Journal
BIOSENSORS-BASEL
Volume 12, Issue 5, Pages -Publisher
MDPI
DOI: 10.3390/bios12050307
Keywords
fluorescence microscopy; two-photon microscopy; multi-color imaging; signal separation
Funding
- National Basic Research Programof China [2017YFA0700500]
- National Natural Science Foundation of China [61620106016, 61835009, 62005171, 61975127]
- Guangdong Natural Science Foundation [2019A1515110380, 2020A1515010679, 2022A1515011954]
- Key Project of Guangdong Provincial Department of Education [2021ZDZX2013]
- Shenzhen Basic Research Project [JCYJ20190808111418696]
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This study demonstrates a multi-color two-photon imaging method with single-wavelength excitation. The problem of spectral crosstalk is effectively solved by selecting suitable filter combinations and applying image subtraction. The results show that two-color and three-color two-photon imaging can be achieved using a single femtosecond laser.
Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues.
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