In vivo Multiphoton Fluorescence Recovery After Photobleaching
Project Collaborators:
Computer generated fluorescence recovery curves
with varying amounts convective flow, relative
to a common diffusion coefficient. The curve of
red squares has no convective flow, that with blue
diamonds has a relatively moderate amount of flow,
and the curve of green circles is flow- dominated.
As the rate of flow increases the recovery speeds
up and changes shape, becoming almost sigmoidal
for dominating levels of flow.
Multi-fluorescence recovery after photobleaching (MPFRAP) is a microscopy technique used to measure the diffusion coefficient of fluorescently tagged macromolecules, and can be applied to both in vitro and in vivo biological systems. The power of MPFRAP lies in its ability to probe thick tissue with 3D resolution. We have been developing improvements to the MPFRAP technique that will broaden its already substantial applicability in vivo, including in the presence of flow, shear flow, and nearby barriers to diffusion. Currently we are implementing a variant of MPFRAP with deliberately decreased resolution, to enable us to average over obstacles to diffusion such as cell walls and neuronal processes. This will allow us to study the diffusive landscape in the brain.
Representative diffusion-only MP-FRAP
recovery curve for FITC-BSA and associated
least-squares fit.
Representative MP-FRAP recovery curve
for FITC-BSA in the presence of flow, and
associated least-squares fits. The solid
line represents the fit made using the
standard diffusion-only recovery model,
and clearly misfits the data. The dashed
line represents the fit made using the
diffusion-convection model we recently
developed, and fits the data beautifully.