Caged Compounds

Photolysis is an experimental tool that enables the release of ligands adjacent to their receptors, overcoming the effects of diffusion that will otherwise dominate the kinetics of the receptor activation. A further consequence of restricted diffusional access is an increased influence of receptor desensitization, metabolism and ligand uptake in determining the size and time-course of the response to a ligand, particularly in complex tissue preparations such as brain slices, or at intracellular receptors. To use photolysis to overcome diffusion barriers, the tissue or cell is equilibrated with solution containing a photolabile precursor of the ligand. Receptor binding and downstream signaling is then initiated by photorelease of the ligand. This technique can be applied to extracellular or intracellular receptors.

Photolysis requires photolabile precursors (cages) of the ligand to be synthesized and evaluated for photolytic efficiency, physiological interference and toxicity. Photolysis is usually achieved using near-UV flashlamp or laser irradiation, or near-IR pulsed lasers for two-photon excitation. The ligands released can be endogenous neurotransmitters, hormones, second messengers, or receptor subtype-specific analogs.

Experimental uses of photolysis in neuroscience and cell biology

• To investigate kinetics of activation of receptors, channels and enzymes in signaling pathways;
• To separate postsynaptic from presynaptic processes, allowing the study of receptors in situ;
• To permit isolation and pharmacological investigation of the steps in a signaling pathway;
• For photostimulation or inhibition to study network connectivity;
• For labeling intracellular compartments with fluorophore to study diffusional exchange.

Kinetics of ligand release

The release of ligand is usually with a brief (10 ns to 1 ms) pulse of near-UV light. The photons absorbed generate excited intermediates that decay to release the ligand during 'dark' reaction steps. These have characteristic rates and quantum yield (the fraction of ligand released for photons absorbed). The efficiency of a cage is measured by the ability to absorb light (i.e. the molar absorption coefficient or the cross-section) multiplied by the quantum yield. To be useful in experiments to study kinetics the dark reactions should be fast compared with the receptor activation and downstream events studied. As an example the rate of the dark release of glutamate from MNI-glutamate following a brief excitation pulse is 2x10⁶ s^-1 (half time 200 ns), much faster than the rate of activation of ionotropic and metabotropic glutamate receptors by high glutamate concentrations. In contrast, the release of DHPG from the cage NPEC-DHPG is slower, approx 10 s^-1 at pH 7.4, but fast enough to be used for activation of metabotropic type 1 GPCR glutamate receptors. The rate of dark reaction steps is inversely proportional to pH, and in the initial publication of NPEC caged L-glutamate this pH dependence was used to speed the photolysis rate at the giant squid synapse to produce postsynaptic activation.

Photolysis can also be achieved with steady illumination, produced for example by the 361 nm Hg line or with near-UV LED sources. In this case the ligand is continually released during the period of illumination.

Calibration of photolysis

Photolysis of the caged fluorophore NPE-HPTS generates a fluorophore, HPTS (pyranine) with excitation and emission similar to FITC. The fluorescence released is readily measured by photometry or fluorescence imaging. The photolysis efficiency of NPE-HPTS has been compared with that of several 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

Xenon flashlamps produce 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).

Coupling of light sources is most often through the epifluorescence condenser in both upright and inverted microscope configurations. Light is attenuated below 350 nm by glass lenses, so silica optics are often used. Microscope objectives transmit 40-50% in the near UV and generally not at all below about 320 nm. A set of suitable dichroic reflectors is needed if experiments require simultaneous fluorescence excitation and detection. These should pass or exclude, as appropriate, the wavelengths for photolysis. It is usually the case that the dichroic reflectors for e.g. FITC or eGFP in commercial microscopes do not reflect near-UV and need to be replaced for photolysis. Chroma part T490DC works well for blue excitation/green emission. Fluorescence excitation filters will not transmit photolysis light and should be installed in the lamphouse not the microscope filter cube.

The couplings from flashlamp to microscope are often a silica multimode fiber or liquid light guide. This has the advantage of removing higher trigger voltage and large (100 Amp) current from the vicinity of the electrophysiological recording, and is mechanically simpler but optically less efficient. The efficiency of cages is not high enough to allow localization of flashlamp light to a small spot, flashlamp photolysis is usually carried out over a full field of diameter about 200 μm. Flashlamps and couplings are available from suppliers such as Cairn Research, Rapp Optoelectronic and TILL Photonics.

LED Sources

Near-UV LEDs with sufficient power for photolysis by long - 100 ms - exposures are available. These are less expensive and more easily controlled than flashlamps but do not have enough energy in brief pulses for fast kinetic studies.

Lasers - localized point or scanning photolysis

A small, diffraction limited spot with sub-micron dimensions is readily generated by an expanded collimated laser beam directed into the back of a microscope objective. Easily controlled diode or DPSS lasers with adequate power, analog modulation of power and exposure, true zero light when OFF and good beam quality are available at wavelengths 355 nm (pulsed) 380 nm and 405 nm (CW). Generally CW is preferred over pulsed ('quasi-CW') to avoid toxicity of high peak powers. Best beam quality is achieved by single-mode fiber coupling. Suitable lasers are available from suppliers such as Point Source, Omicron, Toptica and DPSS Laser.

Extracellular photolysis

Light loss by inner filtering in the cage solution

With extracellular photolysis on upright microscopes, water dipping objectives are generally used. At cage concentrations greater than 0.5 mM significant losses occur due to absorption of near-UV light in the 2-3 mm of solution between objective and cell. This substantially reduces uncaging efficiency at the peak wavelengths 340-360 nm but can be overcome by working at longer wavelengths where light absorption is reduced, e.g. at 405 nm; the lower photolysis efficiency is compensated by increasing the laser power.

Intracellular photolysis

The first successful uses of flash photolysis were to study activation of intracellular processes such as ATP-dependent reactions and Ca²⁺ dependent processes. The receptors are normally inaccessible and access was by permeabilization of the cell membrane. More recently access of the cage to the receptor compartment is achieved by perfusion with whole cell patch clamp, or by microinjection. Fluorescent dyes are often used in conjunction with photolysis to monitor access to the cytosol and the intracellular Ca²⁺ concentration.

Two-photon photolysis

The inner filtering effect discussed above can also be avoided with two-photon photolysis, but with greater susceptibility to phototoxicity and much more expensively. The only potential advantage of two-photon photolysis is the deeper penetration of two-photon excitation due to the inability of scattered IR photons to excite the cage alone. However the poor two-photon excitation of current cages largely negates this. Efficiencies in two-photon excitation are much lower than for near-UV one-photon excitation for all cages currently available. Generally two-photon photolysis cross sections >1 GM are required, whereas currently available cages are <0.1 GM. As a consequence very high cage concentrations are required, increasing the likelihood of interference by the cage. Furthermore, in order to maintain localization of release to the excitation volume the rate of photolysis 'dark' reactions needs to be fast relative to diffusion (>10,000 s^-1). The optimal exposures are brief, similar 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 used to study compartmentalization by measuring diffusion following point release, although the near-UV photolysis is relatively slow at pH7, half-time 10-20 ms, with two-photon excitation the rate of release was found to be much faster (>3000 s^-1), suitable for diffusional studies in small compartments. NPE-HPTS is routinely used for photolysis calibration.

NPEC-DHPG and NPEC-ACPD

The NPEC cages are stable to hydrolysis, unlike many caged neuroactive amino acids that utilize nitrobenzyl or nitrophenyl photochemistry. They are efficient for near-UV photolysis but have slower photolysis, with 'dark' rates about 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. Both activate mGluR1 efficiently and neither interfere with glutamatergic transmission prior to photolysis.