Multispectral Imaging Autofluorescence Microscopy Research Articles

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Lymphadenectomy and extended lymphadenectomy are still controversial issues in surgical oncology. The detection methods for metastatic lymph nodes include lymphoscintigraphy and radiolabelled antibody, but immunohistochemical sentinel lymph node (SLN) identification is commonly used. The potential diagnostic use of cell and tissue autofluorescence (AF) is well known. Here we review our studies on the application of AF-based techniques for diagnosing metastatic lymph nodes. We had previously demonstrated that AF imaging allows discrimination between hyperplastic and primary neoplastic lymph nodes. Our more recent studies show that the combination of autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy can be applied to the diagnosis of secondary neoplastic lymph nodes in gastric and colorectal cancer. These techniques have been validated by histochemical and immunohistochemical methods. Studies are in progress to select the best dye tracer for SLN identification, to improve image quality and to develop a software for automatic analysis of tissue AF to help the decision-making process during surgery.

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Lymphadenectomy and extended lymphadenectomy are still controversial issues in surgical oncology. The detection methods for metastatic lymph nodes include lymphoscintigraphy and radiolabelled antibody, but immunohistochemical sentinel lymph node (SLN) identification is commonly used. The potential diagnostic use of cell and tissue autofluorescence (AF) is well known. Here we review our studies on the application of AF-based techniques for diagnosing metastatic lymph nodes. We had previously demonstrated that AF imaging allows discrimination between hyperplastic and primary neoplastic lymph nodes. Our more recent studies show that the combination of autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy can be applied to the diagnosis of secondary neoplastic lymph nodes in gastric and colorectal cancer. These techniques have been validated by histochemical and immunohistochemical methods. Studies are in progress to select the best dye tracer for SLN identification, to improve image quality and to develop a software for automatic analysis of tissue AF to help the decision-making process during surgery.

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Lymphadenectomy and extended lymphadenectomy are still controversial issues in surgical oncology. The detection methods for metastatic lymph nodes include lymphoscintigraphy and radiolabelled antibody, but immunohistochemical sentinel lymph node (SLN) identification is commonly used. The potential diagnostic use of cell and tissue autofluorescence (AF) is well known. Here we review our studies on the application of AF-based techniques for diagnosing metastatic lymph nodes. We had previously demonstrated that AF imaging allows discrimination between hyperplastic and primary neoplastic lymph nodes. Our more recent studies show that the combination of autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy can be applied to the diagnosis of secondary neoplastic lymph nodes in gastric and colorectal cancer. These techniques have been validated by histochemical and immunohistochemical methods. Studies are in progress to select the best dye tracer for SLN identification, to improve image quality and to develop a software for automatic analysis of tissue AF to help the decision-making process during surgery.

Fibroblast autofluorescence in connective tissue disorders: a future tool for clinical and differential diagnosis?

Marfan syndrome (MFS) is an inherited disorder of connective tissue due to mutations in FBN1 (90%) and TGFBR1 and TGFBR2 (5 to 10%) genes. Clinical and differential diagnosis is difficult because of the inter- and intrafamiliar marked heterogeneity and the variable onset age of clinical manifestations. Among the disorders, in differential diagnosis, thoracic aortic aneurysm (TAA) and Ullrich scleroatonic muscular dystrophy (UCMD) are reported. We evaluate the possibility of utilizing autofluorescence (AF) analysis as a diagnostic tool in the clinical and/or differential diagnosis of MFS and related disorders and in the investigation of the molecular mechanisms involved. Both multispectral imaging autofluorescence microscopy (MIAM) and autofluorescence microspectroscopy (AMS) have been used to characterize AF emission of fibroblasts from patients affected by inherited connective tissue disorders. Our preliminary results show significant differences in AF emission between normal and pathological fibroblasts, suggesting possible improvement in diagnostics of connective tissue disorders by AF analysis.

Multispectral Imaging Autofluorescence Microscopy for the Analysis of Lymph-Node Tissues

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red–green–blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

Multispectral Imaging Autofluorescence Microscopy in Colonic and Gastric Cancer Metastatic Lymph Nodes

The lymphadenectomy and extended lymphadenectomy procedures have been points of controversy in surgical oncology. The methods available for the detection of metastatic lymph nodes are numerous. These include lymphoscintigraphy and radiolabeled antibody detection, but in most cancers the currently used technique is sentinel lymph node identification, performed primarily through the use of immunohistochemistry. We propose the application of autofluorescence (AF)-based techniques for lymph node evaluation in colorectal and gastric tumors. We studied 30 clinical cases: 15 colorectal cancers and 15 gastric cancers. All of the patients were in the advanced stages of the disease and were candidates for adjuvant therapy. Autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy have been used to analyze the AF emission of metastatic lymph node sections, excited with 365-nm wavelength radiation. The AF spectra were recorded in the range of 400-700 nm. Monochrome AF images were acquired sequentially through interference filters peaked at 450, 550, and 650 nm, and then combined together in a single red-green-blue image. The AF pattern and the emission spectrum of metastatic lymph nodes have unique characteristics that can be used to distinguish them from the normal ones. The results, compared with standard histopathologic procedures and with specific staining methods, supplied a satisfactory validation of the proposed technique, revealing the possibility of improving the actual diagnostic procedures for malignant lymph node alterations. With the development of appropriate instrumentation, the proposed technique could be particularly suitable in intrasurgical diagnosis of metastatic lymph nodes.

Can ultrasound counteract bone loss? Effect of low-intensity ultrasound stimulation on a model of osteoclastic precursor

The aim of the present work is to determine whether mechanical stress caused by ultrasound (US) exposure affects osteoclastic precursor cells, thus addressing the hypothesis that mechanical strain-induced perturbation of preosteoclastic cell machinery can contribute to the occurrence of bone turnover alterations. Moreover, cell cytoskeleton was studied because of its supposed involvement in cell mechanotransduction. Our experimental model was the FLG 29.1 human cell line, previously characterized as an osteoclastic precursor model. Cell proliferation was quantified by trypan blue exclusion assay. Cell morpho-functional state was monitored by multispectral imaging autofluorescence microscopy. The expression of cytoskeletal components and markers of proliferation (Ki67) and osteoclastic differentiation (RANK) was analysed by immunocytochemistry. The findings demonstrated that US stimulation affects FLG 29.1 cell growth, depresses the expression of cytoskeletal components and markers of proliferation and differentiation, induces cell damage, thus supporting the hypothesis that US exposure inhibits osteoclastogenesis. These results have been compared with those obtained previously by exposure of FLG 29.1 cells to modelled hypogravity conditions. Finally, the possibility to utilize US stimulation for counteracting osteoporosis has been discussed.

Dependence of leukemic cell autofluorescence patterns on the degree of differentiation.

The characterisation of leukemic cell autofluorescence during differentiation, induced by 12-O-tetradecanoylphorbol 13-acetate and all-trans retinoic acid, was performed by autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy. We have found that a dependence exists between the cell autofluorescence pattern and the degree of cell differentiation. When cells differentiate, their autofluorescence emission changes, following the morphological and functional rearrangement of cell structures. A decrease in emission intensity and a different distribution of endogenous fluorophores are observed. Thus, autofluorescence monitoring on living cells is a potentially useful tool for in vitro study of the differentiation processes. Furthermore, different maturation steps can be distinguished on the basis of the cell fluorescence pattern, leading the way to future application of the technique in diagnostics.

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

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red-green-blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.