Computed tomography-based spectral imaging for fluorescence microscopy

The computed tomography imaging spectrometer (CTIS) is a non-scanning instrument capable of simultaneously acquiring full spectral information (450-750 nm) from every position element within its field of view (75 microm x 75 microm). The current spatial and spectral sampling intervals of the spectrometer are 1.0 microm and 10 nm, respectively. This level of resolution is adequate to resolve signal responses from multiple fluorescence probes located within individual cells or different locations within the same cell. Spectral imaging results are presented from the CTIS combined with a commercial inverted fluorescence microscope. Results demonstrate the capability of the CTIS to monitor the spatiotemporal evolution of pH in rat insulinoma cells loaded with SNARF-1. The ability to analyze full spectral information for two-dimensional (x, y) images allows precise evaluation of heterogeneous physiological responses within cell populations. Due to low signal levels, integration times up to 2 s were required. However, reasonable modifications to the instrument design will provide higher system transmission efficiency with increased temporal and spatial resolution. Specifically, a custom optical design including the use of a larger format detector array is under development for a second-generation system.     

Biodegradable amphiphilic copolymers based on poly(epsilon-caprolactone)-graft chondroitin sulfate as drug carriers

The goal of this study was to develop a new type of core-shell micelles based on biocompatible and biodegradable amphiphilic copolymers, named PCL-CS, using chondroitin sulfate (CS) as a hydrophilic segment and poly(epsilon-caprolactone) (PCL) as a hydrophobic segment. The copolymers, prepared from the various compositions between CS and PCL, were characterized by Fourier transform infrared spectrometer, proton nuclear magnetic resonance spectrometer, and differential scanning calorimeter. The PCL-CS copolymers could be assembled into micelles using a simple emulsion. With the fluorescent probe technique, the critical micelle concentrations were obtained in the range of 1.26 x 10(-3)-8.86 x 10(-3) mg/mL. The spherical images of micelles were visualized in the presence of polyvinyl alcohol (PVA) with the use of the transmission electron microscope. The particle sizes of micelles were all smaller than 300 nm, neither aggregate nor change in hydrodynamic sizes after 15 days staying in solutions containing salts or PVA by dynamic light scattering. The intracellular uptake of KB cells incubated with PCL-CS micelles was evidenced by confocal laser scanning microscope upon loading fluorescein isothiocyanate labeled bovine serum albumin as a probe.     

Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.     

Spectroscopic characterization of streptavidin functionalized quantum dots

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work, we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot 1005-0045, and QD585, lot 0905-0031, from Invitrogen). This is the first step in the development of calibration beads to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited state lifetime measurements, to characterize their spectroscopic behavior, concentrating on the 400- to 500-nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480 nm excitation up to 0.4 at 450 nm and down again to 0.15 at 350 nm. For QD585, QY values were constant at 0.2 between 500 and 400 nm, but they dropped to 0.1 at 350 nm. We attribute the wavelength dependencies to heterogeneity in size and surface defects in the QD525, consistent with characteristics described previously in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots and the quantitative needs of assay development in biological applications.     

Quantitative infrared spectroscopy of lipids in solution: III. Automated data collection and computer aided calculations of lipid analysis.

In the third part of this series, a data acquisition system is described which digitizes the analog signal from a commercial infrared spectrometer and records the data on paper tape. The paper tape is then processed off-line with a microcomputer and an infrared spectrum is printed. The correction routine for absorbance due to the O-H stretching band is discussed as well as the integration limits for calculation of integrated intensities of the C-H stretching band. The computer program is briefly described and statistical data on correlation between integrated intensity and lipid concentration given.     

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS: We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS: Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS: Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.     

Automated screening of 2D crystallization trials using transmission electron microscopy: A high-throughput tool-chain for sample preparation and microscopic analysis

We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow.     

UV‐EXCITED BLUE AUTOFLUORESCENCE OF PSEUDO‐NITZSCHIA MULTISERIES (BACILLARIOPHYCEAE)1

A flow cytometer coupled to a scanning monochromator and a fluorescence microscope were used to characterize the fluorescence spectrum of Pseudo‐nitzschia multiseries (Hasle) Hasle, a pennate diatom that produces the neurotoxin domoic acid, a lethal amnesic. In this research, we characterize the fluorescence spectrum of P. multiseries in vivo over the wavelength range of 360 to 850 nm and show that this diatom autofluoresces blue when excited with UV light (350–365 nm). The autofluorescence characterization of Pseudo‐nitzschia may provide new methods for rapid in situ monitoring of diatom populations and reiterates the usefulness of flow cytometry in the analysis and study of marine phytoplankton.     

A Highlights from MBoC Selection: Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.     

Base pair mismatch recognition using plasmon resonant particle labels

We demonstrate the use of silver plasmon resonant particles (PRPs), as reporter labels, in a microarray-based DNA hybridization assay in which we screen for a known polymorphic site in the breast cancer gene BRCA1. PRPs (40-100 nm in diameter) image as diffraction-limited points of colored light in a standard microscope equipped with dark-field illumination, and can be individually identified and discriminated against background scatter. Rather than overall intensity, the number of PRPs counted in a CCD image by a software algorithm serves as the signal in these assays. In a typical PRP hybridization assay, we achieve a detection sensitivity that is approximately 60 x greater than that achieved by using fluorescent labels. We conclude that single particle counting is robust, generally applicable to a wide variety of assay platforms, and can be integrated into low-cost and quantitative detection systems for single nucleotide polymorphism analysis.     

Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.     

Portable telepathology: methods and tools

Telepathology is becoming easier to implement in most pathology departments. In fact e-mail image transmit can be done from almost any pathologist as a simplistic telepathology system. We tried to develop a way to improve capabilities of communication among pathologists with the idea that the system should be affordable for everybody. We took the premise that any pathology department would have microscopes and computers with Internet connection, and selected a few elements to convert them into a telepathology station. Needs were reduced to a camera to collect images, a universal microscope adapter for the camera, a device to connect the camera to the computer, and a software for the remote image transmit. We found out a microscope adapter (MaxView Plus) that allowed us connect almost any domestic digital camera to any microscope. The video out signal from the camera was sent to the computer through an Aver Media USB connector. At last, we selected a group of portable applications that were assembled into a USB memory device. Portable applications are computer programs that can be carried generally on USB flash drives, but also in any other portable device, and used on any (Windows) computer without installation. Besides, when unplugging the device, none of personal data is left behind. We selected open-source applications, and based the pathology image transmission to VLC Media Player due to its functionality as streaming server, portability and ease of use and configuration. Audio transmission was usually done through normal phone lines. We also employed alternative videoconferencing software, SightSpeed for bi-directional image transmission from microscopes, and conventional cameras allowing visual communication and also image transmit from gross pathology specimens. All these elements allowed us to install and use a telepathology system in a few minutes, fully prepared for real time image broadcast.     

Elemental imaging by EELS and EDXS in the analytical electron microscope

Electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS) can be used to obtain elemental maps from thin biological samples in the analytical electron microscope. The EELS is particularly sensitive for the low-atomic-number elements, including C, N, and O, as well as other elements with favorable ionization cross-sections, such as Fe. The EDXS is useful for a complementary range of atoms, such as P, S, K, and Ca. A system is described for obtaining elemental distributions in an analytical electron microscope operated in the scanning transmission mode at 100–200 keV beam energy. The spatial resolution is typically limited to 10–20 nm when a conventional source is used. A satellite microcomputer controls acquisition of EELS and EDXS data from successive pixels in an image. These data are processed “on-the-fly” by a host computer to remove the noncharacteristic background intensity. Resulting images are stored on disk and can be analyzed by means of an image display system controlled by interactive software. The technique is demonstrated with elemental maps from two samples: alveolar macrophages containing respirable particles; and pancreatic beta cells that secrete insulin.     

Construction of a confocal microscope for real-time x-y and x-z imaging

We describe the construction of a simple 'real-time' laser-scanning confocal microscope, and illustrate its use for rapid imaging of elementary intracellular calcium signaling events. A resonant scanning galvanometer (8 kHz) allows x-y frame acquisition rates of 15 or 30 Hz, and the use of mirrors to scan the laser beam permits use of true, pin-hole confocal detection to provide diffraction-limited spatial resolution. Furthermore, use of a piezoelectric device to rapidly focus the objective lens allows axial (x-z) images to be obtained from thick specimens at similar frame rates. A computer with image acquisition and graphics cards converts the output from the microscope to a standard video signal, which can then be recorded on videotape and analyzed by regular image processing systems. The system is largely made from commercially available components and requires little custom construction of mechanical parts or electronic circuitry. It costs only a small fraction of that of comparable commercial instruments, yet offers greater versatility and similar or better performance.     

新型快速显微多道分光光度系统及其应用

介绍一种新型快速显微多道分光光度系统。利用倒置显微镜、衍射光栅、线阵CCD等构成的显微多道分光光度系统,可对微区样品在350nm-800nm（波长分辨率为0．2nm)光谱波长范围内进行快速光谱检测,最快检测时间为1ms。利用该系统进行的人血红细胞内血红蛋白测量分析表明,该具有高灵敏度、快速、无扰、在位、多光谱显微测量的特点,在生命科学研究中动态监测细胞生命过程的变化。     

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm². The scanning time was less than 9 min.The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm²/s were the parameters used for tractography.There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.     

Scanning secondary ion analytical microscopy with parallel detection.

The secondary ion microscope described here allows to obtain the simultaneous registration of chemical and isotopic distribution maps of several elements composing the sample. The instrument has been specially designed to optimize both sensitivity and selectivity; bombardment with primary Cs+ ions to increase the ionization yields of negative secondary ions, efficient collection of secondary ions at the target surface, matching of the secondary ion beam etendue with the acceptance of the mass spectrometer working at high mass resolution, spectrometer with parallel detection capabilities. The probe diameter can be made as low as 30 nm and ion induced electron images registered at the same time as ion images. Presently, four ion micrographs are obtained simultaneously over a field of view up to 20 x 20 micro m2 containing up to 512 x 512 pixels. Examples are shown with an ion probe diameter of 0.1 microm.     

A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10(4) at 350 nm and 8.29 x 10(3) at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10(3) at 350 nm and 1.26 x 10(5) at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.     

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the ''mislocalization'' phenomenon.