Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.
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.
Ultrafast pump–probe measurements
can discriminate the two forms of melanin found in biological tissue
(eumelanin and pheomelanin), which may be useful for diagnosing and
grading melanoma. However, recent work has shown that bound iron content
changes eumelanin’s pump–probe response, making it more
similar to that of pheomelanin. Here we record the pump–probe
response of these melanins at a wider range of wavelengths than previous
work and show that with shorter pump wavelengths the response crosses
over from being dominated by ground-state bleaching to being dominated
by excited-state absorption. The crossover wavelength is different
for each type of melanin. In our analysis, we found that the mechanism
by which iron modifies eumelanin’s pump–probe response
cannot be attributed to Raman resonances or differences in melanin
aggregation and is more likely caused by iron acting to broaden the
unit spectra of individual chromophores in the heterogeneous melanin
aggregate. We analyze the dependence on optical intensity, finding
that iron-loaded eumelanin undergoes irreversible changes to the pump–probe
response after intense laser exposure. Simultaneously acquired fluorescence
data suggest that the previously reported “activation”
of eumelanin fluorescence may be caused in part by the dissociation
of metal ions or the selective degradation of iron-containing melanin.
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