Optimizing Laser Parameters for Pump-Probe Microscopy of Conjunctival Melanocytic Lesions

Abstract

 

Purpose

 

Previously, we reported using pump-probe microscopy to characterize microscopic spatial variations in melanin pigment expression in 8 conjunctival melanocytic lesions. In preparation for examining a larger data set to draw statistically meaningful conclusions, we seek to find optimal wavelengths (previously 720 nm was used) for using this imaging method to draw out spatial heterogeneity in these lesions.

 

Methods

 

A conjunctival melanoma specimen that exhibited striking spatial variations in pigment chemistry was selected. An unstained 10 μm section was analyzed with pump-probe microscopy. This technique involves a laser scanning microscope with a two-color pulsed laser source to distinguish melanin pigment components based on differences in excited state photodynamics. The specimen was imaged repeatedly at different pump wavelengths. The responses from these stacks were compiled and underwent principal component analysis, which identifies an orthogonal set of signals that best account for the variations in the data. Specimen images were colored according to the top 2 principal components, and compared for contrast between the two. At the optimal wavelength, additional classes of conjunctival lesions were imaged.

 

Results

 

2 components were identified that accounted for most of the variations in the pump-probe signal (Figure 1a). The spatial heterogeneity in the pigment content is most visible at 730 nm (Figure 1b,c). Imaging results from the additional classes of conjunctival melanocytic lesions are shown in Figure 2.

 

Conclusions

 

Previously we found that conjunctival melanomas, compared with benign nevi and PAMs, exhibit more spatial heterogeneity in their melanin pigment composition. Here, we find that imaging conjunctival melanocytic specimens with pump wavelength of 730 nm allows for the most differentiation of the two main pigment components. This setting is most ideal for identifying spatial variation of melanin pigment components.

 

 

Figure 1. A) Principal component analysis of imaging data. B) Unstained specimen showing area imaged by pump-probe laser. C) Specimens colored according to principal components (red and green).

 

 

Figure 2. Imaging results of other conjunctival melanocytic lesions.

 

Keywords: 744 tumors • 474 conjunctiva • 550 imaging/image analysis: clinical