Photolysis is a newly developed tool that facilitates the release of ligands next to their receptors, overcoming the impact of diffusion that will otherwise dictate the kinetics of the receptor activation.
An additional result of limited diffusional access is an amplified influence of receptor desensitization, metabolism and ligand uptake in defining the size and time-course of the response to a ligand, especially in complex tissue preparations, such as brain slices, or at intracellular receptors.
To employ photolysis to beat diffusion barriers, the tissue or cell is equilibrated with a solution containing a photolabile precursor of the ligand. Receptor binding and downstream signaling are then instigated by photo release of the ligand. This method is applicable to extracellular or intracellular receptors.
For successful photolysis, it is necessary that photolabile precursors (cages) of the ligand be synthesized and assessed for photolytic efficiency, physiological interference and toxicity.
Photolysis is typically realized using near-UV flashlamp or laser irradiation, or near-IR pulsed lasers for two-photon excitation. The ligands discharged can be endogenous neurotransmitters, hormones, second messengers, or receptor subtype-specific analogs.
Experimental Uses of Photolysis in Neuroscience and Cell Biology
- To examine kinetics of activation of receptors, channels and enzymes in signaling pathways;
- To distinguish postsynaptic from presynaptic processes, enabling the study of receptors in situ;
- To enable isolation and pharmacological examination of the steps in a signaling pathway;
- For photostimulation or inhibition to examine network connectivity;
- For labeling intracellular compartments with fluorophore to examine diffusional exchange.
Kinetics of Ligand Release
Ligand is typically released with a short (10 ns to 1 ms) pulse of near-UV light. The photons absorbed produce excited intermediates that deteriorate to release the ligand during 'dark' reaction stages. These have distinctive rates and quantum yield (the fraction of ligand released for photons absorbed).
The productivity of a cage is calculated as the ability to absorb light (i.e. the molar absorption coefficient or the cross-section) multiplied by the quantum yield. To be suitable for studies to examine kinetics, the dark reactions should be rapid in comparison to the receptor activation and downstream events analyzed.
For example, the rate of the dark release of glutamate from MNI-glutamate after a short excitation pulse is 2x106 s-1 (half time 200 ns), significantly quicker than the rate of activation of ionotropic and metabotropic glutamate receptors by high glutamate concentrations.
Conversely, the release of DHPG from the cage NPEC-DHPG is slower, at around 10 s-1 at pH 7.4, but still sufficiently quick to be employed for activation of metabotropic type 1 GPCR glutamate receptors.
The rate of dark reaction steps is inversely proportional to pH, and in the preliminary publication of NPEC caged L-glutamate, this pH dependence was employed to speed the photolysis rate at the giant squid synapse to yield postsynaptic activation.
Photolysis can also be attained with stable lighting, produced, for example, by the 361 nm Hg line or with near-UV LED sources. In this instance, the ligand is constantly released during the period of illumination.
Calibration of Photolysis
Photolysis of the caged fluorophore NPE-HPTS produces a fluorophore, HPTS (pyranine), with excitation and emission comparable to FITC. The fluorescence released is freely measured by photometry or fluorescence imaging.
The photolysis efficiency of NPE-HPTS is comparable to that of a number of cages in cuvette measurements with flashlamp (i.e. broad-spectrum) or with laser monochromatic excitation.
Light Sources for Photolysis and Coupling to the Microscope
Xenon flashlamps create a pulse of 0.5-1 ms duration with a white spectrum, from which the near-UV 300-400 nm is extracted with a band-pass filter, such as UG11 or UG5 glass filters (Schott types are available from Thorlabs).
Joining of light sources is most frequently achieved through the epifluorescence condenser in both upright and inverted microscope configurations. Below 350 nm, light is weakened by glass lenses, so silica optics are frequently employed.
Microscope objectives transmit 40-50% in the near UV, and usually not at all at less than 320 nm. A set of appropriate dichroic reflectors is required if experiments necessitate concurrent fluorescence excitation and detection. These should pass or eliminate, as is fitting, the wavelengths for photolysis.
The dichroic reflectors for FITC or eGFP in commercial microscopes, as examples, do not typically reflect near-UV and must be replaced for photolysis. Chroma part T490DC is effective for blue excitation/green emission.
Fluorescence excitation filters will not conduct photolysis light and must be fitted in the lamphouse, rather than the microscope filter cube.
Often, the connections from flashlamp to microscope are a silica multimode fiber or liquid light guide. This offers the benefits of eliminating higher trigger voltage and large (100 Amp) current from the locality of the electrophysiological recording and is a simpler mechanism. However, it is less optically efficient.
