The NIR multimodal optical setup was described in our previous work14 (link),23 (link). It consists of a two-photon multimodal benchtop microscope (Leica SP8, Leica microsystems) able to perform four different optical imaging modalities: (1) One and two-photon fluorescence imaging (2) two-photon FLIM measurement, (3) SHG imaging and (4) one and two-photon Spectral imaging.
Analyses were conducted using the dedicated Leica software (LAS-X) as well as Matlab and imageJ. For FLIM measurements, 3 * 3 images mosaic per sample were analyzed using 810 and 890 nm the excitation wavelengths. The same technique was used also for 2PEF and spectral imaging, where 2PEF images were acquired using 890 nm and the spectral images using 810 nm. The collected fluorescence signal is divided into two detection channels. Each detection channel contain a super sensitive hybrid non descanned (HyD NDD) detector. In the first detection channel (channel 1), a bandpass filter with a bandwidth of 448 ± 20 nm (FF01-448/20-25, semrock, USA) was used to select the NADH fluorescence signal, and in the second detection channel (channel 2) another bandpass filter with a bandwidth of 520 ± 35 nm (FF01-520/35-25, Semrock, New York, USA) was used to select the FAD fluorescence signal.
Analyses were conducted using the dedicated Leica software (LAS-X) as well as Matlab and imageJ. For FLIM measurements, 3 * 3 images mosaic per sample were analyzed using 810 and 890 nm the excitation wavelengths. The same technique was used also for 2PEF and spectral imaging, where 2PEF images were acquired using 890 nm and the spectral images using 810 nm. The collected fluorescence signal is divided into two detection channels. Each detection channel contain a super sensitive hybrid non descanned (HyD NDD) detector. In the first detection channel (channel 1), a bandpass filter with a bandwidth of 448 ± 20 nm (FF01-448/20-25, semrock, USA) was used to select the NADH fluorescence signal, and in the second detection channel (channel 2) another bandpass filter with a bandwidth of 520 ± 35 nm (FF01-520/35-25, Semrock, New York, USA) was used to select the FAD fluorescence signal.
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