Skip to main content
Log in

Electronic light microscopy: present capabilities and future prospects

  • Robert Feulgen Lecture 1995
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Agard DA (1984) Optical sectioning microscopy: cellular architecture in three dimensions. Annu Rev Biophys Bioeng 13: 191–219

    PubMed  Google Scholar 

  • Agard DA, Sedat JW (1983) Three-dimensional architecture of a polytene nucleus. Nature 302:676–681

    PubMed  Google Scholar 

  • Agard DA, Hiraoka Y, Shaw PJ, Sedat JW (1989) Fluorescence microscopy in three dimensions. Methods Cell Biol 30: 353–377

    PubMed  Google Scholar 

  • Aikens R (1993) Properties of low-light-level slow-scan detectors. In: Mason WT (ed) Fluorescent and luminescent probes for biological activity. Academic Press, London, pp 277–286

    Google Scholar 

  • Aikens RS, Agard DA, Sedat JW (1989) Solid-state imagers for microscopy. Methods Cell Biol 29:291–314

    PubMed  Google Scholar 

  • Allen RD (1985a) New observations on cell architecture and dynamics by video-enhanced contrast optical microscopy. Annu Rev Biophys Biophys Chem 14:265–290

    PubMed  Google Scholar 

  • Allen, RD (1985b) Cited in Higgins R (1985) A new window into living cells. Boston Globe 12 August

  • Allen RD, Allen NS (1983) Video-enhanced microscopy with a computer frame memory. J Microscopy 129:3–17

    Google Scholar 

  • Allen RD, Allen NB, Travis JL (1981) Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network ofAllogromia laticollaris. Cell Motility 1:291–302

    PubMed  Google Scholar 

  • Allen RD, Metuzals J, Tasaki I, Brady ST, Gilbert SP (1982) Fast axonal transport in squid giant axon. Science 218:1127–1129

    PubMed  Google Scholar 

  • Allen RD, Weiss DG, Hayden JH, Brown DT, Fujiwake H, Simpson M (1985) Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol 100:736–1752

    PubMed  Google Scholar 

  • Allen TD (1987) Time lapse video microscopy using an animation control unit. J Microsc 147:129–135

    PubMed  Google Scholar 

  • Anderson CM, Georgiou GN, Morrison IE, Stevenson GV, Cherry RJ (1992) Tracking cell surface receptors by fluorescence digital microscopy using a charge-coupled device camera. J Cell Sci 101:415–425

    PubMed  Google Scholar 

  • Appleyard ST, Dunn MJ, Dubowitz V, Scott ML, Pittman SJ, Shotton DM (1984) Monoclonal antibodies detect a spectrinlike protein in normal and dystrophic human skeletal muscle. Proc Natl Acad Sci USA 81:776–780

    PubMed  Google Scholar 

  • Arndt-Jovin DJ, Robert-Nicoud M, Kaufman SJ, Jovin TM (1985) Fluorescence digital imaging microscopy in cell biology. Science 230:247–256

    PubMed  Google Scholar 

  • Beverloo HB, Schadewijk A van, Bonnett J, Geest R van der, Runia R, Verwoerd NP, Vrolijk H, Ploem JS, Tanke HJ (1992) Preparation and microscopic visualisation of multicolor uminescent immunophosphors. Cytometry 13:561–570

    PubMed  Google Scholar 

  • Beverloo HB, Schadewijk A van, Zijlmans HJ, Verwoerd NP, Bonnett J, Vrolijk H, Tanke HJ (1993) A comparison of the detection sensitivity of lymphocyte membrane antigens using fluorescein and phosphor immunoconjugates. J Histochem Cytochem 41:719–725

    PubMed  Google Scholar 

  • Block SM (1990) Optical tweezers: a new tool for biophysics, In: Forskett JK, Grinstein S (eds) Noninvasive techniques in cell biology. (Modern reviews in cell biology, vol 9). Wiley-Liss, New York, pp 375–402

