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URMC / Labs / Brown Lab / Projects / In vivo Multiphoton Fluorescence Recovery After Photobleaching

In vivo Multiphoton Fluorescence Recovery After Photobleaching

Project Collaborators:

Dr. Mitchell Anthamatten, Dr. Edward Brown (Manhattan College)

Computer generated fluorescence recovery curves

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 (MP-FRAP) 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 MP-FRAP lies in its ability to probe thick tissue with 3D resolution. We have been developing improvements to the MP-FRAP technique that will broaden its already substantial applicability in vivo. Recently, we published an article introducing an alternative mathematical recovery model that accounts for the presence of directed flow in a region where diffusion measurements are desired.

This alternative model is especially applicable in leaky interstitial regions, and can be used to measure plasma viscosity, a common gauge of several disease states. Currently, we are probing the effects on photobleaching recovery when MP-FRAP is performed in a constrained environment. We are also working in collaboration with Mitchell Anthamatten (Chemical Engineering) to apply MP-FRAP to a novel question in the field of polymer-structure relationships.

Representative diffusion-only MP-FRAP recovery curve

Representative diffusion-only MP-FRAP
recovery curve for FITC-BSA and associated
least-squares fit.

Representative MP-FRAP recovery curve for FITC-BSA

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.

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