Light Microscopy Imaging Facilities

Abstract

Historically core imaging facilities have been associated with electron microscopes. With the very widespread use of fluorescent markers for both fixed and living cells and tissues the light microscope facility has emerged in its own right. Well‐run core light microscope imaging facilities are focal points within research institutions. Researchers want to do science, acquire images and manipulate data for publication; for them, the microscope and the computing infrastructure within the imaging facility are merely tools. Staff provide organization, continuity, tuition and knowledge and maintenance of equipment, which encompass the needs of both novice and advanced users. Increasingly, imaging facilities are considered from the outset as a component in the design of a new research building, and must recover the costs of operation. This article discusses the need for optical sectioning, the role of the light microscopy facility and points to consider in its design.

Key Concepts:

  • Fluorescent labelling is widely used in life science to study organelles, structures and proteins of interest within cells and tissues.

  • Fluorescent cells and tissues are self‐luminous and light up throughout their thickness. This is often greater than the depth of field (thickness of the in‐focus object plane) of the microscope objective, thus most of the image is composed of scattered light and out‐of‐focus blur which obscures the in‐focus detail and drastically reduces the signal‐to‐noise ratio (SNR).

  • Optical sectioning microscopy ‘cleans up’ the image at each point of focus, so allowing a sharply defined 3D Z‐stack to be constructed and analysed.

  • The light microscopes required to acquire multidimensional images over time, and the software packages needed to analyse the data, are both expensive and complex. Core image facilities provide the instrumentation and specialist staff to assist in this process.

  • The design and operation of a core light microscopy imaging facility which is capable of effectively supporting a research institution requires some forethought.

Keywords: imaging facility; optical sectioning; fluorescence; confocal microscopy; deconvolution; facility design

Figure 1.

Advantages and disadvantages of fluorescence. PMT, photo‐multiplier tube.

Figure 2.

Schematic representation for why we need optical sectioning.

Figure 3.

Schematic representation of the different types of optical sectioning.

Figure 4.

Poster to show facility users which microscope to use.

close

References

Adler J and Pagakis SN (2003) Reducing image distortions due to temperature‐related microscope stage drift. Journal of Microscopy 210(2): 131–137. doi: 10.1046/j.1365‐2818.2003.01160.x.

Anderson KI, Sanderson J and Peychl J (2007) Design and function of a light microscopy facility. In: Shorte SL and Frischknecht F (eds) Imaging Cellular and Molecular Biological Functions. Berlin: Springer. ISBN‐13: 978‐3‐540‐71330‐2.

Andrews PD, Harper IS and Swedlow JR (2002) To 5D and beyond: quantitative fluorescence microscopy in the postgenomic era. Traffic 3(1): 29–36. doi: 10.1034/j.1600‐0854.2002.30105.x.

Axelrod D (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2: 764–774. doi: 10.1034/j.1600‐0854.2001.21104.x.

Chi KR (2008) Imaging and detection: focusing on software. Nature Methods 5(7): 651–658. doi: 10.1038/nmeth0708‐651.

Conchello J‐A and Lichtman JW (2005) Optical sectioning microscopy. Nature Methods 2(12): 920–931. doi: 10.1038/nmeth815.

Davidson MW and Campbell RE (2009) Engineered fluorescent proteins: innovations and applications. Nature Methods 6(10): 713–717. doi: 10.1038/nmeth1009‐713.

Goldberg IG, Allan C, Burel JM et al. (2005) The Open Microscopy Environment (OME) Data Model and XML file: open tools for informatics and quantitative analysis in biological imaging. Genome Biology 6: R47. doi: 10.1186/gb‐2005‐6‐5‐r47 http://genomebiology.com/2005/6/5/R47.

Gräf R, Rietdorf J and Zimmermann T (2005) Live cell spinning disk microscopy. In: Rietdorf J (ed.) Advances in Biochemistry and Engineering Biotechnology, vol. 95, chap. 7, pp. 57–75. Berlin: Springer. ISBN=3‐540‐23698‐8.

Groves JT, Parthasarathy R and Forstner MB (2008) Fluorescence imaging of membrane dynamics. Annual Review of Biomedical Engineering 10: 311–338. doi: 10.1146/annurev.bioeng.10.061807.160431.

Gustafson C, Bug WJ and Nissanov J (2007) NeuroTerrain – a client‐server system for browsing 3D biomedical image data sets. BMC Bioinformatics 8: 40. doi: 10.1186/1471‐2105‐8‐40.

Helmchen F and Denk W (2005) Deep tissue two‐photon microscopy. Nature Methods 2/12: 932–940. doi: 10.1038/nmeth818 Corrigendum: Nature Methods 3(3): 235. doi: 10.1038/nmeth0306‐235.

Kherlopian AR, Song T, Duan Q et al. (2008) A review of imaging techniques for systems biology. BMC Systems Biology 2: 74. doi: 10.1186/1752‐0509‐2‐74.

Lee MF, Kong SK, Fung KP, Lui CP and Lee CY (1996) Practical considerations in acquiring biological signals from confocal microscope: solvent effect and temperature effect. Biological Signals 5(5): 291–300. doi: 10.1159/000109202.

Lichtman JW and Conchello J‐A (2005) Fluorescence microscopy. Nature Methods 2(12): 910–919. doi: 10.1038/nmeth817.

