Difference between revisions of "2010 Lab 7"

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==Spinning Disc Confocal Microscopy==
==Spinning Disc Confocal Microscopy==
[[Image:Confocal microscopy 01.jpg|thumb|Confocal Microscopy 2 Methods]]
[[Image:Confocal microscopy 01.jpg|thumb|Confocal Microscopy 2 Methods]]
[[File:Yokogawa CSU-X1.jpg|thumb|Yokogawa CSU-X1]]
'''Advantage - high speed imaging'''
'''Advantage - high speed imaging'''
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'''Phototoxicity''' - A term describing the cell toxic effects caused by light and fluorescent proteins, thought to be due to the formation of oxygen radicals from the non-radiative energy transfer.
'''Phototoxicity''' - A term describing the cell toxic effects caused by light and fluorescent proteins, thought to be due to the formation of oxygen radicals from the non-radiative energy transfer.
==Total Internal Reflection Fluorescence Microscopy (TIRF)==
==Total Internal Reflection Fluorescence Microscopy (TIRF)==

Revision as of 09:49, 2 April 2010

Confocal Microscopy

Confocal Microscopy 2 Methods
Two-photon microscopy in vivo brain

Example of Spinning Disc Confocal image - Labeled Mitochondria Movie (1 frame/2 seconds)


The laboratory this week will involve a tutorial on microscopy and fluorescent labeling techniques, as well as a visit to the SOMS confocal facility. Before the laboratory try and read some of the linked content describing the confocal microscope and its application in cell biology.

Handout from Confocal Demonstration Confocal Demo Class Handout 2009 PDF

See also your earlier laboratories Lab 2 - Microscopy | Immunochemistry | Preparation/Fixation

Some Initial Thoughts

  • This form of microscopy is sometimes given the acronym CLSM or LSM.
  • There are at least 2 major different technical methods for generating confocal.
  • The actual microscope format (upright, inverted) will also limit the types of analysis.
  • The available fluorochromes and their properties (Peak excitation wavelength, Peak emission wavelength, stability, fading) is continuously being developed.

Diffraction Limit

Diffraction limit
  • Resolving power (resolution) is the ability of an imaging device to measure the angular separation of 2 points in an object
  • Defined by the classical Rayleigh criterion (John William Strutt, 3rd Baron Rayleigh, 1842 –1919)
  • For fluorescence microscopy
    • approx 250nm in the xy-plane
    • approx 500–800nm in the z plane

Fluorescence Microscopy

Standard wide-field illumination uses excitation propagating parallel to the z-axis, collected images have significant contributions from objects located above or below the focal plane. This "out of plane" fluorescence can only be removed by spatial deconvolution of the collected image.

Laser-Scanning Confocal Microscopy

  • the laser beam converges on the focal plane
  • fluorescent radiation from the point of illumination then converges on the conjugate point in the image plane
  • a pinhole can be used to eliminate fluorescence from out-of-focus planes
  • scanning the beam on an xy-raster over a succession of spaced focal planes allows building up a 3D image
  • data is collected with a photomultiplier detector
  • the image is then reconstructed computationally

Spinning Disc Confocal Microscopy

Confocal Microscopy 2 Methods
Yokogawa CSU-X1

Advantage - high speed imaging

  • A rapidly rotating wheel containing a set of microlenses and pinholes in an array
  • The confocal microscope repeatedly scans many points in parallel
  • Leads to increased rate of data acquisition which also decreases phototoxicity

Phototoxicity - A term describing the cell toxic effects caused by light and fluorescent proteins, thought to be due to the formation of oxygen radicals from the non-radiative energy transfer.

Total Internal Reflection Fluorescence Microscopy (TIRF)

Advantage - only at or near the plane of cell contact

  • total internal reflection occurs when a beam of light is incident at a small angle (critical angle) from a medium of higher refractive index onto an interface with a medium of lower refractive index
  • refractive index of a glass coverslip (n∼1.5) onto the aqueous medium of a cell in culture (n∼1.35)
  • an ‘evanescent wave’ is produced in the lower refractive index medium
  • fluorescence microscope excitation illumination is directed that it is totally reflected from the interface between the coverslip and adherent cell
  • fluorophores located within 100–200nm of the coverslip are strongly excited, but not those further away

Evanescent wave - is nearfield standing wave with an intensity that exhibits exponential decay with distance from the boundary at which the wave was formed.

Biomedical Imaging Facility (BMIF)

2010 the confocal microscopes have been relocated to the new Biomedical Imaging Facility (BMIF).

Olympus FV1000 Laser Scanning Microscope

  • The (inverted) FV1000 is equipped with 6 excitation wavelengths that cover the UV/Vis spectrum: 405nm forr UV dyes, 458nm, 488nm, 514nm, 543nm and 633nm. The FV1000 also has a unique SIM scanner to allow FRAP/FLIP experiments with seemless transition between scanning and excitation lasers. The FV1000 uses spectral detection units which can separate the emission wavelengths of separate dyes within 2nm.

Links: BMIF - FV1000 | Olympus - FV1000 Laser Scanning Microscope

Leica TCS SP Laser Scanning Microscope

  • (inverted) three channel excitation: 488nm, 568nm & 647nm.

