The in vitro orthogonal photolysis of the caged compounds was performed in the
Department of Pharmacology, University of Oxford, using a commercially avail- able inverted microscope (IX71, Olympus) incorporating a high numerical aperture (N.A) oil immersion objective (UPlanSApo 100x/1.4, Olympus) and a custom-built
Figure 5.11: Partial NMR spectra reveal the irradiation at 405 nm (a & b) provides selective photolysis of DMNB-GABA (*) to give free GABA (O) in the presence of MPA- Glu (`) which remains unchanged. Irradation at 285 nm (c & d) provides selective photolysis of MPA-Glu (`) in the presence of DMNB-GABA which remains unchanged. Generation of a photolysis by-product (a) was observed observed (c). Spiking the sample with free GABA (d) confirms that no free GABA is formed following irradiation at this wavelength. Figure modified from [1].
Figure 5.12: Quantification as discussed in chapter three of the preliminary photolysis study provided a greater understanding of the selective release of GABA from an equimolar solution of DMNB GABA and MPA glutamate. Figure modified from [1] supplemental information.
electrophysiology stage.
Biological samples were held in a perfusion chamber which provided the neces- sary physiological environment to maintain biological viability for the duration of the experiment while permitting access for the high N.A objective and electrophys- iological equipment. As illustrated in figure 5.13 the microscope was adapted by myself to permit selective orthogonal photolysis of the caged compounds in solution by means of two independent light sources. Selective photolysis of the MPA-Glu was obtained with a mercury flash lamp (JML-C1, Rapp Optoelectronic) in combination with a 255 ±8 nm interference bandpass filter (250FIB25, Knight Optical) mounted externally to the microscope assembly and focused to illuminate the entire sample region in the sample plane by an internal focusing lens. Orthogonal photolysis of the DMNB-GABA was accomplished with a 405 nm CW UV laser diode (PxLS, Toptica photonics) emitting 30 mW. The laser was co-aligned to the optical path of the microscope through the camera port of the microscope utilising a custom built optical cage plate system (Thorlabs). The optical system collimated the output of the diode laser and expanded the beam to match the back aperture of the high N.A objective. The external optics were designed to achieve efficient coupling of the laser into the microscope and comprised a x2 telescope for initial beam expan-
Figure 5.13: The orthogonal wavelength photolysis was performed in a commercial in- verted microscope (Olympus) modified with a 405 nm laser diode (Toptica) coupled to the sample plane and an external UV flashlamp (Rapp Optoelectronic) with a 255 ±8 nm in- terference bandpass filter (Knight Optical) mounted and focused upon the sample plane. In this way the sample plane could be selectively illuminated with either 405 or 255 nm. sion and collimation and a second x1.3 expansion telescope to ensure that the back aperture of the microscope objective was fully filled. A dichroic mirror (530 BK 16, Comar) permitted the introduction of the photolysis beam without interrupt- ing image capture with an electron multiplying charge coupled device (EMCCD) camera (iXon DV887DCS-BV, Andor technology). A mechanical shutter (Uniblitz VS25, Rochester) provided temporal control of the 405 nm beam. The arrangement provided delivery of both wavelengths at the sample plane of the microscope with good control over exposure of the sample.
5.5
In vitro
photorelease of wavelength orthogo-
nal neurotransmitters
My collaborators in the Emptage group at the University of Oxford applied the orthogonally caged compounds to hippocampal pyramidal neurons whose response to photolysis was monitored by whole cell patch clamp recording as illustrated in figure 5.14 . Photolysis experiments were conducted in the commercial upright microscope which I had modified to provide 255 ± 8 nm and 405 nm illumination at the sample plane illustrated in figure 5.13.
Figure 5.14: In vitro application of the technique demonstrated full selective orthogonality between the two compounds descriminated by wavelength. Figures a-d show representative whole-cell patch-clamp recordings of cultured hippocampal pyramidal neurons bathed in a solution containing MPA-Glu (125 μM) and DMNB-GABA (50 μM). DMNB GABA only ilicits a response when illuminated by 405 nm (a) and MPA glutamate only ilicits a response when illuminated under 255 ± 8 nm (b). In a mixture of the compounds one compound can be preferentially activated in the presence of the other using wavelegth to discriminate the selection (c & d). Figure modified form [1].
Initially the compounds were applied individually, it was shown that thein vitro
application and illumination of application of DMNB GABA compound (50μM) at 255 ± 8nm evoked no response whereas exposure to 405 nm generated the expected response (a). The observe large anion current generated upon irradiation at 405 nm is interpreted as photolysis of the DMNB compound, releasing free GABA and subsequent receptor activation. This behaviour was found typical in nine different cells a total of twenty two times.
It was then demonstrated that in vitro applicaiton and illumination of the the
MPA glutamate (125 μM) at 255 ± 8nm generates a response, whereas none is ob- served at 405 nm (b). The observed large cation current response to illumination at 255 ± 8nm is interpreted as photolysis of the MPA glutamate compound, free glutamate generation and subsequent receptor activation. This behaviour was ob- served in nine different cells a total of twenty seven times. This selective wavelength photolysis of both compounds individually established the potential for wavelength selective orthogonal photolysisin vitro.
receptor activation and subsequent 255 ± 8nm elicited glutamate receptor activation (d). It was therefore shown for the first time in vitro, that controllable orthogonal
wavelength-selective photolysis was successfully achieved [1].