Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration

J Neurosci Methods. 2009 May 30;180(1):9-21. doi: 10.1016/j.jneumeth.2009.01.032. Epub 2009 Feb 7.


Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetates / chemistry
  • Animals
  • Calcium Signaling / physiology
  • Calibration
  • Cerebellum / physiology
  • Electrophysiology / methods*
  • Ethylenediamines / chemistry
  • Glutamates / chemistry
  • Glutamates / radiation effects
  • Indoles / chemistry
  • Indoles / radiation effects
  • Lasers / adverse effects*
  • Light / adverse effects*
  • Organ Culture Techniques
  • Patch-Clamp Techniques / methods
  • Photic Stimulation / adverse effects
  • Photic Stimulation / methods
  • Photochemistry / methods*
  • Photolysis / radiation effects*
  • Rats
  • Rats, Sprague-Dawley
  • Synaptic Transmission / physiology


  • 4-methoxy-7-nitroindolinyl-glutamate
  • Acetates
  • Ethylenediamines
  • Glutamates
  • Indoles
  • DM-nitrophen