    Google Scholar 

  • Bolsover SR, Silver RA, Whitaker M (1993) Ratio imaging measurements of intracellular calcium and pH. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 181–210

    Google Scholar 

  • Boyde A (1985) Stereoscopic images in confocal (tandem scanning) microscopy. Science 230:1270–1272

    PubMed  Google Scholar 

  • Boyde A (1987) Colour-coded stereo images from the tandem scanning reflected light microscope (TSRLM). J Microsc 146:137–142

    PubMed  Google Scholar 

  • Boyde A (1993) Real-time direct-view confocal light microscopy. In: Shotton DM (ed.) Electronic light microscopy. Wiley-Liss, New York, pp 289–314

    Google Scholar 

  • Brabander M de, Geuens G, Nuydens R, Moeremans M, Mey J de (1985) Probing microtubule-dependent intracellular motility with nanometer particle video ultramiscroscopy. Cytobios 43:273–283

    PubMed  Google Scholar 

  • Brabander M de, Nuydens R, Geuens G, Moeremans M, Mey J de (1986) The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell. Cell Motility and the Cytoskeleton 6:105–113

    PubMed  Google Scholar 

  • Brabander M de, Nuydens R, Geerts H, Hopkins CR (1988) Dynamic behaviour of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy. Cell Motil Cytoskeleton 9:30–47

    PubMed  Google Scholar 

  • Brabander M de, Nuydens R, Ishihara A, Holifield B, Jacobson K, Geerts H (1991) Lateral diffusion and retrograde movements of cell surface components on single motile cells, observed with nanovid microscopy. J Cell Biol 112:111–124

    PubMed  Google Scholar 

  • Brabander M de, Geerts H, Nuyens R, Nuydens R, Cornelissen F (1993) Nanovid microsopy: imaging and quantification of colloidal gold labels in living cells. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss. New York, pp 141–156

    Google Scholar 

  • Brakenhoff GJ, Visscher K (1990) Novel confocal imaging and visualisation techniques. Trans R Microsc Soc 1:247–250

    Google Scholar 

  • Brakenhoff GJ, Visscher K (1991) Confocal imaging with bilateral scanning and array detectors. Scanning 13[Suppl 1]:65–66

    Google Scholar 

  • Brakenhoff GJ, Voort HTM van der, Spronsen EA van, Linnemans WAM, Nanninga N (1985) Three-dimensional chromatin distribution in neuroblastoma nuclei shown by confocal scanning laser microscopy. Nature 317:748–749.

    PubMed  Google Scholar 

  • Brakenhoff GJ, Visscher K, Gijsbers G (1994a) Fluorescence bleach rate imaging. Proceedings of the International Conference on Confocal and Near Field Microscopy, Munich, April. Society for 3D Imaging Sciences in Microscopy, Amsterdam

    Google Scholar 

  • Brakenhoff GJ, Squier J, Norris T, Bliton C, Athey B (1994b) Real time two-photon confocal microscopy using a femtosecond amplified Ti:sapphire system. Proc Intl Conf Confocal and Near Field Microscopy. Munich (April 1994) Soc 3D Imaging Sciences in Microscopy. Amsterdam

    Google Scholar 

  • Bright GR (1993) Multiparameter imaging of cellular function. In: Mason WT (ed) Fluorescent and luminescent probes for biological activity. Academic Press, London, pp 204–215

    Google Scholar 

  • Bright GR, Fisher GW, Rogowska J, Taylor DL (1989) Fluorescence ratio imaging microscopy. Methods Cell Biol 30: 157–192

    PubMed  Google Scholar 

  • Carazo JM, Marabini R, Vaquerizo C, Frank J (1994) Proceedings of 13th International Conference on Electronic Microscopy, Paris, vol 1, pp 519–520

