Rapid Frequency-Domain FLIM Spinning Disk Confocal Microscope: Lifetime Resolution, Image Improvement and Wavelet Analysis

Abstract

A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples.

Supplementary data

In the case of the conventional wide field frequency-domain lifetime imaging, the homodyne signal recorded at the image intensifier S _ave (Eq. 4 of text) is derived, following Schneider et al. [36] as

$$S_{{\text{avg}}} = \left\langle {F\left( t \right) \cdot D(t)} \right\rangle = \frac{1}{T}\int\limits_0^T {\left( {F_0 + F \cdot \cos \left( {\omega t - \Phi _E - \Phi _F } \right)} \right) \cdot \left( {D_0 + D \cdot \cos \left( {\omega t - \Phi _D } \right)} \right)\operatorname{d} t} $$

(A1)

$$ S_{avg} = \frac{1} {T}\left[ {\int\limits_0^T {F_0 \cdot D_0 dt} + \frac{1} {2}\int\limits_0^T {F \cdot D \cdot \cos \left( {\Delta \Phi _{DE} - \Phi _F } \right)dt} \frac{1} {2}\int\limits_0^T {F \cdot D \cdot \cos \left( {2\omega t - \Delta \Phi _{DE} - \Phi _F } \right)dt} + \int\limits_0^T {F_0 \cdot D \cdot \cos \left( {\omega t - \Phi _D } \right)dt} + \int\limits_0^T {D_0 \cdot F \cdot \cos \left( {\omega t - \Phi _E - \Phi _F } \right)dt} } \right] $$

(A2)

When T is large compared with 1 / ω, as in our case, the last three terms in Eq. A2 vanish due to averaging. Therefore,

$$ S_{avg} = F_0 \cdot D_0 + \frac{{F \cdot D}} {2} \cdot \cos \left( {\Delta \Phi _{DE} - \Phi _F } \right) = S_0 \left( {1 + \frac{M} {2} \cdot \cos \left( {\Delta \Phi _{DE} - \Phi _F } \right)} \right) $$

(A3)

In the case of the spinning disk confocal FLIM, the fluorescence signal emitted is switching between the bright period and the dark period and can be written as in Eq. A4

$$F\left( t \right) = \left\{ {\matrix{ {F_0 + F \cdot \cos \left( {\omega t - \Phi _E - \Phi _F } \right)when} & {n \cdot T_D \le t \le n \cdot T_D + T_B ;{\rm{ }}n = 0,1, \ldots ,T/\left( {T_D + T_B } \right)} \cr 0 & {otherwise} \cr } } \right.$$

(A4)

By putting Eq. A4 back into Eq. A1 and carrying out the calculation proves that this switching behavior reduces the total signal collected by the image intensifier but does not affect the final form of the homodyne signal, i.e. Eq. A3 above is still valid.