Abstract
Purpose: :
Common diseases of the ocular surface such as infection, allergy or dry-eye all lead to an inflammation that is clinically often difficult to differentiate. In addition, experimental studies on ocular surface inflammation in animals require tissue probing and further processing to characterize the type of inflammation. A non-invasive imaging technique is therefore desirable for both clinical diagnosis and intravital experiments. This study was set up to evaluate the use of multidimensional two-photon-microscopy for identifying optical features of key players in ocular surface inflammation such as immune cells, epithelium, connective tissue, blood and lymphatic vessels.
Methods: :
A modified two-photon microscope (DermaInspect) was used. Anesthetized Balb/c-mice were placed under the microscope and investigated in-vivo using excitation wavelengths from 710-850nm. In adition cell suspensions separated by MACS or FACS were analyzed in-vitro. Detection of autofluorescence intensity and spectrum was combined with fluorescence lifetime measurements (FLIM) and Second Harmonic Generation (SHG). Multidimensional data sets were analyzed with Imaris Software.
Results: :
Lymphocytes, macrophages, epithelial cells, goblet cells, collagen fibrils, elastic fibres and erythrocytes were differentiated by unique optical features based on intracellular fluorophores or structural characteristics. Autofluorescence spectra of epithelial cells differ markedly from macrophages and erythrocytes. FLIM enables further differentiation of macrophages from erythrocytes and lymphocytes. Excitation wavelengths of 710-730nm enable visualization of elastic fibres whereas imaging at 800-850nm induces SHG of collagen fibrils.
Conclusions: :
Multidimensional two-photon microscopy enables optical fingerprinting of cells and tissue structures involved in inflammatory processes of the ocular surface without the necessity of tissue probing and the use of artificial dyes. This approach may be used in intravital animal studies to enable repeated non-invasive long term investigations. In future, a clinical use could enable a more precise characterization and grading of ocular surface inflammation.
Keywords: microscopy: light/fluorescence/immunohistochemistry • inflammation • conjunctiva