    Google Scholar 

  • Carlsson K, Åslund N, Mossberg K, Philip J (1994) Simultaneous confocal recording of multiple fluorescent labels with improved channel separation. J Microsc 176:287–299

    PubMed  Google Scholar 

  • Cassimeris L, Pryer NK, Salmon ED (1988) Real-time observations of microtubule dynamics in living cells. J Cell Biol 107:2223–2231

    PubMed  Google Scholar 

  • Castleman KR (1979) Digital Image Processing. Prentice-Hall, Englewood Cliffs, NJ

    Google Scholar 

  • Castleman KR (1993) Resolution and sampling requirements for digital image processing, analysis and display. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 71–93

    Google Scholar 

  • Dauwerse JG, Wiegent J, Raap AK, Breuning MH, Ommen GJB van (1992) Multiple colors by fluorescence in situ hybridisation using ratio-labelled DNA probes create a molecular karyotype. Hum Mol Genet 1:593–598

    PubMed  Google Scholar 

  • DeBiasio R, Bright GR, Ernst LA, Waggoner AS, Taylor DL (1987) Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells. J Cell Biol 105:1613–1622

    PubMed  Google Scholar 

  • Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76

    PubMed  Google Scholar 

  • Draaijer A, Houpt PM (1988) A standard video-rate confocal laser-scanning reflection and fluorescence microscope. Scanning 10:139–145

    Google Scholar 

  • Draaijer A, Houpt PM (1993) High scan-rate confocal laser scanning microscopy. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 273–288

    Google Scholar 

  • Dunn GA, Brown AF (1991) A unified approach to analysing cell motility. J Cell Sci [Suppl] 8:81–102

    Google Scholar 

  • Edidin M, Stroynowski I (1991) Differences between lateral organization of conventional and inositol phospholipid-anchored membrane proteins. A further definition of micrometer scale domains. J Cell Biol 112:1143–1150

    PubMed  Google Scholar 

  • Edidin M, Kuo SC, Sheetz MP (1991) Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers. Science 254:1379–1382

    PubMed  Google Scholar 

  • Eggar MD, Petran M (1967) New reflected-light microscopy for viewing unstained brain and ganglion cells. Science 157: 305–307

    PubMed  Google Scholar 

  • Ellis GW (1985) Microscope illuminator with fiber optic source integrator. J Cell Biol 101:83a

    Google Scholar 

  • Finer JT, Simmons RM, Spudich JA (1994) Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368:113–119

    PubMed  Google Scholar 

  • Forscher P, Smith SJ (1988) Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol 107:1505–1516

    PubMed  Google Scholar 

  • Friedl P, Nobel PB, Zanker KS (1993) Lymphocyte locomotion in three-dimensional collagen gels. Comparison of three quantitative methods for analysing cell trajectories. J Immunol Methods 165:157–165

    PubMed  Google Scholar 

  • Geerts H, Brabander M de, Nuydens R, Geuens S, Moeremans M, Mey J de, Hollenbeck P (1987) Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy. Biophys J 52:775–782

    PubMed  Google Scholar 

  • Gelles J, Schnapp BJ, Sheetz MP (1988) Tracking kinesin-driven movements with nanometer-scale precision. Nature 331: 450–453

    PubMed  Google Scholar 

  • Ghosh RN, Webb WW (1994) Automatic detection and tracking of individual and clustered cell surface low density lipoprotein molecules. Biophys J 66:1301–1318

    PubMed  Google Scholar 

  • Gross D, Webb WW (1986) Molecular counting of low density lipoprotein particles as individuals and small clusters on cell surfaces. Biophys J 49:901–911

    PubMed  Google Scholar 

  • Gruenbaum Y, Hochstrasser M, Mathog D, Saumweber H, Agard DA, Sedat JW (1984) Spatial organisation of theDrosophila nucleus: a three-dimensional cytogenetic study. J Cell Sci [Suppl] 1:223–234

