microscope. Correspondingly, a decrease of the point spread function (PSF) width is obtained. Moreover, the modified SOFI algorithm is capable of resolving point emitters in the presence of simulated noise. Using real biological images we demonstrate that an increase of resolution is obtained in 2D optical sections through densely packed chromatin in cell nuclei and lamin layer at the nuclear envelope. Finally, the approach is extended to 3D wide‐field microscopy, allowing reduction of out‐of‐focus image blurring. Microsc. Res. Tech. 78:958–968, 2015. © 2015 Wiley Periodicals, Inc.     

Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems.     

Simultaneous multiplane imaging‐based localization encoded (SMILE) microscopy for super‐resolution volume imaging

We propose a widefield‐based rapid super‐resolution volume imaging technique. This technique requires encoding single molecules to their respective planes and subsequent identification of the locus of individual molecule (both in the focal plane and off‐focal planes). Experimentally, this is achieved by precise calibration of system PSF size and its natural spread in the off‐focal planes using sub‐diffraction fluorescent beads. The specimen plane touching the coverslip is chosen as the focal plane whereas planes far from coverslip (situated at large penetration depths) represent off‐focal planes. The identification and sorting of single molecules are carried out by setting multiple cut‐offs to the respective PSFs and a 3D super‐resolved volume image is reconstructed. SMILE microscopy technique eliminates the need for multiple z‐plane scanning, minimizes radiation‐dose and enables rapid super‐resolution volume imaging.     

Alignment with sub‐pixel accuracy for images of multi‐modality microscopes using automatic calibration

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in‐depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x–y detector plane using a custom chrome‐on‐glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter‐modal and 30 intra‐modal image pairs, and the success rates are 95 and 100%, respectively, with sub‐pixel accuracy. This methodology is widely applicable to any multi‐modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.     

Reversed scan direction reduces electron beam damage in EBSD maps

The deleterious effects of electron beam damage on high‐resolution electron backscatter diffraction (EBSD) maps of undeformed quartz are significantly reduced by scanning in the direction opposite to that dictated by widely used EBSD acquisition software. Higher quality electron backscatter patterns are produced when the electron beam moves progressively down the sample (the apparent ‘up’ direction in the resulting maps) for all step sizes where beam damage affects EBSD map quality (≤ ～0.4 μm in this study). The relative improvement associated with downward scanning increases as step size is reduced. A comparison of high‐resolution maps made in experimentally deformed quartz demonstrates that downward scanning reduces by a factor of ～2 the lower limit in step size relative to maps scanned in the conventional direction. The electron beam damages quartz at its point of entry, forming ～0.1‐μm diameter bumps visible in Scanning electron microscope (SEM) images. Downward scanning produces better results because it minimizes the flux of electrons through these loci of damaged crystal.     

A novel pixellated solid‐state photon detector for enhancing the everhart–thornley detector

This article presents a pixellated solid‐state photon detector designed specifically to improve certain aspects of the existing Everhart–Thornley detector. The photon detector was constructed and fabricated in an Austriamicrosystems 0.35 µm complementary metal‐oxide‐semiconductor process technology. This integrated circuit consists of an array of high‐responsivity photodiodes coupled to corresponding low‐noise transimpedance amplifiers, a selector‐combiner circuit and a variable‐gain postamplifier. Simulated and experimental results show that the photon detector can achieve a maximum transimpedance gain of 170 dBΩ and minimum bandwidth of 3.6 MHz. It is able to detect signals with optical power as low as 10 nW and produces a minimum signal‐to‐noise ratio (SNR) of 24 dB regardless of gain configuration. The detector has been proven to be able to effectively select and combine signals from different pixels. The key advantages of this detector are smaller dimensions, higher cost effectiveness, lower voltage and power requirements and better integration. The photon detector supports pixel‐selection configurability which may improve overall SNR and also potentially generate images for different analyses. This work has contributed to the future research of system‐level integration of a pixellated solid‐state detector for secondary electron detection in the scanning electron microscope. Microsc. Res. Tech. 76:648–652, 2013. © 2013 Wiley Periodicals, Inc.     

Measuring the PSF from aperture images of arbitrary shape--an algorithm

A new algorithm for determining the point spread function (PSF) of digital imaging systems is presented. The input is an image of an aperture whose shape need not be regular. The aperture shape is refined to an effective sub-pixel resolution and the PSF of the system is determined by de-convolution, assuming uniform illumination and a step function edge. The method has been tested on theoretical aperture images of varying shape and PSF, with and without noise. Depending on the degree of noise, a known PSF can be recovered to an accuracy of between 0.2 and 0.8%. Some typical results are given for a Gatan Image Filter with a 794 YAG multiscan camera on a Philips EM 430 transmission electron microscope at 200 and 300 kV. An example of a de-convoluted convergent beam electron diffraction pattern is included. The algorithm tolerates a small amount of de-focus.     

Background intensity correction for terabyte‐sized time‐lapse images

Several computational challenges associated with large‐scale background image correction of terabyte‐sized fluorescent images are discussed and analysed in this paper. Dark current, flat‐field and background correction models are applied over a mosaic of hundreds of spatially overlapping fields of view (FOVs) taken over the course of several days, during which the background diminishes as cell colonies grow. The motivation of our work comes from the need to quantify the dynamics of OCT‐4 gene expression via a fluorescent reporter in human stem cell colonies. Our approach to background correction is formulated as an optimization problem over two image partitioning schemes and four analytical correction models. The optimization objective function is evaluated in terms of (1) the minimum root mean square (RMS) error remaining after image correction, (2) the maximum signal‐to‐noise ratio (SNR) reached after downsampling and (3) the minimum execution time. Based on the analyses with measured dark current noise and flat‐field images, the most optimal GFP background correction is obtained by using a data partition based on forming a set of submosaic images with a polynomial surface background model. The resulting image after correction is characterized by an RMS of about 8, and an SNR value of a 4 × 4 downsampling above 5 by Rose criterion. The new technique generates an image with half RMS value and double SNR value when compared to an approach that assumes constant background throughout the mosaic. We show that the background noise in terabyte‐sized fluorescent image mosaics can be corrected computationally with the optimized triplet (data partition, model, SNR driven downsampling) such that the total RMS value from background noise does not exceed the magnitude of the measured dark current noise. In this case, the dark current noise serves as a benchmark for the lowest noise level that an imaging system can achieve. In comparison to previous work, the past fluorescent image background correction methods have been designed for single FOV and have not been applied to terabyte‐sized images with large mosaic FOVs, low SNR and diminishing access to background information over time as cell colonies span entirely multiple FOVs. The code is available as open‐source from the following link https://isg.nist.gov/ .