Confocal Laser Scanning Microscopic Photoconversion: A New Method to Stabilize Fluorescently Labeled Cellular Elements for Electron Microscopic Analysis

Raymond J. Colello1, Jordan Tozer1, Scott C. Henderson1

1 Department of Anatomy and Neurobiology, School of Medicine, Virginia Commonwealth University, Richmond, Virginia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.15
DOI:  10.1002/0471142301.ns0215s58
Online Posting Date:  January, 2012
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Abstract

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low‐resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region‐of‐interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. Curr. Protoc. Neurosci. 58:2.15.1‐2.15.12. © 2012 by John Wiley & Sons, Inc.

Keywords: photo‐oxidation; diaminobenzidine; ultrastructure; electron microscopy; immunolabeling; confocal microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Photoconversion and Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Photoconversion and Microscopy

  Materials
  • Postnatal day 14 (P14) Sprague Dawley rat pups
  • Pentobarbital anesthetic
  • 0.1 M phosphate‐buffered saline (PBS; see recipe)
  • Tissue‐Tek
  • Isopentane
  • Fixation solution (see recipe)
  • Blocking solution (see recipe)
  • Rabbit anti‐GFAP (DAKO, cat. no. Z0334)
  • 5% normal horse serum (NHS)
  • Alexa Fluor 568 (AF568) goat anti‐rabbit IgG (Invitrogen, cat. no. A‐11036)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI), optional
  • 0.1 M Tris buffer, pH 8.2 (see recipe)
  • 3,3′‐diaminobenzidine (DAB) solution (see recipe)
  • Basic dissection kit including:
    • Forceps
    • Scissors
    • Scalpel
    • Microdissection scissors
    • Rongeurs
  • Leica cryostat with tungsten knife
  • 0.17‐mm (no. 1.5) glass coverslips, coated with poly‐L‐lysine (see recipe) or Superfrost (Sigma) glass microscope slides
  • PAP pen, optional
  • Olympus IX70 conventional microscope with a 100 W mercury lamp and 20× (0.4 NA) and 40× (0.55 NA) objective lenses
  • Micropipets
  • Leica TCS‐SP2 (AOBS) inverted confocal laser scanning microscope equipped with a 405‐nm diode laser, a multi‐line Ar laser (458, 488, and 514 nm), three separate HeNe lasers (543, 594, and 633 nm), reflected and transmitted light PMTs, and several objective lenses including: 20× (0.7 NA) dry, 40× (1.25 NA) oil, and 63× (1.4 NA) oil
  • Zeiss LSM 510 META NLO upright 2‐photon/confocal laser scanning microscope equipped with a multi‐line Ar laser (458, 488, and 514 nm), a 561‐nm diode‐pumped solid‐state laser, a 633‐nm HeNe laser, a Spectra‐Physics Mai‐Tai Ti:sapphire laser, reflected and transmitted light PMTs and several objective lenses including a 10× (0.3 NA) dry lens used in this protocol
  • Additional reagents and equipment for anesthetizing the animal (see Donovan and Brown, )
NOTE: Both confocal laser scanning microscopes were equipped with a galvanometer driven point scanning system and an acousto‐optical tunable filter (AOTF) that enabled scan zoom and ROI scanning (i.e., regionally defined beam blanking).
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Figures

