Methods

Methods

Direct r(t) diagnostic

Background-corrected anisotropy traces and registered, neighbourhood-pooled maps.

This is a diagnostic only. Division and IRF convolution do not commute, so the trace is not used to estimate a rotational correlation time.

Global polarized-decay fit

The preferred method, following the channel equations and direct global analysis in Lakowicz, chapter 11, section 11.2.2.

It jointly models the raw parallel and perpendicular photon decays with separate measured IRFs, a fixed known fluorescence lifetime, fixed G and relative exposure factors, periodic excitation, one rotational correlation time, a common IRF timing shift, and separate channel backgrounds. Residuals are fitted on Poisson deviance, and a parameter that reaches its bound is reported rather than accepted silently.