As the efficiency of cages is not great enough to permit localization of flashlamp light to a small spot, flashlamp photolysis is typically employed over a full field with a diameter of around 200 μm. Flashlamps and couplings are commercially available from suppliers, such as Cairn Research, Rapp Optoelectronic and TILL Photonics.
Near-UV LEDs with adequate power for photolysis by long exposures of around 100 ms are obtainable. These are more cost-effective and easier to control than flashlamps. However, in brief pulses, they offer insufficient energy for rapid kinetic studies.
Lasers - Localized Point or Scanning Photolysis
A small, diffraction-limited spot with sub-micron dimensions is readily produced by an expanded collimated laser beam aimed at the reverse of a microscope objective.
Simple to control diode or DPSS lasers with sufficient power, analog modulation of power and exposure, true zero light when OFF and decent beam quality can be sourced at wavelengths of 355 nm (pulsed) 380 nm and 405 nm (CW).
To circumvent the toxicity of high peak powers, CW is typically preferable against pulsed ('quasi-CW'). Top beam quality is attained through single-mode fiber coupling. Appropriate lasers can be ordered from suppliers such as Point Source, Omicron, Toptica and DPSS Laser.
Light Loss by Inner Filtering In the Cage Solution
Water dipping objectives are typically employed with extracellular photolysis on upright microscopes. At cage concentrations above 0.5 mM, substantial losses arise as a result of absorption of near-UV light in the 2-3 mm of solution between objective and cell. This significantly decreases uncaging efficiency at the peak wavelengths of 340-360 nm.
However, this can be overcome by functioning at longer wavelengths where light absorption is lessened. For example, at 405 nm, the laser power is increased to compensate for the lower photolysis efficiency.
The earliest effective uses of flash photolysis were to examine activation of intracellular processes, such as ATP-dependent reactions and Ca2+dependent processes. The receptors are usually unreachable, and access was managed through permeabilization of the cell membrane.
In more recent studies, access of the cage to the receptor compartment has been managed through perfusion with whole cell patch clamp, or by microinjection. Fluorescent dyes are frequently employed alongside photolysis to observe access to the cytosol and the intracellular Ca2+ concentration.
The inner filtering effect outlined above can also be circumvented with two-photon photolysis, although this does result in increased susceptibility to phototoxicity and is far more costly.
The sole possible benefit of two-photon photolysis is the deeper penetration of two-photon excitation, as a result of the inability of scattered IR photons to excite the cage alone.
However, this is mostly negated by the meager two-photon excitation of current cages. In all currently available cages, effectiveness in two-photon excitation is considerably lower than for near-UV one-photon excitation.
In general, two-photon photolysis cross sections >1 GM are necessary, while presently available cages are <0.1 GM. As such, extremely high cage concentrations are needed, resulting in a greater probability of interference by the cage.
Moreover, in order to uphold localization of release to the excitation volume, the rate of photolysis 'dark' reactions must be rapid in relation to diffusion (>10,000 s-1). The ideal exposures are short, comparable to the diffusional exchange time constant of 150-300 μs for the two-photon spot volume.
Caged Fluorophore NPE-HPTS
NPE-HPTS releases the fluorophore HPTS (pyranine), a ratiometric pH indicator with pK 7.25. It has been employed to study compartmentalization by measuring diffusion after point release.
While the near-UV photolysis is comparatively slow at pH7, half-time 10-20 ms, with two-photon excitation, the rate of release was found to be much more rapid (>3000 s-1), making it appropriate for diffusional studies in small compartments.
NPE-HPTS is customarily employed for photolysis calibration.
Caged GABA reagents have been demonstrated to hinder activation of GABAA receptors. DPNI-GABA was developed to reduce this interference. It has a significantly higher IC50, 0.5 mM, than the original NI-GABA NS. It has been employed for kinetic and mapping studies of GABA receptors in situ.
NPEC-DHPG and NPEC-ACPD
Unlike a lot of caged neuroactive amino acids that make use of nitrobenzyl or nitrophenyl photochemistry, the NPEC cages are stable to hydrolysis. They are effective for near-UV photolysis, but have slower photolysis, with 'dark' rates of approximately 10-20 s-1 at pH 7.4.
The DHPG and ACPD cages have been tested at mGluR1 receptors and for interference with synaptic transmission. Each activates mGluR1 competently and neither causes interference with glutamatergic transmission ahead of photolysis.
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