Imaging microscopic pigment chemistry in conjunctival melanocytic lesions using pump-probe laser microscopy.
OBJECTIVE
To report the application of a novel imaging technique, pump-probe microscopy, to analyze patterns of pigment chemistry of conjunctival melanocytic lesion biopsies.


METHODS
Histopathologic specimens of eight previously excised conjunctival melanocytic lesions were analyzed with pump-probe microscopy. The technique uses a laser scanning microscope with a two-color pulsed laser source to distinguish hemoglobin, eumelanin, and pheomelanin pigment based on differences in transient excited state and ground state photodynamics. The pump-probe signatures of conjunctival melanins were compared with cutaneous melanins. The distributions of hemoglobin, eumelanin, and pheomelanin were analyzed, and pump-probe images were correlated with adjacent hematoxylin and eosin (H&E)-stained sections.


RESULTS
The pump-probe signatures of conjunctival melanins are similar, but not identical to cutaneous melanins. In addition, there are qualitative and quantitative differences in the structure and pigment chemistry of conjunctival benign nevi, primary acquired melanosis of the conjunctiva (PAM), and conjunctival melanomas. The pump-probe images correlated well with histopathologic features observed in the adjacent H&E-stained sections, and provided a label-free means of discerning conjunctival anatomic features and pathologic benign or malignant tissue.


CONCLUSIONS
Pump-probe laser microscopy shows promise as an adjuvant diagnostic tool in evaluation of ocular melanocytic lesions based on morphologic correlation with the histopathology results and pigment chemistry. This initial study suggests systematic differences in pigmentation patterns among conjunctival benign nevi, primary acquired melanosis, and melanomas. In addition, pump-probe microscopy has the potential for use as a noninvasive "in vivo" optical biopsy technique to aid clinical and surgical management of conjunctival melanocytic lesions.
Department of Chemistry, Duke University, Durham, North Carolina.
Jesse W
JW
Wilson
Lejla
L
Vajzovic
Francisco E
FE
Robles
Thomas J
TJ
Cummings
Prithvi
P
Mruthyunjaya
Warren S
WS
Warren
D003230:Conjunctival Neoplasms / Q000737:chemistry* / Q000175:diagnosis; D006454:Hemoglobins / Q000032:analysis*; D006801:Humans; D007834:Lasers; D008543:Melanins / Q000032:analysis*; D008545:Melanoma / Q000737:chemistry* / Q000175:diagnosis; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton
D006454:Hemoglobins; D008543:Melanins
benign conjunctival nevus; conjunctival melanoma; multiphoton microscopy; primary acquired melanosis; pump-probe microscopy