Talk:2010 Lab 7

From CellBiology
  • Selective plane illumination microscopy techniques in developmental biology. Huisken J, Stainier DY. Development. 2009 Jun;136(12):1963-75. Review. PMID: 19465594
  • Quantitative time-lapse fluorescence microscopy in single cells. Muzzey D, van Oudenaarden A. Annu Rev Cell Dev Biol. 2009;25:301-27. Review. PMID: 19575655
  • Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. Sekar RB, Periasamy A. J Cell Biol. 2003 Mar 3;160(5):629-33. Review. PMID: 12615908

Multiphoton fluorescence microscopy

  • similar to confocal laser scanning microscopy by using focused laser beams scanned in a raster pattern to generate images
  • both have an optical sectioning effect
  • multiphoton microscopes do not contain pinhole apertures
    • pinhole apertures give confocal microscopes their optical sectioning quality

Two-photon Microscopy

Advantage - very high depth of imaging

http://en.wikipedia.org/wiki/Two-photon_excitation_microscopy

Fluorescent proteins

Fluorescent proteins: a cell biologist's user guide

GFP from jellyfish to expression in mammalian cells. (a) The jellyfish Aequorea victoria. Image provided by Sierra Blakely. (b) GFP targeted to the endoplasmic reticulum of a mammalian fibroblast. (c) Ribbon diagram of the β-barrel structure typical of FPs. Image produced by Richard Wheeler from PDB:1EMA rendered in PyMOL. (d) Co-expression of an endoplasmic reticulum targeted red fluorescent protein and a Golgi complex targeted GFP in a mammalian fibroblast. Scale bars = 10μm.

Table 1 from above reference

Protein Reference Notes

TagBFP [37] New bright photostable blue FP

Cerulean [38] Improved form of the cyan FP (CFP)

Monomeric EGFP [1,15,20,21] The original optimized green FP and best characterized FP for FP-fusion protein design

Venus or Citrine [39,40] Improved forms of yellow FP (YFP)

mCherry or mKate2 [3,22] Popular red FPs

PA–GFP and PA–mCherry [41,42] Photoactivatable FPs

Fusion Proteins

GRP94–GFP [43] Luminal ER Chaperone

GFP–NMDAR1 [44] Ion channel.

GFP–Tub1 [45] Yeast α-tubulin

GFP–clathrin [46] Secretory pathway coat protein


Confocal Terms

(Terms List below from http://www.cell.com/trends/cell-biology/fulltext/S0962-8924(09)00192-5)


Diffraction limit: the resolution of an optical system, as defined by the classical Rayleigh criterion. In practical terms, the diffraction limit for fluorescence microscopy is ∼250nm in the xy-plane and ∼500–800nm in the z plane.

DiNa: ‘differential evanescence nanometry’, a method based on TIR and wide-field illumination to determine the axial separation of the centroids of two different fluorophore distributions with a precision of ∼10nm. This method was used in real time for the differential localization of AP-2 and epsin with respect to clathrin during the formation of clathrin-coated pits and plaques [22].

Laser-scanning confocal microscopy: in a confocal microscope, the illuminating beam converges on the focal plane; fluorescent radiation from the point of illumination will then converge on the conjugate point in the image plane, and a pinhole can be used to eliminate fluorescence from out-of-focus planes. Scanning the beam on an xy-raster over a succession of suitably spaced focal planes allows one to build up a 3D image. Data are collected with a photomultiplier detector and the image reconstructed computationally.

Spinning disc confocal microscopy: by arranging a set of microlenses and pinholes in an appropriate array on a rapidly rotating wheel, a confocal microscope can be constructed that repeatedly scans many points in parallel, thereby greatly increasing the rate of data acquisition, while decreasing phototoxicity. An image for each focal plane is collected on a standard CCD camera. With currently available laser illumination, it is possible to acquire single frame images in a few milliseconds. This makes spinning disc confocal microscopy a relatively straightforward way to achieve high temporal and spatial resolution for dynamic structures.

Total internal reflection fluorescence (TIRF) microscopy: total internal reflection (TIR) occurs when a beam of light is incident at a suitably small angle from a medium of higher refractive index onto an interface with a medium of lower refractive index. In particular, light is totally reflected when incident from a glass coverslip (n∼1.5) onto the aqueous medium of a cell in culture (n∼1.35). There is nonetheless an ‘evanescent wave’ produced in the lower refractive index medium, with an intensity that falls off exponentially as the distance from the interface grows. Thus, if the excitation illumination in a fluorescence microscope is so 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 those further away are not. Thus, the images of the layer at or near the cell surface contain little or no interference from fluorescent molecules elsewhere in the cytosol.

Wide-field illumination fluorescence microscopy: visualization using excitation illumination propagating parallel to the z-axis. The intensity of fluorescent molecules is directly proportional to their number within the illuminated volume. Images generated in this way have significant contributions from objects located above or below the focal plane. Spatial deconvolution is required for accurate representation of the image.


Histology and Microscopy Unit (HMU) | Microscopy Services

Confocal Microscopes

Biomedical Imaging Facility:

Room LG12, Lower Ground Lowy Cancer Research Centre (C25) Kensington UNSW Sydney NSW 2052

Tel: +61 (2) 9385 1721 Fax: +61 (2) 9385 1720 Email: bmif@unsw.edu.au

Relocated to BMIF, please contact

Dr Chris Marjo (c.marjo@unsw.edu.au) for all training and booking enquiries.

Henry Haeberle (h.haeberle@unsw.edu.au) Microscopist 9385 1726

Olympus FV1000 Laser Scanning Microscope (inverted). The 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.

Relocated to BMIF, please contact Dr Chris Marjo (c.marjo@unsw.edu.au) for all training and booking enquiries. The FV1000 is set up for live cell imaging, with a 37oC incubation chamber, 5% CO2 connection, anti-vibration table and zero drift compensator to keep cells in focus during the scanning period.


Medicine Cell Biology course (ANAT3231) for science students has previously run a practical lab where students are able to see demonstrations of confocal microscopy. I would like to run again this year a visit to the new confocal facility on Wed 28th April 4-6 pm.