When monitoring fast kinetic changes using microfluorescence or electrophysiology it is often important to avoid the time lag and mixing artefacts associated with conventional perfusion systems or the direct application of reagents. One useful approach is to introduce protected “caged” reagents in advance and then to photolytically release them using a brief and intense UV flash. This offers the combined benefits of near-instantaneous introduction of compounds, improved homogeneity and easy synchronisation with recording equipment.

The key elements of a flash photolysis system are described below:

The energy required to cleave caged compounds is usually provided by a xenon arc flash lamp or a UV laser. In both cases the the source should be capable of producing highly energetic pulses in the region of 1 msec in duration and must have the facility to be externally triggered using a TTL trigger pulse. The light source would typically be optically filtered with a broadband UV transmitting filter.

The photolysis light is usually introduced to the sample through a high Numerical Aperture (NA) microscope objective via an epifluorescence port. If combining photolysis with fluorescence measurements then a dual port coupling is required so that both the the flash and continuous light can be introduced via the same route. In such cases a dichroic beamsplitter would typically be used to combine ultraviolet flash light with visible fluorescence excitation. A useful option here is to have a framing aperture so that the area of flash irradiation can be clearly defined independently of the continuous fluorescence light.

Alternatively a small silica fibre or quartz lens system can be used to apply the flash light directly to the specimen.

Flash photolysis is usually combined with microfluorescence or electrophysiological recordings and consequently most systems will be mounted onto research grade epifluorescence microscopes. The main consideration when using photolysis is that the epifluorescence port and objective are optically effiecient and have high ultraviolet transmission.

Flash photolysis light is usually diverted into the objective lens using a dichroic mirror at 45 degrees immediately prior to the objective. The dichroic mirror reflects the shorter wavelength light towards the sample and passes the longer wavelength fluorescence to the detector. When using photolysis it is usually necessary to use specialist dichroic mirrors with extended reflectance in the ultraviolet.