RESUMO
We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein-protein interactions via Förster resonance energy transfer.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Lasers , Rodaminas , Peixe-ZebraRESUMO
Ovarian cancer is a leading gynecological malignancy associated with high mortality. The development of acquired drug resistance is the primary cause of chemotherapy failure in the treatment of ovarian cancer. To examine the mechanism underlying paclitaxel resistance in ovarian cancer and attempt to reverse it, the present study induced a TAX-resistant ovarian cancer cell line, SKOV3/TAX. Cathepsin L (CTSL) has been found to be overexpressed in ovarian cancer. The aim of the present study was to investigate the possible involvement of CTSL in the development of TAX resistance in ovarian cancer. CTSL expression was knocked down in SKOV3 ovarian cancer cells and their phenotypic changes were analyzed. The effects of silenced CTSL on the resistant cell line were investigated by proliferation and apoptosis analysis compared with control SKOV3 cells. CTSL was more highly expressed in SKOV3/TAX cells compared with SKOV3 cells. Paclitaxel treatment downregulated the expression of CTSL in SKOV-3 but not in the paclitaxel-resistant SKOV3/TAX cells. CTSL small hairpin RNA (shRNA) knockdown significantly potentiated apoptosis induced by paclitaxel compared with SKOV3/TAX cells transfected with control shRNA, suggesting that CTSL contributes to paclitaxel resistance in ovarian cancer cells and that CTSL silencing can enhance paclitaxel-mediated cell apoptosis. Thus, CTSL should be explored as a candidate of therapeutic target for modulating paclitaxel sensitivity in ovarian cancer.