Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging
| Autor: | Vitor B. Pelegati, Liliana Aparecida Lucci De Angelo Andrade, Fátima Böttcher-Luiz, André A. de Thomaz, Carlos L. Cesar, Diogo B. Almeida, Javier Adur, Mariana Ozello Baratti |
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| Rok vydání: | 2011 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Histology Materials science Microscope Confocal Second-harmonic imaging microscopy law.invention Optics law Microscopy Onions Image Processing Computer-Assisted Humans Instrumentation Solanum tuberosum Ovarian Neoplasms Microscopy Confocal business.industry Second-harmonic generation Laser Adenocarcinoma Mucinous Medical Laboratory Technology Microscopy Fluorescence Female Anatomy business Ultrashort pulse |
| Zdroj: | Microscopy research and technique. 75(10) |
| ISSN: | 1097-0029 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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