Label-Free Delineation of Human Uveal Melanoma Infiltration With Pump-Probe Microscopy

Abstract

Uveal melanoma (UM) is the most frequent primary intraocular malignancy in adults, characterized by melanin depositions in melanocytes located in the uveal tract in the eyes. Differentiation of melanin species (eumelanin and pheomelanin) is crucial in the diagnosis and management of UM, yet it remains inaccessible for conventional histology. Here, we report that femtosecond time-resolved pump-probe microscopy could provide label-free and chemical-specific detection of melanin species in human UM based on their distinct transient relaxation dynamics at the subpicosecond timescale. The method is capable of delineating the interface between melanoma and paracancerous regions on various tissue conditions, including frozen sections, paraffin sections, and fresh tissues. Moreover, transcriptome sequencing was conducted to confirm the active eumelanin synthesis in UM. Our results may hold potential for sensitive detection of tumor boundaries and biomedical research on melanin metabolism in UM.

Keywords: label-free imaging; melanin; multiphoton microscopy; pump-probe microscopy; uveal melanoma.

Figure 1

Experimental design and transient optical behaviors of melanin in uveal melanoma (UM). (A) Optical transitions of pump-probe processes. (B) Optical layout of the pump-probe microscope. EOM, electro-optic modulator; DM. dichroic mirror; τ, interpulse delay between pump and probe; GM, galvo mirror; BP, band-pass filter; PD, photodiode; LIA, lock-in amplifier; ESA, Excited-State-Absorption; GSB, Ground-state bleach; Fs, femtosecond; ps, picosecond. (C, D)Typical pump-probe images of cellular debris excised from melanoma and paracancerous tissues, taken at 0 and 500 fs interpulse delays. (E) Time-resolved transient absorption (TA) dynamics of the two melanin species measured at the representative areas 1 and 2 [arrowheads in panels (C, D)]. Scale bar: 20 μm.

Figure 2

Differentiation of melanoma and paracancerous regions with pump-probe microscopy on frozen tissue sections. (A) Pump-probe images and corresponding H&E staining of a whole tissue section. (B) Magnified images in panel (A) (dashed rectangle) with the melanoma/paracancer interface defined by melanin species distribution, which agrees with the histological interface found in the adjacent H&E section. (C) Magnified paracancerous region [arrowhead in panel (A)] demonstrated predominantly pheomelanin. Red: eumelanin; cyan: pheomelanin. Scale bar: 50 μm.

Figure 3

Pump-probe imaging of paraffin-embedded sections. (A) A normal choroid tissue section. (B) A melanoma tissue section excised from a uveal melanoma (UM) patient. (C) A tissue section from the interface area with clear eumelanin/pheomelanin contrast. (D) Magnified view of the interface (dashed square in panel C), eumelanin (asterisk) and pheomelanin (arrowhead) are hardly resolvable in H&E. Red: eumelanin; cyan: pheomelanin. Scale bar: 50 μm.

Figure 4

Pump-probe imaging of fresh human tissues. (A) Large-area view of a fresh uveal melanoma (UM) tissue. (B) Normal choroid with abundant pheomelanin. (C) The interface between paracancerous regions and melanoma (dashed line). (D) A UM case showing heterogeneous eumelanin distribution. (E) Magnified view of the eumelanin deposition, while the melanocytes are rich in pheomelanin. Red: eumelanin; cyan: pheomelanin. Scale bar: 50 μm.

Figure 5

Imaging melanoma cell lines. (A) Pump-probe imaging of B16 cells. (B) Transient absorption dynamics of intracellular pheomelanin (a) and eumelanin (b). (C) Pump-probe imaging of B16 cells. (D) Transient absorption dynamics of the melanin species. Red: eumelanin; cyan: pheomelanin. Scale bar: 10 μm.

Figure 6

Differential analysis of gene expression profiles in uveal melanoma (UM) and paracancerous tissues. (A) Cluster heatmap of total differentially expressed genes shown in heatmap graph with q value <0.05 and |log₂Foldchange >1| in 3 UM samples and the corresponding paracancerous tissues. (B) The melanogenesis-related genes (including MLANA, OCA2, PAX3, PMEL, MLPH) were picked from the cluster analysis in panel (A), showing the highly expressed genes involved in melanin synthesis in UM specimens; the q values of these genes were 0.035 for MLANA, 0.013 for OCA2, 0.013 for PAX3, 0.027 for PMEL, and 0.021 for MLPH. (C) Gene enrichment analysis. The Y-axis in the figure corresponds to the functions or the pathways. The X-axis corresponds to the ratio of differential genes in a specific pathway to all genes contained in the pathway; the larger the value, the higher the percentage of differential genes in the pathway. The dots on the bubble chart indicate the number of differential genes; larger dots indicate more specific differential genes in the pathway.