Two‐Photon Imaging in Live Rodents

Leonardo Belluscio1

1 National Institutes of Health/NINDS, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.9
DOI:  10.1002/0471142301.ns0209s32
Online Posting Date:  August, 2005
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Two‐photon imaging is an innovative optical technique that has quickly become state of the art for imaging fluorescent signals in a variety of organisms. With many advantages over conventional confocal microscopy, such as greater image resolution, deeper access (∼400 µm), and much less photo damage, two‐photon microscopy has already proven to be an extremely useful tool for imaging live cells or tissue. Due to its tremendous versatility, recent efforts have adapted this technique to allow visualization of fluorescent cells directly in living animals. This unit describes a basic procedure for performing two‐photon imaging in vivo as applied to the dorsal surface of the brain in live anesthetized mice or rats. The protocol outlines a surgical preparation to enable the capture of stable, high‐resolution (<1 µm) images of fluorescently labeled neurons in intact brain with very little detrimental effect to either cells or tissue.

Keywords: two‐photon; confocal imaging in vivo; mice; rats; brain; GFP

PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: In Vivo Imaging Using Two‐Photon Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: In Vivo Imaging Using Two‐Photon Microscopy

  • Low‐melting agarose (Sigma, Fisher Scientific)
  • Artificial cerebrospinal fluid (ACSF, see recipe)
  • N 2O/O 2 tank
  • Mouse subject
  • Appropriate anesthesia (see appendix 4B)
  • Atropine
  • PBS‐soaked gauze pads (see recipe for PBS)
  • Lidocaine ointment (if using ear‐bar mount)
  • 70% (v/v) ethanol
  • Betadine
  • Dental cement (if using head‐post mount)
  • Ophthalmic surgical spears or gelfoam
  • Lactated Ringer's solution (Abbott Laboratories), optional
  • Eye lubricant (e.g., Lacrilube)
  • 10‐ to 100‐µl Hamilton syringe
  • 27‐G disposable needles
  • Scissors (0.5‐, 1‐, and 6‐cm blades)
  • Scalpel holder and blades (no. 10, 11, and 15)
  • Forceps
  • Slide cover glass (0.5‐mm thick)
  • Diamond‐tipped glass cutter
  • Hair clipper
  • Anesthesia machine containing dual gas flow meters (O 2 and N 2O; Biomedical Research Instruments, Roboz, SurgiVet, Harvard Apparatus)
  • Anesthesia vaporizer (Biomedical Research Instruments, Roboz, SurgiVet, Harvard Apparatus)
  • Stereotaxic apparatus (Jensen Studio, Stoelting):
    • Head‐post mount (see Fig ) or
    • Ear‐bar mount (see Fig )
  • Pulse oximeter (Biomedical Research Instruments, Roboz, SurgiVet, Harvard Apparatus)
  • Animal heating pad
  • Cotton swabs
  • 6‐0 silk sutures
  • Dental drill with a very small burr‐shaped <1‐mm bit on the tip (a less expensive alternative is a Dremel tool, available at most hardware stores)
  • Two‐photon microscope (Coherent, Spectra‐Physics), upright model with a minimum of two objectives, a 10× air objective and a 40× water objective (IR‐rated if possible)
  • Dissecting scope (∼10× to 100×; Zeiss, Leica Microsystems, Bio‐Rad, Olympus, Nikon)
  • Fiberoptic light source
PDF or HTML at Wiley Online Library



