Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas

Abstract

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm(2) is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425-515 nm spectral emission) to 39.8% (620-655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.

Figure 1. Multispectral fluorescence intensity and FLIM images acquired from normal skin and BCCs.

(a–i) Fluorescence intensity and false color FLIM images from a single field of view acquired at a depth of 110 µm with all spectral channels taken near a dermal papilla from normal skin. (j–n) FLIM images taken from the green channel only of different depths within a sample of normal skin. (o–u) FLIM images taken from the green channel illustrating visual architectural features seen in BCC using MPT. (v) FLIM image taken from the blue channel of a BCC. (w,x) paired FLIM images taken from the green and blue channels respectively of a BCC nest. KEY SG-Stratum Granulosum, SS- Stratum Spinosum, BCL-Basal Cell Layer, DP-Dermal Papilla. Scale bar 25 µm.

Figure 2. Histograms of morphology and fluorescence lifetime parameters for cells/ROIs in normal skin and BCCs.

(a–c) Histograms of cellular morphology features demonstrating the difference between normal skin (coded Blue) and BCCs (Red) (d–i) Histograms of τ₁, τ₂, spectral contribution for all spectral channels for BCC and normal skin. Curves are color-coded according to channel “colors”.

Figure 3. Exemplar segmented fluorescence intensity images, fitted fluorescence decay and fluorophore emission spectra.

(a) Total fluorescence intensity image, same image with (b) manually and (c) automatically defined cellular regions of interest overlayed. (d) Top – exemplar fluorescence decay from one region of interest (black), biexponential fit to data (green) and instrument response function (blue). (d) Bottom – normalized residuals. (e) The emission spectra from endogenous fluorophores plotted in relation to the four spectral detection channels.

Figure 4. False color FLIM image from the green channel of a BCC consisting of 12×8 fields of view covering an area of 1.86×1.24 mm².

Bar 0.2 mm.