Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging |
| |
Authors: | Pelegati Vitor B Adur Javier De Thomaz André A Almeida Diogo B Baratti Mariana O Andrade Liliana A L A Bottcher-Luiz Fátima Cesar Carlos L |
| |
Affiliation: | 1. Biomedical Lasers Application Laboratory. Optics and Photonics Research Center, “Gleb Wataghin” Institute of Physics. State University of Campinas (UNICAMP). Departamento de Eletr?nica Qu?ntica. Rua Sergio Buarque de Holanda, 777. Cidade Universitária “Zeferino Vaz”. Campinas 13083‐859. S?o Paulo. Brazil;2. Microscopy Laboratory Applied to Molecular and Cellular Studies. School of Bioengineering. National University of Entre Ríos (UNER). Ruta 11, km 10, Oro Verde 3101. Entre Ríos. Argentina;3. Microscopy Laboratory Applied to Molecular and Cellular Studies. School of Bioengineering. National University of Entre Ríos (UNER). Ruta 11, km 10, Oro Verde 3101. Entre Ríos. ArgentinaV.B.P. and J.A. contributed equally to this work.;4. Department of Pathology. State University of Campinas (UNICAMP). Faculdade de Ciências Médicas, Departamento de Anatomia Patológica. Cidade Universitária “Zeferino Vaz”. Campinas 13083‐970. S?o Paulo. Brazil;5. Department of Obstetrics and Gynecology. State University of Campinas (UNICAMP). Faculdade de Ciências Médicas, Departamento de Tocoginecologia. Cidade Universitária “Zeferino Vaz”. CAISM. Laborat?rio de Citogenética e Cultivo Celular. 13083‐970 Campinas. S?o Paulo. Brazil |
| |
Abstract: | In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc. |
| |
Keywords: | two‐photon excitation fluorescence second and third harmonic generation fluorescence lifetime |
本文献已被 PubMed 等数据库收录! |
|