Literature Cited

   Andrasfalvy, B.K., Smith, M.A., Borchardt, T., Sprengel, R., and Magee, J.C. 2003. Impaired regulation of synaptic strength in hippocampal neurons from GluR1‐deficient mice. J. Physiol. 552:35‐45.
   Charpak, S., Mertz, J., Beaurepaire, E., Moreaux, L., and Delaney, K. 2001. Odor‐evoked calcium signals in dendrites of rat mitral cells. Proc. Natl. Acad. Sci. U.S.A. 98:1230‐1234.
   Denk, W., Delaney, K.R., Gelperin, A., Kleinfeld, D., Strowbridge, B.W., Tank, D.W., and Yuste, R. 1994. Anatomical and functional imaging of neurons using two‐photon laser scanning microscopy. J. Neurosci. Meth. 54:151‐162.
   Denk, W., Strickler, J.H., and Webb, W.W. 1990. Two‐photon laser scanning fluorescence microscopy. Science 248:73‐76.
   Gan, W.B., Grutzendler, J., Wong, W.T., Wong, R.O., and Lichtman, J.W. 2000. Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27:219‐225.
   Goppert‐Mayer, M. 1931. Uber Elementarakte mit zwei Quantensprungen. Ann. Phys. Leipzig 9:273‐295.
   Grutzendler, J., Kasthuri, N., and Gan, W.B. 2002. Long‐term dendritic spine stability in the adult cortex. Nature 420:812‐816.
   Hadjantonakis, A.K. and Nagy, A. 2001. The color of mice: In the light of GFP‐variant reporters. Histochem. Cell Biol. 115:49‐58.
   Helmchen, F., Fee, M.S., Tank, D.W., and Denk, W. 2001. A miniature head‐mounted two‐photon microscope. High‐resolution brain imaging in freely moving animals. Neuron 31:903‐912.
   Huang, H.V. 1996. Sindbis virus vectors for expression in animal cells. Curr. Opin. Biotechl. 7:531‐535.
   Lichtman, J.W. and Sanes, J.R. 2003. Watching the neuromuscular junction. J. Neurocytol. 32:767‐775.
   Lo, D.C., McAllister, A.K., and Katz, L.C. 1994. Neuronal transfection in brain slices using particle‐mediated gene transfer. Neuron 13:1263‐1268.
   Margrie, T.W., Meyer, A.H., Caputi, A., Monyer, H., Hasan, M.T., Schaefer, A.T., Denk, W., and Brecht, M. 2003. Targeted whole‐cell recordings in the mammalian brain in vivo. Neuron 39:911‐918.
   Mizrahi, A. and Katz, L.C. 2003. Dendritic stability in the adult olfactory bulb. Nat. Neurosci. 6:1201‐1207.
   Slack, R.S. and Miller, F.D. 1996. Viral vectors for modulating gene expression in neurons. Curr. Opin. Neurobiol. 6:576‐583.
   Trachtenberg, J.T., Chen, B.E., Knott, G.W., Feng, G., Sanes, J.R., Welker, E., and Svoboda, K. 2002. Long‐term in vivo imaging of experience‐dependent synaptic plasticity in adult cortex. Nature 420:788‐794.
   Wachowiak, M., Denk, W., and Friedrich, R.W. 2004. Functional organization of sensory input to the olfactory bulb glomerulus analyzed by two‐photon calcium imaging. Proc. Natl. Acad. Sci. U.S.A. 101:9097‐9102.
   Xu, C., Zipfel, W., Shear, J.B., Williams, R.M., and Webb, W.W. 1996. Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. U.S.A. 93:10763‐10768.
   Yuste, R. and Denk, W. 1995. Dendritic spines as basic functional units of neuronal integration. Nature 375:682‐684.
   Zhao, H., Otaki, J.M., and Firestein, S. 1996. Adenovirus‐mediated gene transfer in olfactory neurons in vivo. J. Neurobiol. 30:521‐530.
Key References
   Pawley, J. 1997. Handbook of Biological Confocal Microscopy, 2nd ed. Plenum Press, New York.
  Chapter 28, titled “Two‐photon molecular excitation in laser scanning microscopy,” written by W. Denk, D.W. Piston, and W.W. Webb, pp. 445‐458, is useful for basic theory and procedural imaging of slices.
   Diaspro, A. 2002. Confocal and Two‐Photon Microscopy. Wiley‐Liss, New York.
  An easy to understand yet very comprehensive reference book for two‐photon theory and application.
   Inoue, S. and Spring, K. 1997. Video Microscopy: The Fundamentals. Plenum, New York.
  A classic that still has many basic and useful principles for imaging and microscopy in general.
   Kohn, D.F., Wixson, S.K., Benson, G.J., and White, W.J. 1997. Anesthesia and Analgesia in Laboratory Animals. Elsevier, New York.
  A useful reference for anesthetic use in small animals.
   Yuste, R., Lanni, F., and Konnerth, A. 2000. Imaging Neurons: A Laboratory Manual. CSHL Press, New York.
  An excellent manual for performing many different types of imaging experiments and also contains a lot of useful theoretical information in an easy‐to‐read text.
Internet Resources
  A useful index site for information or tutorial that is maintained by the Developmental Resource for Biophysical Imaging Opto‐Electronics (DRBIO) research team associated with Watt Webb's group at Cornell University.‐
  This site contains multi‐photon spectral information for a variety of fluorophores.
  A tutorial site maintained by Nikon that is interactive in demonstrating the function and quality difference between a confocal image and a simple wide‐field fluorescent image.
  A useful index site for general information on both microscopes and microscopy.
  A Web site for Molecular Probes, a major manufacturer of fluorescent probes.
  An NIH site for the Office of Laboratory Animal Welfare (OLAW) contains information on policy and use of animals in research.
  An NIH site for the Institute for Laboratory Animal Research (ILAR) contains more practical information on guidelines and use of animals in research.
PDF or HTML at Wiley Online Library