    Google Scholar 

  • Hahn K, Kolega J, Montibeller J, DeBiasio R, Post P, Myers J, Taylor DL (1993) Fluorescent analogues: optical biosensors of the chemical and molecular dynamics of macromolecules in living cells. In: Mason WT (ed) Fluorescent and luminescent probes for biological activity. Academic Press, London, pp 349–359

    Google Scholar 

  • Haugland PR (1992) Handbook of fluorescent probes and research chemicals, 5th edn. Molecular Probes, Eugene, Oregon

    Google Scholar 

  • Hell S, Witting S, Schickfus M von, Wijnaendts van Resandt RW, Hunklinger S, Smolka E, Neiger M (1991) A confocal beam scanning white-light microscope. J Microsc 163: 179–187

    Google Scholar 

  • Hiraoka Y, Sedat JW, Agard DA (1987) The use of a charge-coupled device for quantitative optical microscopy of biological structures. Science 238:36–41

    PubMed  Google Scholar 

  • Holmes T, Bhattacharyya S, Cooper J, Hanzel D, Krishnamurthi V, Lin W, Roysam B, Szarowski D, Turner J (1994) Blind deconvolution in widefield fluorescence, confocal and transmission bright-field microscopy. Proceedings of the International Conference on Confocal and Near Field Microscopy, Munich, April. Society for 3D Imaging Sciences in Microscopy, Amsterdam

    Google Scholar 

  • Inoué S (1981) Video image processing greatly enhances contrast, quality and speed in polarization-based microscopy. J Cell Biol 89:346–356

    PubMed  Google Scholar 

  • Inoué S (1986) Video microscopy. Plenum Press, New York

    Google Scholar 

  • Karrasch S, Hegerl R, Hoh JH, Baumeister W, Engel A (1994) Atomic force microscopy produces faithful high-resolution images of protein surfaces in an aqueous environment. Proc Natl Acad Sci USA 91:836–838

    PubMed  Google Scholar 

  • Kashar B (1985) Asymmetric illumination contrast: a method of image formation for video light microscopy. Science 227: 766–768

    PubMed  Google Scholar 

  • Kashima S (1995) Development of laser scanning microscopy using a near ultraviolet laser. Scanning 17:66–69

    Google Scholar 

  • Kuo SC, Sheetz MP (1993) Force of single kinesin molecules measured with optical tweezers. Science 260:232–234

    PubMed  Google Scholar 

  • Kuznetsov SA, Langford GM, Weiss DG (1992) Actin-dependent organelle movement in squid axoplasm. Nature 356:722–725

    PubMed  Google Scholar 

  • Lee GM, Ishihara A, Jacobson KA (1991) Direct observation of Brownian motion of lipids in membranes. Proc Natl Acad Sci USA 88:6274–6278

    PubMed  Google Scholar 

  • Lin CH, Thompson CA, Forscher P (1994) Cytoskeletal reorganization underlying growth cone motility. Curr Opin Neurobiol 4:640–647

    PubMed  Google Scholar 

  • Lindek S, Stelzer EHK, Hell SW (1995) Two new high-resolution confocal fluorescence microscopies (4Pi, Theta) with one- and two-photon excitation. In: Pawley J (ed) Handbook of biological confocal microscopy, 3rd edn. Plenum Press, New York, pp 417–430

    Google Scholar 

  • Marabini R, Vaquerizo C, Fernandez JJ, Carazo JM, Frank J (1995) Proposal for a distributed volume database for the different types of microscopy. Biophys J (in press)

  • Mason WT (ed) (1993) Fluorescent and luminescent probes for biological activity. A practical guide to technology for quantitative real-time analysis. Academic Press, London

    Google Scholar 

  • Minsky M (1957) US patent No 3013467. Microscopy apparatus, 19 December 1961 (filed 7 November 1957)

  • Mitchison TJ (1989) Poleward microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J Cell Biol 109:637–652

