ELECTRONIC LIGHT-MICROSCOPY - PRESENT CAPABILITIES AND FUTURE-PROSPECTS

Authors
Citation
Dm. Shotton, ELECTRONIC LIGHT-MICROSCOPY - PRESENT CAPABILITIES AND FUTURE-PROSPECTS, HISTOCHEM C, 104(2), 1995, pp. 97-137
Citations number
156
Categorie Soggetti
Cell Biology",Microscopy
Journal title
HISTOCHEMISTRY AND CELL BIOLOGY
ISSN journal
09486143 → ACNP
Volume
104
Issue
2
Year of publication
1995
Pages
97 - 137
Database
ISI
SICI code
0948-6143(1995)104:2<97:EL-PCA>2.0.ZU;2-5
Abstract
Electronic light microscopy involves the combination of microscopic te chniques with electronic imaging and digital image processing, resulti ng in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images a nd a discussion of the sampling requirements for the accurate digital recording of optical images. I discuss the three most important imagin g modalities in electronic light microscopy - video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning micr oscopy - considering their capabilities, their applications, and recen t developments that will increase their potential. Video-enhanced cont rast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloid al gold particles, an order of magnitude smaller than the resolution l imit of the light microscope. It has revolutionised the study of cellu lar motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the techniq ue of optical trapping (optical tweezers), it permits exquisitely sens itive force and distance measurements to be made on motor proteins. Di gital fluorescence microscopy enables low-light-level imaging of fluor escently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particula rly multiple bandpass dichroic mirrors, and developments in multiparam eter imaging, which is becoming particularly important for in situ hyb ridisation studies and automated image cytometry, fluorescence ratio i maging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-f ocus blur deconvolution and image restoration are becoming increasingl y important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, b ut suffers from a number of limitations. I discuss advances in confoca l techniques that address the problems of temporal resolution, spheric al and chromatic aberration, wavelength flexibility and cross-talk bet ween fluorescent channels, and describe new optics to enhance axial re solution 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 stor age of, and public Internet access to, multidimensional image data fro m all forms of biological microscopy. Submission of images to the BioI mage database would be made in coordination with the scientific public ation of research results based upon these data. In the context of ele ctronic light microscopy, this would be particularly useful for three- dimensional images of cellular structure and video sequences of dynami c cellular processes, which are otherwise hard to communicate. However , it has the wider significance of allowing correlative studies on dat a obtained from many different microscopies and from sequence and crys tallographic investigations. It also opens the door to interactive hyp ermedia access to the multidimensional image data, and multimedia publ ishing ventures based upon this.