Hostname: page-component-78c5997874-xbtfd Total loading time: 0 Render date: 2024-11-19T11:31:38.863Z Has data issue: false hasContentIssue false
  • English
  • Français

International Test Results for Objective Lens Quality, Resolution, Spectral Accuracy and Spectral Separation for Confocal Laser Scanning Microscopes

Published online by Cambridge University Press:  08 October 2013

Richard W. Cole ,
Marc Thibault ,
Carol J. Bayles ,
Brady Eason ,
Anne-Marie Girard ,
Tushare Jinadasa ,
Cynthia Opansky ,
Katherine Schulz  and
Claire M. Brown
Richard W. Cole
Affiliation:
New York State Department of Health, Wadsworth Center, P.O. Box 509, Albany, NY 12201, USA
Marc Thibault
Affiliation:
Montreal General Hospital, C9, 1650 Cedar, Montreal H3G 1A4, Canada
Carol J. Bayles
Affiliation:
Life Sciences Core Laboratory Center, Cornell University, Weill Hall, Ithaca, NY 14853, USA
Brady Eason
Affiliation:
Life Sciences Complex Advanced BioImaging Facility (ABIF), McGill University, 3649 Prom, Sir William Osler, Bellini Building, Room 137, Montreal, QC H3G 0B1, Canada
Anne-Marie Girard
Affiliation:
Center for Genome Research and Biocomputing, Oregon State University, 3021 Agriculture and Life Sciences Building, Corvallis, OR 97331, USA
Tushare Jinadasa
Affiliation:
Life Sciences Complex Advanced BioImaging Facility (ABIF), McGill University, 3649 Prom, Sir William Osler, Bellini Building, Room 137, Montreal, QC H3G 0B1, Canada
Cynthia Opansky
Affiliation:
Blood Center of Wisconsin, Blood Research Institute, 8733 Watertown Plank Road, Milwaukee, WI 53226, USA
Katherine Schulz
Affiliation:
Blood Center of Wisconsin, Blood Research Institute, 8733 Watertown Plank Road, Milwaukee, WI 53226, USA
Claire M. Brown*
Affiliation:
Life Sciences Complex Advanced BioImaging Facility (ABIF), McGill University, 3649 Prom, Sir William Osler, Bellini Building, Room 137, Montreal, QC H3G 0B1, Canada
*
*Corresponding author.claire.brown@mcgill.ca
Get access