    PubMed  Google Scholar 

  • Mitchison TJ, Salmon ED (1992) Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis. J Cell Biol 119:569–582

    PubMed  Google Scholar 

  • Nederlof PM, Robinson D, Abuknesha R, Wiegant J, Hopman AHN, Tanke HJ, Raap AK (1989) Three colour fluorescence in situ hybridisation for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10:20–27

    PubMed  Google Scholar 

  • Nederlof PM, Flier S van der, Wiegant J, Raap AK, Tanke HJ, Ploem JS (1990) Multiple fluorescence in situ hybridisation. Cytometry 11:126–131

    PubMed  Google Scholar 

  • Nederlof PM, Flier S van der, Verwoerd NP, Vrolijk J, Raap AK, Tanke HJ (1992a) Quantification of fluorescence in situ hybridisation signals by image cytometry. Cytometry 13: 846–852

    PubMed  Google Scholar 

  • Nederlof PM, Flier S van der, Vrolijk J, Tanke HJ, Raap AK (1992b) Fluorescence ratio measurements of double-labelled probes for multiple in situ hybridisation by digital imaging microscopy. Cytometry 13:839–845

    PubMed  Google Scholar 

  • Pawley J (ed) (1989) Handbook of biological confocal microscopy. IMR Press, University of Wisconsin, Madison

    Google Scholar 

  • Pawley J (ed) (1989) Handbook of biological confocal microscopy, 2nd edn. Plenum Press, New York

    Google Scholar 

  • Pawley J (ed) (1995) Handbook of biological confocal microscopy, 3rd edn. Plenum Press, New York

    Google Scholar 

  • Petroll WM, Jester JV, Cavanagh HD (1994) In vivo confocal imaging: general principles and applications. Scanning 16: 131–149

    PubMed  Google Scholar 

  • Ploem JS (1982) Automated methods in immunofluorescence studies. In: Wick EA (ed) Immunofluorescence technology: selected theoretical and clinical aspects. Elsevier Biomedical Press, Amsterdam

    Google Scholar 

  • Ploem JS (1993) Fluorescence microscopy. In: Mason WT (ed) Fluorescent and luminescent probes for biological activity. Academic Press, London, pp. 1–11

    Google Scholar 

  • Ploem JS, Tanke HJ (1987) Introduction to fluorescence microscopy. (Royal Microscopical Society Microscopy Handbook, vol 10). Oxford University Press, Oxford

    Google Scholar 

  • Qian H, Sheetz MP, Elson EL (1991) Single particle tracking. Analysis of diffusion and flow in 2D systems. Biophys J 60:910–921

    PubMed  Google Scholar 

  • Reynolds GT (1968) Image intensification applied to microscope systems. Adv Optical Electron Microsc 2:1–40

    Google Scholar 

  • Reynolds GT (1972) Image intensification applied to biological problems. Quartery Rev Biophys 5:295–347

    Google Scholar 

  • Reynolds GT, Taylor DL (1980) Image intensification applied to light microscopy. BioScience 30:586–592

    Google Scholar 

  • Rigaut JP, Vassy J (1991) High-resolution three-dimensional images from confocal scanning laser microscopy. Anal Quant Cytol Histol 13:223–232

    PubMed  Google Scholar 

  • Sammak PJ, Borisy GG (1988) Direct observation of microtubule dynamics in living cells. Nature 332:724–726

    PubMed  Google Scholar 

  • Sawin KE, Theriot JA, Mitchison TJ (1993) Photoactivation of fluorescence as a probe for cytoskeletal dynamics in mitosis and cell motility. In: Mason WT (ed) Fluorescent and luminescent probes for biological activity. Academic Press, London, pp 405–419

    Google Scholar 

  • Saxton MJ (1993) Lateral diffusion in an archipelago: single particle diffusion. Biophys J 64:1766–1780

    PubMed  Google Scholar 

  • Schnapp BJ (1987) Viewing single microtubules by video light microscopy. Methods Enzymol 134:561–573

