2011 Lab 7

From CellBiology

Confocal Microscopy

Confocal Microscopy 2 Methods
Two-photon microscopy in vivo brain

GFP3.jpg

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

Introduction

The laboratory this week will involve a tutorial on microscopy and fluorescent labeling techniques, as well as a visit to the Biomedical Imaging Facility (BMIF). Before the laboratory try and read some of the linked content describing the confocal microscope and its application in cell biology. See also your earlier laboratories Lab 2 - Microscopy | Immunochemistry | Preparation/Fixation

Links: Biomedical Imaging Facility (BMIF) | BMIF - Equipment | About the Analytical Centre


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

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
  • increased rate of data acquisition which also decreases photobleaching and phototoxicity


Photobleaching - photochemical destruction of a fluorophore due to light exposure. Usually unwanted, but some studies use this property to observe dynamic processes within cells.

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.

B. burgdorferi intravital microscopy.jpg

B._burgdorferi intravital microscopy (mouse skin) PMID: 18833295

Total Internal Reflection Fluorescence Microscopy (TIRF)

TIRF microscopy

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.

Poliovirus release cell surface.jpg

Poliovirus release cell surface PMID: 17622193

Photoactivated Localization Microscopy (PALM)

Bacteria PAL-M imaging
  • target proteins are labeled with photoactivatable proteins which are nonfluorescent until activated by near-UV light
  • near-UV light of low intensity activates only one protein per diffraction-limited region (~250 nm)
  • Following activation, each individual protein is then excited and imaged.
  • the center of each molecular point spread function indicates the location of each protein
    • Serial cycles of activation and excitation are repeated until all fusion proteins are bleached. Since individual proteins are imaged, we can count the number of proteins and computationally assemble the locations of all proteins into a composite, high-resolution image.
  • location of each protein can be determined to a precision of 2–25 nm, or approximately 10–100× better than the diffraction limit

(Text modified from PMID:19547746)

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 Photoactivated Localization Microscope (PAL-M)

TIRF microscopy
  • images with effective lateral resolutions down to 20nm
  • 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

GFP3.jpg

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

  • RFP, mRuby, Cherry
  • mRuby

Green to Red Fluorescent Protein

Gap junction dynamics green-to-red fluorescence.jpg

Gap junction (GJ) dynamics revealed by photoconverting expressed Cx43-Dendra2


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

Beyond Confocal

Moving beyond the diffraction barrier of light microscopy and single-molecule localization microscopy (SMLM). Know your acronyms! Some text below from Cell - Super-Resolution

Links: Q&A: Single-molecule localization microscopy for biological imaging | Cell - Super-Resolution

PhotoActivated Localization Microscopy

(PALM) single-molecule imaging techniques are used to measure the position of individual molecules within the diffraction-limited region.


Stochastic Optical Reconstruction Microscopy

(STORM) uses photo-switchable fluorescent probes that reversibly cycle between fluorescent and dark states upon exposure to light of specific wavelengths.

Stimulated Emission Depletion Microscopy

(STED) fluorescent molecules are effectively "switched off" by a process called "stimulated emission."

Links: Department of NanoBiophotonics | STED Dyes

Structured Illumination Microscopy

(SIM, SSIM) a laser beam passes through a grate, creating a striped interference pattern on the sample. Fine structures in the sample combine with the pattern and shift the high-frequency spatial information of the sample into to lower frequencies that are detectable by the light microscope.

References

Textbooks

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

Books

PubMed

  • 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

Reviews

  • 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

Articles

  • 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

Links

2012 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 - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | 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 | 2012 Revision | Development


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



2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9

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