Abstract

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

Keywords

laser scanning confocalresolutionquality assuranceobjective lenspoint spread function (PSF)spectral imagingspectral unmixingspectral accuracy
Type
Techniques and Instrumentation Development
Information
Microscopy and Microanalysis , Volume 19 , Issue 6 , December 2013 , pp. 1653 - 1668
Copyright
Copyright © Microscopy Society of America 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F. & Beltram, F. (2009). Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair. J Photochem Photobiol B 85, 287–297.CrossRefGoogle ScholarPubMed
Azizi, F. & Wahl, P. (1997). Fluorescence recovery after photobleaching (FRAP) of a fluorescent transferrin internalized in the late transferrin endocytic compartment of living A431 cells: Experiments. Biochim Biophys Acta 1327, 75–88.Google Scholar
Berg, R.H. (2004). Evaluation of spectral imaging for plant cell analysis. J Microsc 214, 174–181.Google Scholar
Beyer, H. (1985). Handbuch der Mikroskopie. Berlin: VEB Verlag Technik.Google Scholar
Brown, C.M., Dalal, R.B., Hebert, B., Digman, M.A., Horwitz, A.R. & Gratton, E. (2008). Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229, 78–91.Google Scholar
Centonze, V. & Pawley, J.B. (2006). Tutorial on practical confocal microscopy and use of the confocal test specimen. In Handbook of Biological Confocal Microscopy, Pawley, J.B. (Ed.), pp. 627–647. New York: Springer.Google Scholar
Cogswell, C.J., Sheppard, C.J.R., Moss, M.C. & Howard, C.V. (1990). A method for evaluating microscope objectives to optimize performance of confocal systems. J Microsc 158, 177–185.CrossRefGoogle Scholar
Cole, R.W., Jinadasa, T. & Brown, C.M. (2011). Resolution and quality control of confocal microscopy optics. Nature Prot 6(12), 1929–1941; doi:10.1038/nprot.2011.407.CrossRefGoogle Scholar
Cox, G. & Sheppard, C.J. (2004). Practical limits of resolution in confocal and non-linear microscopy. Microsc Res Tech 63, 18–22.Google Scholar
Digman, M.A., Sengupta, P., Wiseman, P.W., Brown, C.M., Horwitz, A.R. & Gratton, E. (2005). Fluctuation correlation spectroscopy with a laser-scanning microscope: Exploiting the hidden time structure. Biophys J 88, L33–L36.CrossRefGoogle ScholarPubMed
Dusch, E., Dorval, T., Vincent, N., Wachsmuth, M. & Genovesio, A. (2007). Three-dimensional point spread function model for line-scanning confocal microscope with high-aperture objective. J Microsc 228, 132–138.Google Scholar
Garini, Y., Young, I.T. & McNamara, G. (2006). Spectral imaging: Principles and applications. Cytometry A 69, 735–747.Google Scholar
Gibson, S.F. & Lanni, F. (1992). Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy. J Opt Soc Am A 9, 154–166.Google Scholar
Goodwin, P.C. (2007). Evaluating optical aberration using fluorescent microspheres: Methods, analysis, and corrective actions. Methods Cell Biol 81, 397–413.Google Scholar
Hibbs, A.R., MacDonald, G. & Garsha, K. (2006). Practical confocal microscopy. In Handbook of Biological Confocal Microscopy, Pawley, J.B. (Ed.), pp. 650–672. New York: Springer.Google Scholar
Hiraoka, Y., Sedat, J.W. & Agard, D.A. (1990). Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy. Biophys J 57, 325–333.Google Scholar
Hutter, H. (2004). Five-colour in vivo imaging of neurons in Caenorhabditis elegans . J Microsc 215, 213–218.Google Scholar
Icenogle, R.D. & Elson, E.L. (1983). Fluorescence correlation spectroscopy and photobleaching recovery of multiple binding reactions. I. Theory and FCS measurements. Biopolymers 22, 1919–1948.Google Scholar
Jares-Erijman, E.A. & Jovin, T.M. (2003). FRET imaging. Nat Biotechnol 21, 1387–1395.Google Scholar
Jares-Erijman, E.A. & Jovin, T.M. (2006). Imaging molecular interactions in living cells by FRET microscopy. Curr Opin Chem Biol 10, 409–416.Google Scholar
Juskaitis, R. (2006). Measuring the real point spread function of high numerical aperture microscope objective lenses. In Handbook of Biological Confocal Microscopy, Pawley, J.B. (Ed.), pp. 239–250. New York: Springer.CrossRefGoogle Scholar
Juskaitis, R., Neil, M.A.A. & Wilson, T. (1999). Characterizing high quality microscope objectives: A new approach. P Soc Photo-Opt Ins 3605, 140–143.Google Scholar
Keller, H.E. (2006). Objective lenses for confocal microscopy. In Handbook of Biological Confocal Microscopy, Pawley, J.B. (Ed.), pp. 145–160. New York: Springer.Google Scholar
Lerner, J.M. (2006). Imaging spectrometer fundamentals for researchers in the biosciences—A tutorial. Cytometry A 69, 712–734.Google Scholar