    Google Scholar 

  • Schnapp BJ, Vale RD, Sheetz MP, Reese TS (1985) Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell 40:455–462

    PubMed  Google Scholar 

  • Schulze E, Kirschner M (1988) New features of microtubule behaviour observed in vivo. Nature 334:356–359

    PubMed  Google Scholar 

  • Semper AE, Fitzsimons RB, Shotton DM (1988) Ultrastructural identification of type 1 fibres in human skeletal muscle. Immunogold labelling of thin cryosections with a monoclonal antibody aganist slow myosin. J Neurol Sci 83:93–108

    PubMed  Google Scholar 

  • Shaw PJ (1990) Three-dimensional optical microscopy using tilted views. J Microsc 158:165–172

    PubMed  Google Scholar 

  • Shaw PJ (1993) Computer reconstruction in three-dimensional fluorescence microscopy. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 211–230

    Google Scholar 

  • Shaw PJ, Rawlins DJ (1991) The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D Data. J Microsc 163:151–165

    Google Scholar 

  • Shaw PJ, Agard DA, Hiraoka Y, Sedat JW (1989) Tilted view reconstruction in optical microscopy: three dimensional reconstruction ofDrosophila melanogaster embryo nuclei. Biophys J 55:101–110

    PubMed  Google Scholar 

  • Sheetz MP, Turney S, Quin H, Elson EL (1989) Nanometer-level analysis demonstrates that lipid flow does not drive membrane glycoprotein movements. Nature 340:284–288

    PubMed  Google Scholar 

  • Shotton DM (1983) The proteins of the erythrocyte membrane. In: Harris JR (ed) The electron microscopy of proteins, vol 4. Academic Press, London, pp 205–230

    Google Scholar 

  • Shotton DM (1988a) Video enhanced light microscopy and its applications in cell biology. J Cell Sci 89:129–150

    PubMed  Google Scholar 

  • Shotton DM (1988b) The current renaissance in light microscopy. II. Blur-free optical sectioning of biological specimens by confocal scanning fluorescence microscopy. Proc R Microsc Soc 23:289–297

    Google Scholar 

  • Shotton DM (1989) Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 94:175–206

    Google Scholar 

  • Shotton DM (1991) Video and opto-digital imaging microscopy. In: Cherry RJ (ed) New techniques of optical microscopy and microspectroscopy. Macmillan, London, pp 1–47

    Google Scholar 

  • Shotton DM (ed) (1993a) Electronic light microscopy: the principles and practice of intensified fluorescence, video-enhanced contrast and confocal scanning optical microscopy. Wiley-Liss, New York

    Google Scholar 

  • Shotton DM (1993b) Electronic acquisition of light microscope images In: Shotton DM (ed) Electronic light microscopy Wiley-Liss, New York, pp 1–38

    Google Scholar 

  • Shotton DM (1993c) Digital image processing and image display in electronic light microscopy. In: Shottong DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 39–70

    Google Scholar 

  • Shotton DM, Attaran A (1995) Video microscopic analysis of perforin-andFas-mediated cell death: kinetic and morphological analysis of necrotic and apoprotic death of fibroblast target cells by cytotoxic T cells. Proc R Microsc Soc 30:134

    Google Scholar 

  • Shotton DM, White N (1989) Confocal scanning microscopy: three-dimensional biological imaging. Trends Biochem Sci 14: 435–439

    PubMed  Google Scholar 

  • Song L, Vrolijk J, Verwoerd NP, Bonnet J, Beverloo HB, Nederlof PM, Tanke HJ (1990) An imaging system for time-resolved fluorescence microscopy. Trans R Microsc Soc 1:467–470

    Google Scholar 

  • Speel EJM, Jansen MPHM, Ramaekers FCS, Hopman AHN (1994) A novel triple-color detection procedure for bright-field microscopy, combining in situ hybridisation with immunocytochemistry. J Histochem Cytochem 42:1299–1307

