Abstract
Pump-probe microscopy provides molecular information by probing transient, excited state dynamic properties of pigmented samples. Analysis of the transient response is typically conducted using principal component analysis or multi-exponential fitting, however these methods are not always practical or feasible. Here, we show an adaptation of phasor analysis to provide an intuitive, robust, and efficient method for analyzing and displaying pump-probe images, thereby alleviating some of the challenges associated with differentiating multiple pigments. A theoretical treatment is given to understand how the complex transient signals map onto the phasor plot. Analyses of cutaneous and ocular pigmented tissue samples, as well as historical pigments in art demonstrate the utility of this approach.
Highlights
IntroductionNonlinear pump-probe microscopy ( known as transient absorption microscopy) is an emerging technique that achieves high molecular specificity of absorptive pigments [1,2]
Nonlinear pump-probe microscopy is an emerging technique that achieves high molecular specificity of absorptive pigments [1,2]
If the data are analyzed using orthogonal projections, such as in principal component analysis (PCA), some contributions from Hb will typically be projected onto eumelanin, and some contributions from surgical ink will be projected onto pheomelanin
Summary
Nonlinear pump-probe microscopy ( known as transient absorption microscopy) is an emerging technique that achieves high molecular specificity of absorptive pigments [1,2]. Pump-probe microscopy signals (acquired as a function of the time delay between the pump and probe pulses) display positive and negative (i.e., bipolar) multi-exponential dynamics resulting from a broad range of physical mechanisms. This rich structure provides the sensitive molecular specificity of the method. In FLIM, these challenges have been addressed by using multi-exponential fitting; but this requires high signal to noise ratios for reliable separation of components [11], and a priori knowledge of the number of exponentials These requirements are often not met in pump-probe microscopy, and multi-exponential fitting is not always a feasible solution. We demonstrate applications to pigment analysis of melanoma biopsies and historical artwork
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