Lerner, J.M. & Zucker, R.M. (2004). Calibration and validation of confocal spectral imaging systems. Cytometry A 62, 8–34.Google Scholar
Lopez, A., Dupou, L., Altibelli, A., Trotard, J. & Tocanne, J.F. (1988). Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Critical comparison of analytical solutions, and a new mathematical method for calculation of diffusion coefficient D. Biophys J 53, 963–970.CrossRefGoogle Scholar
McNally, J.G., Karpova, T., Cooper, J. & Conchello, J.A. (1999). Three-dimensional imaging by deconvolution microscopy. Methods 19, 373–385.Google Scholar
Murphy, D.B. & Davidson, M.W. (2013). Fundamentals of Light Microscopy and Electronic Imaging. Hoboken, NJ: Wiley-Blackwell.Google Scholar
Murray, J.M., Appleton, P.L., Swedlow, J.R. & Waters, J.C. (2007). Evaluating performance in three-dimensional fluorescence microscopy. J Microsc 228, 390–405.CrossRefGoogle ScholarPubMed
Nasse, M.J., Woehl, J.C. & Huant, S. (2007). High-resolution mapping of the three-dimensional point spread function in the near-focus region of a confocal microscope. Appl Phys Lett 90, 90–93.Google Scholar
Pawley, J. (2000). The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. Biotechniques 28, 884–886, 888.CrossRefGoogle ScholarPubMed
Pawley, J. (2006). Handbook of Biological Confocal Microscopy. New York: Plenum.Google Scholar
Periasamy, A., Wallrabe, H., Chen, Y. & Barroso, M. (2008). Chapter 22: Quantitation of protein-protein interactions: Confocal FRET microscopy. Methods Cell Biol 89, 569–598.Google Scholar
Piston, D.W. & Kremers, G.J. (2007). Fluorescent protein FRET: The good, the bad and the ugly. Trends Biochem Sci 32, 407–414.CrossRefGoogle ScholarPubMed
Reiss, S.M. (2010). Quality and standards: Making bioimaging measure up. BioOptics World 3, 14–18.Google Scholar
Scalettar, B.A., Swedlow, J.R., Sedat, J.W. & Agard, D.A. (1996). Dispersion, aberration and deconvolution in multi-wavelength fluorescence images. J Microsc 182, 50–60.CrossRefGoogle ScholarPubMed
Sekar, R.B. & Periasamy, A. (2003). Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160, 629–633.Google Scholar
Shaw, P. (1994). Deconvolution in 3-D optical microscopy. Histochem J 26, 687–694.Google Scholar
Stack, R.F., Bayles, C.J., Girard, A.M., Martin, K., Opansky, C., Schulz, K. & Cole, R.W. (2011). Quality assurance testing for modern optical imaging systems. Microsc Microanal 17, 598–606.Google Scholar
Stavreva, D.A. & McNally, J.G. (2004). Fluorescence recovery after photobleaching (FRAP) methods for visualizing protein dynamics in living mammalian cell nuclei. Methods Enzymol 375, 443–455.CrossRefGoogle ScholarPubMed
Stelzer, E.H.K. (1998). Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: Fundamental limits to resolution in fluorescence light microscopy. J Microsc 189, 15–24.Google Scholar
Swedlow, J.R. (2007). Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol 81, 447–465.Google Scholar
Swedlow, J.R. & Platani, M. (2002). Live cell imaging using wide-field microscopy and deconvolution. Cell Struct Funct 27, 335–341.Google Scholar
van Royen, M.E., Farla, P., Mattern, K.A., Geverts, B., Trapman, J. & Houtsmuller, A.B. (2009). Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells. Methods Mol Biol 464, 363–385.CrossRefGoogle ScholarPubMed
Vogel, S.S., Thaler, C. & Koushik, S.V. (2006). Fanciful FRET. Sci STKE 2006, re2. Google Scholar
Wallace, W., Schaefer, L.H. & Swedlow, J.R. (2001). A workingperson's guide to deconvolution in light microscopy. Biotechniques 31, 1076–1078, 1080, 1082 passim.Google Scholar
Wilhelm, S., Gröbler, B., Gluch, M. & Heinz, H. (1997). Confocal Laser Scanning Microscopy. Jenna, Germany: Carl Zeiss.Google Scholar
Zahorian, S.A., Zuckerwar, A.J. & Karnjanadecha, M. (2012). Dual transmission model and related spectral content of the fetal heart sounds. Comput Methods Programs Biomed 108, 20–27.Google Scholar
Zheng, C.Y., Petralia, R.S., Wang, Y.X. & Kachar, B. (2011). Fluorescence recovery after photobleaching (FRAP) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons. J Vis Exp 50, 2568. Google Scholar
Zucker, R.M. & Lerner, J.M. (2005). Wavelength and alignment tests for confocal spectral imaging systems. Microsc Res Tech 68, 307–319.CrossRefGoogle ScholarPubMed
Zucker, R.M., Rigby, P., Clements, I., Salmon, W. & Chua, M. (2007). Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads. Cytometry A 71, 174–189.Google Scholar
Supplementary material: PDF

Cole Supplementary Material

Supplementary Materials

Download Cole Supplementary Material(PDF)
PDF 6.7 MB