Confocal microscopy: a tool to visualise differentiation of stem cells into cardiomyocytes

Confocal microscopy offers important advantages compared to conventional epifluorescence microscopy. It works as an "optical microtome" leading to a accurate image resolution of a defined focal plane. Furthermore, the addition of a Nipkow disk on the confocal microscope greatly accelerates the image acquisition, up to 30 frames per second. Nevertheless, the software-assisted mathematical restoration of images acquired using a wide-field microscope allows to get images with a resolution similar to the one obtained in confocal microscopy. These imaging technologies allowed us to monitor on line cardiac differentiation of murine embryonic stem (ES) cells within 3D structures called embryoid bodies. The high rate acquisition of images using the confocal microscope equipped with a Nipkow disk allows to monitor calcium spiking in differentiating cardiomyocytes within embryoid bodies.     

Construction of a two-photon microscope for video-rate Ca imaging

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.     

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.     

Power and limits of laser scanning confocal microscopy

In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out-of-focus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and nondestructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three-dimensional reconstitution of nuclear components using a cubic representation and a ray-tracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3-D data set: self-shadowing effects of thick specimens, spherical aberrations due to the sub-optimum use of the objective lenses and photobleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for 3-D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers and a possible means to attenuate photodamage to biological specimens.     

High-speed multicolor microscopy of repeating dynamic processes

Images of multiply labeled fluorescent samples provide unique insights into the localization of molecules, cells, and tissues. The ability to image multiple channels simultaneously at high speed without cross talk is limited to a few colors and requires dedicated multichannel or multispectral detection procedures. Simpler microscopes, in which each color is imaged sequentially, produce a much lower frame rate. Here, we describe a technique to image, at high frame rate, multiply labeled samples that have a repeating motion. We capture images in a single channel at a time over one full occurrence of the motion then repeat acquisition for other channels over subsequent occurrences. We finally build a high-speed multichannel image sequence by combining the images after applying a normalized mutual information-based time registration procedure. We show that this technique is amenable to image the beating heart of a double-labeled embryonic quail in three channels (brightfield, yellow, and mCherry fluorescent proteins) using a fluorescence wide-field microscope equipped with a single monochrome camera and without fast channel switching optics. We experimentally evaluate the accuracy of our method on image series from a two-channel confocal microscope.     

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca²⁺ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record lines-cans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.     

Measuring fast dynamics in solutions and cells with a laser scanning microscope

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.     

Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons

A video-rate (30 frames/s) scanning two-photon excitation microscope has been successfully tested. The microscope, based on a Nikon RCM 8000, incorporates a femtosecond pulsed laser with wavelength tunable from 690 to 1050 nm, prechirper optics for laser pulse-width compression, resonant galvanometer for video-rate point scanning, and a pair of nonconfocal detectors for fast emission ratioing. An increase in fluorescent emission of 1.75-fold is consistently obtained with the use of the prechirper optics. The nonconfocal detectors provide another 2.25-fold increase in detection efficiency. Ratio imaging and optical sectioning can therefore be performed more efficiently without confocal optics. Faster frame rates, at 60, 120, and 240 frames/s, can be achieved with proportionally reduced scan lines per frame. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at specimen with the genetically modified green fluorescent proteins. Preliminary results obtained using this system confirm that the yellow "cameleons" exhibit similar optical properties as under one-photon excitation conditions. Dynamic two-photon images of cardiac myocytes and ratio images of yellow cameleon-2.1, -3.1, and -3.1nu are also presented.     

Combined confocal and wide-field high-resolution cytometry of fluorescent in situ hybridization-stained cells

BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance.     

