Light sheet microscopy for real-time developmental biology

Within only a few short years, light sheet microscopy has contributed substantially to the emerging field of real-time developmental biology. Low photo-toxicity and high-speed multiview acquisition have made selective plane illumination microscopy (SPIM) a popular choice for studies of organ morphogenesis and function in zebrafish, Drosophila, and other model organisms. A multitude of different light sheet microscopes have emerged for the noninvasive imaging of specimens ranging from single molecules to cells, tissues, and entire embryos. In particular, developmental biology can benefit from the ability to watch developmental events occur in real time in an entire embryo, thereby advancing our understanding on how cells form tissues and organs. However, it presents a new challenge to our existing data and image processing tools. This review gives an overview of where we stand as light sheet microscopy branches out, explores new areas, and becomes more specialized.     

Application of real-time confocal microscopy for observation of living cells in tissue specimens.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) has been a fundamental technique in cell biology. However, most investigations have used cultured or isolated cells as an experimental model, and consequently can provide only limited insight into the mechanisms that operate in tissue in situ. Useful information may be obtained by studying intact tissue specimens. High-speed confocal microscopes that can acquire digital images at video rate have recently been developed. These confocal microscopes which can acquire data in real-time enable [Ca2+]i dynamics of individual cells in intact tissue specimens to be observed. The present paper examines the use of fluorescent microscopy and confocal microscopy for [Ca2+]i imaging of living tissue. We analyzed the dynamics of the duodenal gland, lacrimal gland, intestinal smooth muscles, arterioles, myenteric plexus, and dorsal root ganglion. In these specimens, individual cells exhibited different [Ca2+]i dynamics, and the responses to transmitters/modulators were heterogeneous. In conclusion, real-time imaging provides a useful tool for observing dynamic changes in cells in situ, and it may lead to improve understanding tissue physiology.     

Atomic force microscopy imaging of dried or living bacteria.

Atomic force microscopy (AFM) was used to obtain micrographs of dried bacteria in air, and of living ones in their culture medium. Images of dried bacteria were very similar to images obtained elsewhere by the much more complicated cryoetching preparation technique for transmission electron microscopy. Living bacteria were immobilized on a poly-L-lysine film, and directly observed in their culture medium at a resolution unattainable by any other technique applicable to living material. The images were similar to those obtained in scanning electron microscopy where the specimen must be fixed, dried and coated with conductive material, and as a result, no longer viable.     

Scanning electron microscopy of alcohol-fixed cytopathology specimens

Cells in specimens fixed in alcohol and stained by the Papanicolaou method for routine cytodiagnosis were subsequently studied by scanning electron microscopy (SEM) to determine if they manifested topologic features described in specimens prepared for optimal SEM observation. In normal bronchial washings, ciliated columnar cells were obvious, and microridges were detected in several squamous cells. Microvilli, although sometimes coarse and blunted, were present in cells of adenocarcinoma, squamous carcinoma and malignant mesothelioma in fluid specimens. Benign histiocytes in bronchial washings, neuroblastoma cells in a smear of bone marrow aspirate and lymphocytic lymphoma cells in spinal fluid had ruffled surfaces. Should topologic features prove to be diagnostically significant, SEM may be of value in further studying equivocal specimens even though they were previously prepared for routine light microscopic observation.     

Scanning electron microscopy of plasma etched implant specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Structured illumination microscopy of a living cell

Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells. This article has been submitted as a contribution to the Festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday.     

Quantitative reflection contrast microscopy of living cells

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.     

Multispectral imaging fluorescence microscopy for living cells

Multispectral imaging technologies have been widely used in fields of astronomy and remote sensing. Interdisciplinary approaches developed in, for example, the National Aeronautics and Space Administration (NASA, USA), the Jet Propulsion Laboratory (JPL, USA), or the Communications Research Laboratory (CRL, Japan) have extended the application areas of these technologies from planetary systems to cellular systems. Here we overview multispectral imaging systems that have been devised for microscope applications. We introduce these systems with particular interest in live cell imaging. Finally we demonstrate examples of spectral imaging of living cells using commercially available systems with no need for user engineering.     

The freezing rate of freeze-etch specimens for electron microscopy

Experiments on the rapid freezing of freeze-etch sized specimens have shown this rate between 0 and ?100 °C to be approximately 1000 °C/sec. This is much faster than the rate of 100 °C/sec estimated by Moor for specimens cooled in liquid Freon 12.Heat transfer from the rapidly immersed specimen and mount appears to be mainly through forced convection. Such a mechanism would make the initial rate highly variable as it would be sensitive to liquid velocity. If this occurs it will be impossible to obtain consistent results for freezingrate studies unless a stable method is evolved for both injecting and containing the specimen.     

Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy

Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.     

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.