McNally JG, Karpova T, Cooper J and Conchello J‐A (1999) Three‐dimensional imaging by deconvolution microscopy. Methods 19: 373–385. doi: 10.1006/meth.1999.0873.

Megason SG and Fraser SE (2007) Imaging in systems biology. Cell 130(5): 784–795. doi: 10.1016/j.cell.2007.08.031.

Murray JM (2005) Confocal microscopy, deconvolution, and structured illumination methods. In: Goldman RD and Spector DL (eds) Live Cell Imaging: A Laboratory Manual, chap. 14, pp. 239–279. New York: CSHL Press. ISBN‐10: 0‐87969‐683‐4.

Nakano A (2002) Spinning‐disk confocal microscopy – a cutting‐edge tool for imaging of membrane Traffic. Cell Structure and Function 27(5): 349–355. doi: 10.1247/csf.27.349.

Nature Editorial (2007) Under the microscope. Nature 447(7141): 116. doi: 10.1038/447116a.

Niesner RA, Andresen V and Gunzer M (2008) Intravital two‐photon microscopy: focus on speed and time resolved modalities. Immunological Reviews 221: 7–25. doi: 10.1111/j.1600‐065X.2008.00582.x.

Parton RM and Davis I (2004) Lifting the fog: Image restoration by deconvolution. In: Celis JL (ed.) Cell Biology: A Laboratory Handbook, 3rd edn, chap. 19. San Diego: Academic Press. ISBN‐13: 978‐0‐12‐164733‐1.

Peng H (2008) Bioimage informatics: a new area of engineering biology. Bioinformatics 24(17): 1827–1836. doi: 10.1093/bioinformatics/btn346.

Shaner NC, Steinbach PA and Tsien RY (2005) A guide to choosing fluorescent proteins. Nature Methods 2(12): 905–909. doi: 10.1038/nmeth819.

Shaner NC, Patterson GH and Davidson MW (2007) Advances in fluorescent protein technology. Journal of Cell Science 120(24): 4247–4260. doi: 10.1242/10.1242/jcs005801.

Stutzmann G and Parker I (2005) Dynamic multiphoton imaging: a live view from cells to systems. Physiology 20: 15–21. doi: 10.1152/physiol.00028.2004 1548‐9213/05.

Swedlow JR, Hu K, Andrews PD, Roos DS and Murray JM (2002) Measuring tubulin content in Toxoplasma gondii: a comparison of laser‐scanning confocal and wide‐field fluorescence microscopy. Proceedings of the National Academy of Sciences of the USA 99/4: 2014–2019. doi: 10.1073/pnas.022554999.

Waters JC and Swedlow JR (2007) Interpreting fluorescence microscopy images and measurements. In: Zuk D (ed.) Evaluating Techniques in Biomedical Research. Cambridge, MA: Cell Press. http://www.cellpress.com/misc/ETBR.

Waters JC (2009) Accuracy and precision in quantitative fluorescence microscopy. Journal of Cell Biology 185(7): 1135–1148. doi: 10.1083/jcb.200903097.

Further Reading

Fernández‐Suárez M and Ting AY (2008) Fluorescent probes for super‐resolution imaging in living cells. Nature Reviews. Molecular Cell Biology 9(12): 929–943. doi: 10.1038/nrm2531.

Frigault MM, Lacoste J, Swift JL and Brown CM (2009) Live‐cell microscopy – tips and tools. Journal of Cell Science 122: 753–767. doi: 10.1242/jcs.033837.

Hibbs AR (2004) Confocal Microscopy for Biologists. Berlin: Plenum Publishers. ISBN‐13: 978‐0306484681.

Jepson MA (2006) Confocal or Wide‐field? A guide to selecting appropriate methods for imaging. In: Stephens D (ed.) Cell Imaging, chap. 2, pp. 17–48. Oxon: Scion Publishing. ISBN‐13: 978‐1904842267.

Khodjakov A and Reider CL (2006) Imaging the division process in living tissue culture cells. Methods 38(1): 2–16. doi: 10.1016/j.ymeth.2005.07.007.

Klaunberg BA and Davis JA (2008) Considerations for laboratory animal imaging center design and setup. Institute of Laboratory Animal Research Journal 49(1): 4–16.

Miyawaki A (2008) Green fluorescent protein glows gold. Cell 135(6): 987–990. doi: 10.1016/j.cell.2008.11.025.

Murphy JA (2002) Designing an electron microscopy facility: step by step procedure. Microscopy Today 10: 36–39. With checklist URL: http://www.sjdccd.cc.ca.us/dept/electmicro/images/Murphy_Design_CheckLsts.pdf.

North AJ (2006) Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. Journal of Cell Biology 172(1): 9–18. doi: 10.1083/jcb.200507103.

Pearson H (2007) The good, the bad and the ugly. Nature 447(7141): 138–140. doi: 10.1038/447138a.

Petty HR (2007) Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology. Microscopy Research & Technique 70(8): 687–709.

Spector DL and Goldman RD (eds) (2005) Basic Methods in Microscopy. Oxon: Scion Publishing. ISBN‐13: 978‐0879697518.

Stephens D (ed.) (2006) Cell Imaging. Bloxham: Scion Publishing Ltd. ISBN 1‐904842‐046.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Sanderson, Jeremy(Jan 2010) Light Microscopy Imaging Facilities. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022189]