Software for drift compensation, particle tracking and particle analysis of high-speed atomic force microscopy image series

Atomic force microscopy (AFM) image acquisition is performed by raster-scanning a faint tip with respect to the sample by the use of a piezoelectric stage that is guided by a feedback system. This process implies that the resulting images feature particularities that distinguish them from images acquired by other techniques, such as the drift of the piezoelectric elements, the unequal image contrast along the fast- and the slow-scan axes, the physical contact between the tip of nondefinable geometry and the sample, and the feedback parameters. Recently, high-speed AFM (HS-AFM) has been introduced, which allows image acquisition about three orders of magnitude faster (500-100 ms frame rate) than conventional AFM (500 s to 100 s frame rate). HS-AFM produces image sequences, large data sets, which report biological sample dynamics. To analyze these movies, we have developed a software package that (i) adjusts individual scan lines and images to a common contrast and z-scale, (ii) filters specifically those scan lines where increased or insufficient force was applied, (iii) corrects for piezo-scanner drift, (iv) defines particle localization and angular orientation, and (v) performs particle tracking to analyze the lateral and rotation displacement of single molecules.     

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.     

isoSTED microscopy with water-immersion lenses and background reduction

Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few 10 nm at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO₂ supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm³. Because fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method that allows us to identify this unspecific background signal and to remove it from the image.     

Statistical evaluation of confocal microscopy images

BACKGROUND: The coefficient of variation (CV) is defined as the standard deviation (sigma) of the fluorescent intensity of a population of beads or pixels expressed as a proportion or percentage of the mean (mu) intensity (CV = sigma/mu). The field of flow cytometry has used the CV of a population of bead intensities to determine if the flow cytometer is aligned correctly and performing properly. In a similar manner, the analysis of CV has been applied to the confocal laser scanning microscope (CLSM) to determine machine performance and sensitivity. METHODS: Instead of measuring 10,000 beads using a flow cytometer and determining the CV of this distribution of intensities, thousands of pixels are measured from within one homogeneous Spherotech 10-microm bead. Similar to a typical flow cytometry population that consists of 10,000 beads, a CLSM scanned image consists of a distribution of pixel intensities representing a population of approximately 100,000 pixels. In order to perform this test properly, it is important to have a population of homogeneous particles. A biological particle usually has heterogeneous pixel intensities that correspond to the details in the biological image and thus shows more variability as a test particle. RESULTS: The bead CV consisting of a population of pixel intensities is dependent on a number of machine variables that include frame averaging, photomultiplier tube (PMT) voltage, PMT noise, and laser power. The relationship among these variables suggests that the machine should be operated with lower PMT values in order to generate superior image quality. If this cannot be achieved, frame averaging will be necessary to reduce the CV and improve image quality. There is more image noise at higher PMT settings, making it is necessary to average more frames to reduce the CV values and improve image quality. The sensitivity of a system is related to system noise, laser light efficiency, and proper system alignment. It is possible to compare different systems for system performance and sensitivity if the laser power is maintained at a constant value. Using this bead CV test, 1 mW of 488 nm laser light measured on the scan head yielded a CV value of 4% with a Leica TCS-SP1 (75-mW argon-krypton laser) and a CV value of 1.3% with a Zeiss 510 (25-mW argon laser). A biological particle shows the same relationship between laser power, averaging, PMT voltage, and CV as do the beads. However, because the biological particle has heterogeneous pixel intensities, there is more particle variability, which does not make as useful as a test particle. CONCLUSIONS: This CV analysis of a 10-microm Spherotech fluorescent bead can help determine the sensitivity in a confocal microscope and the system performance. The relationship among the factors that influence image quality is explained from a statistical endpoint. The data obtained from this test provides a systematic method of reducing noise and increasing image clarity. Many components of a CLSM, including laser power, laser stability, PMT functionality, and alignment, influence the CV and determine if the equipment is performing properly. Preliminary results have shown that the bead CV can be used to compare different confocal microscopy systems with regard to performance and sensitivity. The test appears to be analogous to CV tests made on the flow cytometer to assess instrument performance and sensitivity. Published 2001 Wiley-Liss, Inc.     

Localization and high-resolution imaging of cortical neurotransmitter compartments using confocal laser scanning microscopy: GABA and glutamate interactions in rat cortex.

