2017 Lab 7
- 1 Confocal Microscopy
- 1.1 Introduction
- 1.2 BMIF Research Techniques Available
- 1.3 Diffraction Limit
- 1.4 Fluorescence Microscopy
- 1.5 Laser-Scanning Confocal Microscopy
- 1.6 Spinning Disc Confocal Microscopy
- 1.7 Total Internal Reflection Fluorescence Microscopy (TIRF)
- 1.8 Photoactivated Localization Microscopy (PALM)
- 1.9 Biomedical Imaging Facility (BMIF)
- 1.10 Theory of Confocal Microscopy
- 1.11 Confocal Microscopy Practical Issues
- 1.12 Confocal Microscopy - Fluorochromes
- 1.13 Beyond Confocal
- 1.14 Lab 7 Individual Assessment
- 1.15 References
- 1.15.1 Textbooks
- 1.15.2 Books
- 1.15.3 PubMed
- 1.16 Links
- 1.17 2017 Course Content
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.
Dr Michael Carnell (BMIF research associate)
BMIF Research Techniques Available
- 2-photon and Intravital Microscopy
- Biological / Soft Material Atomic Force Microscopy (AFM)
- Confocal Microscopy
- Epifluorescence for live cell imaging
- Fluorescence Correlation Spectroscopy (FCS)
- Fluorometer and Lifetime Spectroscopy
- Spinning Disc Microscopy
- Stimulated Emission Depletion (STED) Microscopy
- Stochastic Optical Reconstruction Microscopy (STORM)
- Superresolution Fluorescence Microscopy including photo-activation localisation microscopy (PALM)
- Time-Resolved Single Molecule Imaging including Fluorescence Lifetime Imaging Microscopy (FLIM)
- Total Internal Reflection Fluorescence (TIRF) Microscopy
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.
- 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
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
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 (mouse skin) PMID: 18833295
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.
Poliovirus release cell surface PMID: 17622193
Photoactivated Localization Microscopy (PALM)
- 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.
Leica TCS SP Laser Scanning Microscope
- (inverted) three channel excitation: 488nm, 568nm & 647nm.
Zeiss Observer X.1
- 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
Zeiss Photoactivated Localization Microscope (PAL-M)
- images with effective lateral resolutions down to 20nm
- Total Internal Reflection Fluorescence microscope (TIRFM)
- Laser widefield illumination TIRF system.
Theory of Confocal Microscopy
- Olympus online educational resource - Theory of Confocal Microscopy | spinning disk confocal
- Molecular expressions - Laser Scanning Confocal Microscopy
- Zeiss - Education in Microscopy and Digital Imaging
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
- RFP, mRuby, Cherry
Green to Red Fluorescent Protein
Gap junction (GJ) dynamics revealed by photoconverting expressed Cx43-Dendra2
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."
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.
Lab 7 Individual Assessment
- Complete the microscopy lab worksheet questions.
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
- The Confocal Microscope Produces Optical Sections by Excluding Out-of-Focus Light
- Conventional and confocal fluorescence microscopy compared
- Figure 9-45. Dynamics of GFP tagging
Molecular Cell Biology
Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999
- Confocal Scanning and Deconvolution Microscopy Provide Sharper Images of Three-Dimensional Objects
- Fluorescence Microscopy Can Localize and Quantify Specific Molecules in Cells
- Revealing Specific Proteins in Living Cells
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
- "confocal microscopy" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf
- "fluorescence microscopy" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf
- 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
Steve Paddock Over the rainbow: 25 years of confocal imaging. BioTechniques: 2008, 44(5);643-4, 646, 648 PubMed 18474039
David Feng, David Marshburn, Dennis Jen, Richard J Weinberg, Russell M Taylor, Alain Burette Stepping into the third dimension. J. Neurosci.: 2007, 27(47);12757-60 PubMed 18032646
Laurent Kreplak, Karsten Richter, Ueli Aebi, Harald Herrmann Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy. Methods Cell Biol.: 2008, 88;273-97 PubMed 18617039
Ben N G Giepmans Bridging fluorescence microscopy and electron microscopy. Histochem. Cell Biol.: 2008, 130(2);211-7 PubMed 18575880
Claire M Brown Fluorescence microscopy--avoiding the pitfalls. J. Cell. Sci.: 2007, 120(Pt 10);1703-5 PubMed 17502480
Alberto Diaspro, Paolo Bianchini, Giuseppe Vicidomini, Mario Faretta, Paola Ramoino, Cesare Usai Multi-photon excitation microscopy. Biomed Eng Online: 2006, 5;36 PubMed 16756664
Alison J North Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. J. Cell Biol.: 2006, 172(1);9-18 PubMed 16390995
Nathan C Shaner, Paul A Steinbach, Roger Y Tsien A guide to choosing fluorescent proteins. Nat. Methods: 2005, 2(12);905-9 PubMed 16299475
Akihiko Nakano Spinning-disk confocal microscopy -- a cutting-edge tool for imaging of membrane traffic. Cell Struct. Funct.: 2002, 27(5);349-55 PubMed 12502889
Bruce H Reed, Stephanie C McMillan, Roopali Chaudhary The preparation of Drosophila embryos for live-imaging using the hanging drop protocol. J Vis Exp: 2009, (25); PubMed 19287353
Georgeann S O'Brien, Sandra Rieger, Seanna M Martin, Ann M Cavanaugh, Carlos Portera-Cailliau, Alvaro Sagasti Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. J Vis Exp: 2009, (24); PubMed 19229185
Emilie Flaberg, Per Sabelström, Christer Strandh, Laszlo Szekely Extended Field Laser Confocal Microscopy (EFLCM): combining automated Gigapixel image capture with in silico virtual microscopy. BMC Med Imaging: 2008, 8;13 PubMed 18627634
E B van Munster, J Goedhart, G J Kremers, E M M Manders, T W J Gadella Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy. Cytometry A: 2007, 71(4);207-14 PubMed 17266147
Jessica M Teddy, Rusty Lansford, Paul M Kulesa Four-color, 4-D time-lapse confocal imaging of chick embryos. BioTechniques: 2005, 39(5);703-10 PubMed 16312219
| Biotechniques M Ono, T Murakami, A Kudo, M Isshiki, H Sawada, A Segawa Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy. J. Histochem. Cytochem.: 2001, 49(3);305-12 PubMed 11181733
| See also Mountants and Antifades PDF
External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.
- Confocal Microscope Systems - A Comparison Of Technologies PDF
- Improvision - UltraVIEW VoX 4D confocal imaging
- Biovision - CARV II confocal imager from BD Biosciences
- Salk Institute - Table of Fluorochromes
- Mountants and Antifades PDF -
2017 Course Content
Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | 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 | 2017 Revision
Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G