    PubMed  Google Scholar 

  • Spring KR, Lowy RJ (1989) Characteristics of low-light-level fluorescence imagestelevision cameras. Methods Cell Biol 29:270–291

    Google Scholar 

  • Spudich JA (1994) How molecular motors work. Nature 372:515–518

    PubMed  Google Scholar 

  • Stelzer EHK, Lindek S, Albrecht S, Pick R, Ritter G, Salmon N, Stricker R (1995) A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy. J Microsc 179:1–10

    Google Scholar 

  • Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365:7211–727

    Google Scholar 

  • Tanke HJ, Raap AK (1992) A new approach to microscopic identification and quantitation of nucleic acids. Eur J Histochem 36:27–28

    PubMed  Google Scholar 

  • Taylor DL, Wang Y-L (1980) Fluorescently labelled molecules as probes of the structure and function of living cells. Nature 284:405–410

    PubMed  Google Scholar 

  • Taylor DL, Wang Y-L (1989) Fluorescence microscopy of living cells in culture. B. Quantitative fluorescence microscopy-imaging and spectroscopy. (Methods Cell Biol, vol 30). Academic Press, London

    Google Scholar 

  • Taylor DL, Amato PA, Luby-Phelps K, McNeil P (1984) Emerging techniques: fluorescence analog cytochemistry. Trends Biochem Sci 9:88–91

    Google Scholar 

  • Tsay T-T, Inman R, Wray B, Herman B, Jacobson K (1990) Characterization of low-light-level cameras for digitised video microscopy. J Microsc 160:141–159

    PubMed  Google Scholar 

  • Tsien RY (1990) Laser scanning confocal fluorescence microscopy at video rate(30 frames/sec) with dual-wavelength emission ratioing for quantitative imaging of intracellular messengers. Proc R Microsc Soc 25:S53

    Google Scholar 

  • Tsien RY, Harootunian AT (1990) Practical design criteria for a dynamic ratio imaging system. Cell Calcium 11:93–109

    PubMed  Google Scholar 

  • Tsien RY, Poenie M (1986) Fluorescence ratio imaging: a new window into intracellular ionic signaling. Trends Biochem Sci 11:450–455

    Google Scholar 

  • Vale RD, Reese TS, Sheetz MP (1985a) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42:39–50

    PubMed  Google Scholar 

  • Vale RD, Schnapp BJ, Mitchison T, Steuer E, Reese TS, Sheetz MP (1985b) Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro. Cell 43:623–632

    PubMed  Google Scholar 

  • Vale RD, Schnapp BJ, Reese TS, Sheetz MP (1985c) Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon. Cell 40:449–454

    PubMed  Google Scholar 

  • Vale RD, Schnapp BJ, Reese TS, Sheetz MP (1985d) Organelle, bead and microtubule translocations promoted by soluble factors from the squid giant axon. Cell 40:559–569

    PubMed  Google Scholar 

  • Verwoerd NP, Hennick EJ, Bonnet J, Geest CR van der, Tanke HJ (1994a) Use of ferro-electric liquid crystal shutters for time-resolved microscopy. Cytometry 16:113–117

    PubMed  Google Scholar 

  • Vrolijk J, Sloos WC, Darroudi F, Natarajan AT, Tanke HJ (1994a) A system for fluorescence metaphase finding and scoring of chromosomal translocations visualised by in situ hybridisation. Int J Radiat Biol 66:287–295

    PubMed  Google Scholar 

  • Vrolijk J, Sloos WC, Verwoerd NP, Tanke HJ (1994b) Application of a non-cooled video-rate CCD camera for detection of fluorescence in situ hybridisation signals. Cytometry 15:2–11

    PubMed  Google Scholar 

  • Waggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof N, Taylor D (1989) Multiple spectral parameter imaging. Methods Cell Biol 30:449–478

