Christopher James Lloyd

Optic neuropathy in methylmalonic acidemia and propionic acidemia

Background Methylmalonic acidemia (MMA) and propionic acidemia (PA) are rare hereditary disorders of protein metabolism, manifesting early in life with ketoacidosis and encephalopathy and often resulting in chronic complications. Optic neuropathy (ON) has been increasingly recognised in both conditions, mostly through isolated case reports or small cases series. We here report the clinical features and visual outcomes of a case series of paediatric patients with a diagnosis of MMA or PA. Methods Retrospective observational case series. A database of patients attending the Willink Biochemical Genetics unit in Manchester was interrogated. Fifty-three patients had a diagnosis of either isolated MMA or PA, of which 12 had been referred for ophthalmic review. Results Seven patients had clinical findings compatible with ON. Visual outcomes in these patients were poor, with slow clinical progression or stability over time in five cases with follow-up. Presentation was acute in a context of metabolic crisis in two of the cases. Four patients with ON had electrodiagnostics showing absent pattern evoked potentials, with one showing a preserved flash response. All four showed marked attenuation of the dark-adapted electroretinogram with better preservation of the light-adapted response. Conclusions Our study suggests that ON is under-reported in patients with MMA and PA. Clinical presentation can be acute or insidious, and episodes of acute metabolic decompensation appear to trigger visual loss. Photoreceptor involvement may coexist. Active clinical surveillance of affected patients is important as comorbidities and cognitive impairment may delay diagnosis.

Quantitative fluorescence microscopy of macromolecules in gel and biological tissue

Quantitative fluorescence microscopy provides valuable insight into drug delivery and pharmacokinetics. The technique is based on analysis of statistical fluctuations in fluorescence that arises as fluorophores pass through a small volume illuminated by a focused laser beam, and has been applied to measure particle motion and binding interactions in solutions, on surfaces and inside the cells. We examined the use of fluorescence correlation spectroscopy combined with a microscope (FCSM) to assess the transport of fluorescent beads and macromolecules in aqueous solutions, gels and living biological tissue. Obstructed diffusion of fluorescent beads in gels of various densities was tested to get a sensible estimate of diffusion in the interstitial tissue matrix consistent with previous reports. Fluorescently labelled liposomes as an artificial drug or gene carrying vehicles were used for pharmacokinetic tests of drug delivery in living tissue. The results indicate that FCS is an accurate and valuable tool for measuring the physical properties of gene vectors in vitro and for characterizing interactions with tissue in vivo.

Temporal autocorrelation function for a diffusing-wave spectroscopy experiment with a point source and backscattering detection

The autocorrelation function of the backscattered intensity in a diffusing-wave spectroscopy experiment that uses a point source is calculated by use of the diffusive-wave model. We show that in this approximation the calculated autocorrelation function decays faster than if the plane-source approximation were used. The design of a probe that implements this geometry is presented together with preliminary results that show the utility of the probe as a sizing tool in concentrated dispersions.

Two-photon fluorescence correlation microscopy for biophysical studies

Two-photon correlation spectroscopy enables the deeper insight into the living tissue in comparison to single photon spectroscopy and visualize the biophysical processes occurring there. We present the basic features of an experimental set up based on a research Nikon microscopy and preliminary experimental results of suitable dye with two- photon excitation for pharmacokinetic studies. Despite some technical problems the proposed design differs by its simplicity, requires relatively low-cost optics and has a reasonably low dispersion on the optical elements.

Application of fluorescence correlation spectroscopy for drug delivery to tumor tissue

Quantitative fluorescence microscopy methods can provide valuable insight into drug delivery and pharmacokinetics. We are investigating the use of single photon fluorescence correlation spectroscopy (FCS) to measure particle concentration and mobility in living tissue. In this study we examined whether a relatively large illumination volume could be used to probe the state of macromolecules in free solution and in tissue. The FCS set-up is based upon an upright research microscope, diode laser with 635 nm wavelength, an avalanche photodiode/single photon counting module, and PC based correlation electronics. Diffusion coefficients were e4xtracted from measured autocorrelation functions. We used fluorescent monodisperse beads with diameter 20 and 200 nm to calibrate the excitation volume. Particle diffusion coefficients measured by FCS were compared with conventional light-scattering measurements. We then applied the technique to measure fluorescently labeled liposome distribution in tissue and tissue models. We found that the difference in quantum brightness and diffusion times of liposomes and free dye may be used to detect changes due to liposome interaction with living cancer cells.