  •   FigureFigure 2.15.1 Comparison of cell selectivity between conventional widefield and confocal laser scanning microscopy. (A) P14 rat astrocytes immunolabeled for GFAP, viewed with widefield fluorescence microscopy and a 20× objective lens before photoconversion. The circle corresponds to the region selected to be photoconverted. (B) Area shown in panel A viewed after photoconversion. The dark circle corresponds to the field photobleached by fluorescent light using the 40× objective lens. (C) Brightfield microscopy of the area shown in panel A, after photoconversion. Note the photobleached area in B corresponds to the area in panel C where previously fluorescent astrocytes now contain a brown reaction product. (D) P14 rat astrocytes immunolabeled for GFAP, viewed with confocal laser scanning microscopy before photoconversion. The square denotes an approximation of the ROI selected for photoconversion and corresponds to the squares in E‐G. (E) Astrocytes shown after photoconversion using the 594‐nm HeNe laser at 100% power. Note the fluorescence is bleached only from the region of interest selected. (F) Differential interference contrast (DIC) image shown taken after photoconversion. Note the appearance of reaction product in the area selected. The astrocytic processes are readily visible. (G) Fluorescent image of adult rat hippocampal dentate gyrus stained for synaptophysin and BrdU. (H) BrdU‐positive cells after photoconversion with the 488‐nm line of the Ar laser set at 100%. White arrow corresponds to the same BrdU cell in both panels G and H. (I) Transmission electron micrograph of a photoconverted BrdU‐positive cell showing DAB reaction product (white arrows). Scale bars: A‐C = 0.5 mm; D‐G = 20 µm..
  •   FigureFigure 2.15.2 Increased selectivity exhibited by confocal laser scanning microscopy. (A) Fluorescent image of P14 rat brain with astrocytes immunolabeled for GFAP and cell nuclei labeled with DAPI (arrow) before photoconversion. (B) The same image shown after photoconverting only the nucleus (arrow) using the 405‐nm diode laser set at 30% power. Using this wavelength coupled with the ROI scanning to selectively photoconvert the nucleus spares the astrocytic processes from the reaction. (C) DIC image of the same area showing the reaction product deposited after photoconversion (arrow). (D) Fluorescent image of P14 rat astrocytes immunolabeled for GFAP before photoconversion. (E) The same image after cells were “ tagged ” using the 594‐nm HeNe laser set at 100% power (arrows). All three “tags” were created simultaneously. (F) DIC image showing the DAB reaction product localized to the ROI selected (arrows). Scale bars: A‐C = 5 µm; D, E, F = 1 µm.
  •   FigureFigure 2.15.3 Photoconversion times. Chart comparing the photoconversion time of AF568 using the Olympus widefield microscope, the Leica CLSM, and the Zeiss two‐photon confocal microscope.

Videos

Literature Cited

   Burghardt, R.C. and Droleskey, R. 2006. Transmission electron microscopy. Curr. Protoc. Microbiol. 3:2B.1.1‐2B.1.39.
   Deerinck, T.J., Martone, M.E., Lev‐Ram, V., Green, D.P., Tsien, R.Y., Spector, D.L., Huang, S., and Ellisman, M.H. 1994. Fluorescence photooxidation with eosin: A method for high resolution immunolocalization an in situ hybridization detection for light and electron microscopy. J. Cell Biol. 126:901‐910.
   De‐Miguel, F.F., Muller, K.J., Adams, W.B., and Nicholls, J.G. 2002. Axotomy of single fluorescent nerve fibers in developing mammalian spinal cord by photoconversion of diaminobenzidine. J. Neursci. Methods 117:73‐79.
   Donovan, J. and Brown, P. 1998. Anesthesia. Curr. Protoc. Immunol. 27:1.4.1‐1.4.5.
   Kacza, J., Hartig, W., and Seeger, J. 1997. Oxygen‐enriched photoconversion of fluorescent dyes by means of a closed conversion chamber. J. Neursci. Methods 71:225‐232.
   Lubke, J. 1993. Photoconversion of diaminobenzidine with different fluorescent neuronal markers into a light and electron microscopic dense reaction product. Microsc. Res. Tech. 24:2‐14.
   Maranto, A. 1982. Neuronal mapping: A photooxidation of reaction makes Lucifer yellow useful for electron microscopy. Science 217:953‐955.
   Mark, M., Teletin, M., Antal, C., Wendling, O., Auwerx, J., Heikkinen, S., Khetchoumian, K., Argmann, C.A., and Dgheem, M. 2007. Histopathology in mouse metabolic investigations. Curr. Protoc. Mol. Biol. 78:29B.4.1‐29B.4.32.
   Sandell, J.H. and Masland, R.H. 1988. Photoconversion of some fluorescent markers to a diaminobenzidine product. J. Histochem. Cytochem. 36:555‐559.
   Singleton, C.D. and Casagrande, V.A. 1996. A reliable and sensitive method for fluorescent photoconversion. J. Neursci. Methods 64:47‐54.
   Tozer, J.T., Henderson, S.C., Sun, D., and Colello, R.J. 2007. Photoconversion using confocal laser scanning microscopy: a new tool for the ultrastructural analysis of fluorescently labeled cellular elements. J. Neursci. Methods 164:240‐246.
Key References
   Maranto, 1982. See above.
   Sandell and Masland, 1988. See above.
   Tozer et al., 2007. See above.
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