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.     

Electronic light microscopy: present capabilities and future prospects

Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy-video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy-considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, but suffers from a number of limitations. I discuss advances in confocal techniques that address the problems of temporal resolution, spherical and chromatic aberration, wavelength flexibility and cross-talk between fluorescent channels, and describe new optics to enhance axial resolution and the use of two-photon excitation to reduce photobleaching. Finally, I consider the desirability of establishing a digital image database, the BioImage database, which would permit the archival storage of, and public Internet access to, multidimensional image data from all forms of biological microscopy. Submission of images to the BioImage database would be made in coordination with the scientific publication of research results based upon these data. In the context of electronic light microscopy, this would be particularly useful for three-dimensional images of cellular structure and video sequences of dynamic cellular processes, which are otherwise hard to communicate. However, it has the wider significance of allowing correlative studies on data obtained from many different microscopies and from sequence and crystallographic investigations. It also opens the door to interactive hypermedia access to the multidimensional image data, and multimedia publishing ventures based upon this.Presented at the XXXVII Symposium of the Society for Histochemistry, 23 September 1995, Rigi Kaltbad, Switzerland     

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.     

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.     

Confocal microscopy: principles and applications to the field of reproductive biology

Confocal microscopy allows analysis of fluorescent labeled thick specimens without physical sectioning. Optical sections are generated by eliminating out-of-focus fluorescence and displayed as digitalized images. It allows 3-dimensional reconstruction (XYZ) and time-analysis (XYT), thus providing unique chance to link morphology with cell function. Since images are obtained by scanning, excess illumination of the specimen and quick decrease of the fluorescent signal are avoided. Resolution obtained with a Laser Scanning Confocal Microscopy (LSCM) is theoretically better than that of a conventional microscope. The preparation of the specimen may be based on standard techniques, such as immunocytochemistry applied to fixed cells, or on staining of living cells, following the use of different fluorescent probes at the same time (colocalization). In our laboratory, we use the LSCM system Fluoview version 2.1 (Olympus) to study reproductive biology of animals and humans. We work on stainings of oocytes and blastocysts (mouse, bovine, human), and human ovarian tissues. We study mitochondrial distribution, cortical granule migration, calcium oscillations and spindle quality to link culture conditions and oocyte quality. Staining of F-actin is used to check transzonal projections (in zona pellucida) or to detect abnormalities following experimental treatment. Blastocyst quality is analyzed in sequential optical sections for microfilament organization and counting of total cell number (staining with phalloidin (actin) and picogreen (DNA). Trophectoderm and inner cell mass distribution (differential staining), apoptotic cells (TUNEL method) and viable cells (live/dead test) are also evaluated. Confocal imaging can be helpful for rapid determination of follicle density (staining with AM Calcein) and follicle morphology (picogreen) in ovarian cortical biopsies. The current review describes the principles of confocal microscopy and illustrates its applications to the field of reproductive biology by a large collection of pictures.     

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets¹, monitor dynamics in living cells^2-4, and visualize the three dimensional evolution of entire organisms^5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo⁷ and are currently being applied to disease imaging and diagnosis in clinical settings^8,9.Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole¹, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.Download video file.^{(90M, mov)}     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.     

Confocal imaging of exine as a tool for grass pollen analysis

A new method which utilises confocal optical imaging has been developed which can be expected to improve grass pollen analysis. Confocal microscopy, in reflection mode, was used to examine the exine morphology of unacetolysed pollen grains from the following species of common wild grasses: Paspalum dilatatum, Setaria gracilis, Bromus catharticus, Daclylis glomerata, Lolium perenne, Poa pratensis, and Phalaris aquatica. Variations in the surface texture patterns, similar to those hitherto only seen by scanning electron microscopy, were visualised. In contrast to the latter method, specimen preparation for this confocal microscopy based technique was characterised by its simplicity, permitting the use of fresh and chemically untreated pollen grains. This confocal imaging technique, with its capacity for optical sectioning of specimens, offered the additional advantage of allowing the examination of the sub‐surface exine layers as well as the surface morphology of the pollen grains. Furthermore, three‐dimensional reconstruction of these optical sections enabled visualisation of the identified sculptural and structural exine elements and layers. A number of differences in these patterns were found, which indicate that confocal microscopy, in combination with image analysis, may enable finer taxonomic distinctions to be made than those currently provided by other light microscope based methods.     

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate.     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

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.     

High-performance confocal system for microscopic or endoscopic applications

We designed a high-performance confocal system that can be easily adapted to an existing light microscope or coupled with an endoscope for remote imaging. The system employs spatially and temporally patterned illumination produced by one of several mechanisms, including a micromirror array video projection device driven by a computer video source or a microlens array scanned by a piezo actuator in the microscope illumination path. A series of subsampled "component" video images are acquired from a solid-state video camera. Confocal images are digitally reconstructed using "virtual pinhole" synthetic aperture techniques applied to the collection of component images. Unlike conventional confocal techniques that raster scan a single detector and illumination point, our system samples multiple locations in parallel, with particular advantages for monitoring fast dynamic processes. We compared methods of patterned illumination and confocal image reconstruction by characterizing the point spread function, contrast, and intensity of imaged objects. Sample 3D reconstructions include a diatom and a Golgi-stained nerve cell collected in transmission.     

Single cell and tissue specific methods for evaluation of radiation and microgravity effects

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Utility of cytoplasmic fluorescent proteins for live-cell imaging of Magnaporthe grisea in planta

The subcellular expression patterns and fluorescence intensities of cytoplasm-targeted, constitutively expressed blue-, cyano-, green-, yellow- and red-fluorescent protein were assessed in a number of transformants of the blast pathogen, Magnaporthe grisea. All transformants grew normally, remained pathogenic on barley, and, except for those expressing blue fluorescent protein, exhibited significant cytoplasmic fluorescence. The exceptionally intense brightness of some strains proved very useful for laser scanning confocal microscope imaging during invasion of host tissues. Acquisition of three-dimensional data sets from intact, individual, pathogen encounter sites in planta were generated during the time course of pathogenesis using non-invasive optical sectioning methods. Confocal and multiphoton microscopy imaging in conjunction with fluorescent protein expression allowed for the real time documentation of fungal colonization within plant cells and tissues with remarkable ease. These methods constitute valuable new tools for the investigation of plant disease.     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.