FLIM measurements on live cultured cells were performed using an LSM 880 (Carl Zeiss) laser-scanning microscope equipped with a FLIM module Simple Tau 152 TCSPC (Becker & Hickl GmbH). Two-photon fluorescence of NAD(P)H was excited with a femtosecond Ti:Sa laser (repetition rate 80 MHz, pulse duration 140 fs) at a wavelength of 750 nm and registered in the range 455 to 500 nm. A water immersion objective (С-Apochromat 40×/1.2 W Corr, Zeiss, Inc.) was used for image acquisition. The average power applied to the samples was ∼6 mW. Image collection time was 60 s. During image acquisition, the cells were maintained at 37 °C and 5% CO2.
Simple tau 152 tcspc
Manufactured by Becker & Hickl
Sourced in Germany
The Simple Tau 152 TCSPC is a time-correlated single-photon counting (TCSPC) system. It is capable of measuring fluorescence lifetimes and other time-resolved optical phenomena with high temporal resolution. The device provides a compact and user-friendly platform for performing TCSPC experiments.
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3 protocols using simple tau 152 tcspc
1
Two-photon FLIM of Live Cells
2
Multimodal Fluorescence Imaging of Cells and Tumors
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An LSM 710 (Carl Zeiss, Germany) fluorescence confocal laser-scanning microscope equipped with a femtosecond Ti:Sa laser with a repetition rate of 80 MHz and pulse duration of 140 fs, and an FLIM module based on time-correlated single photon counting - Simple Tau 152 TCSPC (Becker & Hickl GmbH, Germany), were used to obtain one- and two-photon fluorescence and FLIM images of the cultured cells. A water immersion objective С-Apochromat 40x/1.2 NA W Korr was used for image acquisition. During image acquisition, the cells were maintained at 37 °C and 5% CO2.
For two-photon fluorescence microscopy and FLIM of tumors in vivo an MPTflex (JenLab GmbH, Germane) multiphoton tomograph, equipped with a tunable 80 MHz, 200 fs MaiTai Ti:Sa laser and a TCSPC-based FLIM module (Becker & Hickl GmbH, Germany) were used. The images were acquired through a 40x/1.3 NA oil immersion EC Plan-Neofluar objective.
ImageJ 1.39p software (NIH, USA) was used for fluorescence image processing. Analysis of the FLIM data was performed using SPCImage software (Becker & Hickl GmbH, Germany).
For two-photon fluorescence microscopy and FLIM of tumors in vivo an MPTflex (JenLab GmbH, Germane) multiphoton tomograph, equipped with a tunable 80 MHz, 200 fs MaiTai Ti:Sa laser and a TCSPC-based FLIM module (Becker & Hickl GmbH, Germany) were used. The images were acquired through a 40x/1.3 NA oil immersion EC Plan-Neofluar objective.
ImageJ 1.39p software (NIH, USA) was used for fluorescence image processing. Analysis of the FLIM data was performed using SPCImage software (Becker & Hickl GmbH, Germany).
3
Multimodal Fluorescence Imaging of 3D Cell Cultures
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Fluorescence intensity and lifetime images of the 3D cell cultures were acquired using an LSM 880 (Carl Zeiss, Jena, Germany) laser scanning microscope equipped with a FLIM module Simple Tau 152 TCSPC (Becker & Hickl GmbH, Berlin, Germany). A water immersion objective C-Apochromat 40×/1.2 NA W Korr was used for image acquisition. During image acquisition, the cells were maintained at 37 °C and 5% CO2.
Two-photon fluorescence of NAD(P)H was excited with a femtosecond Ti:Sa laser (MaiTai, Spectra-Physics, Milpitas, CA, USA, repetition rate 80 MHz, pulse duration 140 fs) at 750 nm wavelength and registered in the range of 455–500 nm. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. Image collection time was 60 s. Two-photon excitation of DOX fluorescence was performed at 780 nm and emission was registered using 690/50 filter. The photons were collected for 90 s. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. In one-photon mode, DOX fluorescence was excited at 488 nm and registered in the range of 540–650 nm.
FLIM images of NAD(P)H and DOX were acquired sequentially from the same 5–7 randomly selected fields of view in each culture dish.
Two-photon fluorescence of NAD(P)H was excited with a femtosecond Ti:Sa laser (MaiTai, Spectra-Physics, Milpitas, CA, USA, repetition rate 80 MHz, pulse duration 140 fs) at 750 nm wavelength and registered in the range of 455–500 nm. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. Image collection time was 60 s. Two-photon excitation of DOX fluorescence was performed at 780 nm and emission was registered using 690/50 filter. The photons were collected for 90 s. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. In one-photon mode, DOX fluorescence was excited at 488 nm and registered in the range of 540–650 nm.
FLIM images of NAD(P)H and DOX were acquired sequentially from the same 5–7 randomly selected fields of view in each culture dish.
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