Links: BMIF - BMIF - FV1000 | Leica - TCS SP Laser Scanning Microscope | Leica - Gallery

Zeiss Observer X.1

Yokogawa CSU-X1
  • Total Internal Reflection Fluorescence microscope (TIRFM)
  • (inverted) confocal microscope with Yokogawa Spinning disk CSU-X1 module and a TIRF3 module.
  • fluorescence microscopy technique
  • activated fluorophores are illuminated during image acquisition
  • all of them are bleached and then a new subpopulation is photoactivated to begin the next cycle

Links: BMIF - Zeiss Observer | Zeiss PDF | Microscopy and Analysis 2008 | Wiki PALM | Wiki TIRF

Zeiss PAL-M

TIRF microscopy
  • Total Internal Reflection Fluorescence microscope (TIRFM)
  • Laser widefield illumination TIRF system.

Links: BMIF - PALM | Zeiss PAL-M | Wiki TIRF | WIKI PALM

Theory of Confocal Microscopy

Confocal optical pathways

Confocal Microscopy Practical Issues

  • Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. North AJ. J Cell Biol. 2006 Jan 2;172(1):9-18. Review. PMID: 16390995

Confocal Microscopy - Fluorochromes

The confocal technique, along with fluorescence microscopy, is dependent upon the availability of compounds that can be excited with a specific wavelength (peak excitation wavelength nm) of light and then emit light at a different wavelength (peak emission wavelength nm). This area of biotechnology has been rapidly changing in response to developments in chemistry, electronics, optics and biodiscovery of new fluorescent compounds. Table of Fluorochromes

Organelle Specific Fluorescent Dyes

There are a number of organelle specific fluorescent dyes that target organelles within the living cell.

Green Fluorescent Protein


Green fluorescent protein (GFP) was the first fluorescent protein discovered and applied to allow endogenous tagging of proteins and then be able to analyse localization and dynamics within the living cell. The discoverers were awarded the 2008 Nobel Prize in Chemistry.

From the original protein a number of variants have now been developed with different spectral properties (excitation/emission) as well as stabilities.

Red Fluorescent Protein


Links: Nobel Prize 2008 Nobel Prize in Chemistry | Illustrated Presentation | Tsien Lab, UCSD | Lab - Images | Tsien Lab - Movies | Fluorescent proteins: a cell biologist's user guide



Essential Cell Biology

  • * Essential Cell Biology Chapter 1 Cells Under the Microscope

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

Molecular Imaging and Contrast Agent Database

Bethesda (MD): National Library of Medicine (US), NCBI; 2004-2009

  • About MICAD
    • "The Molecular Imaging and Contrast Agent Database (MICAD) is an online source of information on in vivo molecular imaging agents based on recommendations from the extramural community."

Search Online Textbooks



  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed


  • Over the rainbow: 25 years of confocal imaging. Paddock S. Biotechniques. 2008 Apr;44(5):643-4, 646, 648. Review. PMID: 18474039
  • Stepping into the third dimension. Feng D, Marshburn D, Jen D, Weinberg RJ, Taylor RM 2nd, Burette A. J Neurosci. 2007 Nov 21;27(47):12757-60. Review. PMID: 18032646
  • Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy. Kreplak L, Richter K, Aebi U, Herrmann H. Methods Cell Biol. 2008;88:273-97. Review. PMID: 18617039
  • Bridging fluorescence microscopy and electron microscopy. Giepmans BN. Histochem Cell Biol. 2008 Aug;130(2):211-7. PMID: 18575880
  • Fluorescence microscopy--avoiding the pitfalls. Brown CM. J Cell Sci. 2007 May 15;120(Pt 10):1703-5. Review. Erratum in: J Cell Sci. 2007 Oct 1;120(Pt 19):3488. PMID: 17502480
  • Multi-photon excitation microscopy. Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C. Biomed Eng Online. 2006 Jun 6;5:36. Review. PMID: 16756664
  • Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. North AJ. J Cell Biol. 2006 Jan 2;172(1):9-18. Review. PMID: 16390995
  • A guide to choosing fluorescent proteins. Shaner NC, Steinbach PA, Tsien RY. Nat Methods. 2005 Dec;2(12):905-9. Review. PMID: 16299475
  • Spinning-disk confocal microscopy -- a cutting-edge tool for imaging of membrane traffic. Nakano A. Cell Struct Funct. 2002 Oct;27(5):349-55. Review. PMID: 12502889


  • The preparation of Drosophila embryos for live-imaging using the hanging drop protocol. Reed BH, McMillan SC, Chaudhary R. J Vis Exp. 2009 Mar 13;(25). pii: 1206. doi: 10.3791/1206. PMID: 19287353
  • Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. Sagasti A, O'Brien GS, Rieger S, Martin SM, Cavanaugh AM, Portera-Cailliau C. J Vis Exp. 2009 Feb 16;(24). pii: 1129. doi: 10.3791/1129. PMID: 19229185
  • Extended Field Laser Confocal Microscopy (EFLCM): combining automated Gigapixel image capture with in silico virtual microscopy. Flaberg E, Sabelström P, Strandh C, Szekely L. BMC Med Imaging. 2008 Jul 16;8:13. PMID: 18627634
  • Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy. van Munster EB, Goedhart J, Kremers GJ, Manders EM, Gadella TW Jr. Cytometry A. 2007 Apr;71(4):207-14. PMID: 17266147
  • Four-color, 4-D time-lapse confocal imaging of chick embryos. Teddy JM, Lansford R, Kulesa PM. Biotechniques. 2005 Nov;39(5):703-10.

PMID: 16312219 | Biotechniques

  • Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy. Ono M, Murakami T, Kudo A, Isshiki M, Sawada H, Segawa A. J Histochem Cytochem. 2001 Mar;49(3):305-12. PMID: 11181733 See also Mountants and Antifades PDF


2010 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | Revision

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G