    PubMed  Google Scholar 

  • Walker RA, O Brian ET, Pryer NK, Soboeiro MF, Voter WA, Erickson HP, Salmon ED (1988) Dynamic instability of individual microtubules analysed by video light microscopy: rate constants and transition frequencies. J Cell Biol 107:1437–1448

    PubMed  Google Scholar 

  • Walker RA, Inou S, Salmon ED (1989) Asymmetric behaviour of severed microtubule ends after ultraviolet microbeam irradiation of individual microtubules in vitro. J Cell Biol 108:931–937

    PubMed  Google Scholar 

  • Walz T, Smith BL, Agre P, Engel A (1994) The three-dimensional structure of human erythrocyte aquaporin CHIP. EMBO J 13:2985–2993

    PubMed  Google Scholar 

  • Wang Y-L (1985) Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol 107:1505–1516

    Google Scholar 

  • Wang Y-L (1989) Fluorescence analog cytochemistry: tracing functional protein components in living cells. Methods Cell Biol 29:1–12

    Google Scholar 

  • Wang Y-L, Taylor DL (1989) Fluorescence microscopy of living cells in culture A. Fluorescent analogs, labeling cells and basic microscopy. (Methods Cell Biol, vol 29). Academic Press, London

    Google Scholar 

  • Wang Y-L, Heiple J, Taylor DL (1982) Fluorescence analog cytochemistry of contractile proteins. Methods Cell Biol 25:1–11

    Google Scholar 

  • Walson TF (1990) Real-time confocal microscopy of high speed dental burr/tooth cutting interactions. J Microsc 157:51–60

    PubMed  Google Scholar 

  • Webb WW (1990) Two photon excitation in laser scanning fluorescence microscopy. Trans R Microscop Soc 1:445–450

    Google Scholar 

  • Weiss DG, Maile W (1993) Principles, practice and applications of video-enhanced contrast microscopy. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 105–140

    Google Scholar 

  • Weiss DG, Maile W, Wick RA (1989) Video microscopy. In: Lacey AJ (ed) Light microscopy in biology. A practical approach. IRL Press, Oxford, pp 221–278

    Google Scholar 

  • White N, Fricker MD, Shotton DM (1991) Quantitative 3D visualisation of biological CLSM images. Scanning 13 [Suppl] 1: 51–53

    Google Scholar 

  • Wijnaendts van Resandt RW, Marsman HJB, Kaplan R, Davoust J, Stelzer EHK, Stricker R (1985) Optical fluorescence microscopy in three dimensions: microtomoscopy. J Microsc 138: 29–34

    Google Scholar 

  • Williams RM, Piston DW, Webb WW (1994) Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FACEB J 8:804–813

    Google Scholar 

  • Willingham MC, Pastan IH (1978) The visualisation of fluorescent proteins in living cells by video intensification microscopy (VIM). Cell 13:501–507

    PubMed  Google Scholar 

  • Willingham MC, Pastan IH (1983) Image intensification techniques for detection of proteins in cultured cells. Methods Enzymol 98:266–283 and 635

    PubMed  Google Scholar 

  • Wilson T (1990) Confocal microscopy. Academic Press, London

    Google Scholar 

  • Wilson T (1993) Image formation in confocal microscopy. In: Shotton DM (ed) Electronic light microscopy. Wiley-Liss, New York, pp 231–246

    Google Scholar 

  • Wilson T, Hewlett SJ (1990) Imaging in scanning microscopes with slit-shaped detectors. J Microsc 160:115–139

    PubMed  Google Scholar 

  • Wilson T, Sheppard CJR (1984) Theory and practice of scanning optical microscopy. Academic Press, London

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shotton, D.M. Electronic light microscopy: present capabilities and future prospects. Histochem Cell Biol 104, 97–137 (1995). https://doi.org/10.1007/BF01451571

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01451571

Keywords

Navigation