This report compares the application of confocal laser scanning fluorescence microscopy with standard epifluorescence microscopy for the simultaneous localization of the neurotransmitters gamma-aminobutyric acid and glutamate in rat cerebral cortex. With this approach, sections of fixed rat brain are treated with primary antibodies against gamma-aminobutyric acid (rabbit-derived) and glutamate (mouse-derived), followed by treatment with fluorescein isothiocyanate-tagged donkey anti-rabbit and rhodamine-tagged goat anti-mouse secondary antibodies, respectively. The results demonstrate that images from immunofluorescence localizations with a confocal laser scanning microscope have superior resolution and contrast as a result of significant reductions of background flare caused by emission from out-of-focus structures in the field of view. The confocal microscope achieves this improved image quality by optically sectioning through a specimen at narrow planes of focus and then compiling a composite image of an object of interest. The composite image can be further enhanced by using various image processing options. The combined use of double immunofluorescence and confocal laser scanning microscopy provides an important means to simultaneously study the anatomical relationships of pre- and post-synaptic elements in a complex neural system.     

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100-300mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.     

Virtual slit scanning microscopy

We present a novel slit scanning confocal microscope with a CCD camera image sensor and a virtual slit aperture for descanning that can be adjusted during post-processing. A very efficient data structure and mathematical criteria for aligning the virtual aperture guarantee the ease of use. We further introduce a method to reduce the anisotropic lateral resolution of slit scanning microscopes. System performance is evaluated against a spinning disk confocal microscope on identical specimens. The virtual slit scanning microscope works as the spinning disk type and outperforms on thick specimens.     

Hyperspectral imaging: a novel approach for microscopic analysis

BACKGROUND: The usefulness of the light microscope has been dramatically enhanced by recent developments in hardware and software. However, current technologies lack the ability to capture and analyze a high-resolution image representing a broad diversity of spectral signatures in a single-pass view. We show that hyperspectral imaging offers such a technology. METHODS AND RESULTS We developed a prototype hyperspectral imaging microscope capable of collecting the complete emission spectrum from a microscope slide. A standard epifluorescence microscope was optically coupled to an imaging spectrograph, with output recorded by a CCD camera. Software was developed for image acquisition and computer display of resultant X--Y images with spectral information. Individual images were captured representing Y-wavelength planes, with the stage successively moved in the X direction, allowing an image cube to be constructed from the compilation of generated scan files. This prototype instrument was tested with samples relevant to cytogenetic, histologic, cell fusion, microarray scanning, and materials science applications. CONCLUSIONS: Hyperspectral imaging microscopy permits the capture and identification of different spectral signatures present in an optical field during a single-pass evaluation, including molecules with overlapping but distinct emission spectra. This instrument can reduce dependence on custom optical filters and, in future imaging applications, should facilitate the use of new fluorophores or the simultaneous use of similar fluorophores.     

Visualization of silver-enhanced reaction products from protein- and immuno-colloidal gold probes by laser scanning confocal microscopy in leflection mode

We have employed a laser scanning confocal microscope in reflection mode to directly and indirectly visualize sites of deposition of silver-enhanced reaction products from colloidal gold probes. A direct approach was used for the localization of alpha-fetoprotein receptors in human myoblasts by incubating primary cultures with an alpha-fetoprotein-gold conjugate. For an indirect approach, cultured CEM cells, derived from a human T-lymphoma cell line, were incubated with a mouse monoclonal antibody to mature T-cells, followed by a gold-labelled antibody to mouse immunglobulins. Multiple optical sections of each sample were collected by reflection laser scanning confocal microscopy and combined into three-dimensional renderings. A (non-confocal) transmission image was generated of each field for comparative purposes. The increasing use of reflection laser scanning confocal microscopy combined with colloidal gold conjugates as biological markes will probably be of considerable advantage in cytochemical analysis.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc^Min/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.