Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues

Although histochemical and immunohistochemical methods are the standard pro cedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the intro duction of tissue autofluorescence analyses. We used microspectrofluorometr y and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of diffe rent origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interferenc e filters (50 mm bandwidth) peaked at 450, 550 and 658 mm. Monochrome image s were combined together in a single red-green-blue color image. Most of th e fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as fro m reduced form of intracellular nicotinamide adenine dinucleotide (phosphat e). Autofluorescence imaging shows morphological differences between neopla stic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated b y high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, IMAM permits to d iscriminate between non-neoplastic and neoplastic tissues on the basis of t heir autofluorescence pattern. Multispectral imaging of tissue autofluoresc ence may present some advantages with respect to standard histochemical mic roscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon speci men resection; and (3) supplies a representation of the biological structur e organization linked to endogenous fluorophores.