To overcome limitations of indirect immunofluorescence, a new method is presented to employ the ostensible disadvantage of cross-labeling secondary antibodies by separation of the fluorescence signals via spectral FLIM-FRET. The undesirable cross-labeling among secondary antibodies leads to the generation of new characteristic FRET emission spectra including a change in the donor lifetime. We used a spectrally resolved FLIM detection system with pulse interleaved excitation. The combined spectral FLIM-FRET and pattern-matching analysis forms an excellent tool for use in indirect immunofluorescence that overcomes the undesirable effect of secondary antibody cross-labeling by assigning separate color channels to cross-labeled fluorescent antibodies.
In this research work, we present a novel indirect immunolabeling method for labeling up to three different antigens using just two primary and fluorophore tagged secondary antibodies. We propose a viable solution to overcome the limitations imposed by limited variability in primary and secondary antibody type by leveraging cross-labeling phenomenon. Cross-labeling among two fluorophore conjugated secondary antibodies leads to FRET effects, resulting in changed spectral and fluorescence lifetime properties of donor molecule. To detect and quantify these changes in photophysical properties of immunolabeled species, we developed an eight-channel spectrally resolved fluorescence lifetime imaging (sFLIM) system. We demonstrate the capabilities of our approach in case of multi-targeted immunostaining in A549 cells. Efficient excitation of samples is achieved using two pulsed laser of wavelengths 485 nm and 561 nm operating in alternating/interleaved manner. Acquired multi-dimensional sFLIM data was pre-processed and analysed using state-of-the-art pattern-matching algorithm1 which takes into account the information of fluorescence emission spectra as well as lifetime. The sFLIM detection system together with pattern-matching analysis enables separation of cross-linked labels from single labeled species.
Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential for imaging dynamic processes in life science. The rapidFLIM approach dramatically reduces acquisition times through a combination of fast beam scanning, hybrid photomultiplier detectors, which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high spatial resolution offered in confocal microscopy. Depending on image size, rapidFLIM enables following dynamic processes like protein interactions, chemical reactions or highly mobile species in live cell imaging with a rate of several frames per second.
The separation of overlapping fluorescence emissions in biological samples has been improved in the last years by using spectral confocal microscopy in combination with linear unmixing. However, the separation of multiple labels in biological samples remains challenging, especially when strong tissue autofluorescence (AF) overshadows specifically labeled structures. Combining the spectral approach with fluorescence lifetime measurements based on a simultaneous acquisition of both spectral and lifetime parameters could significantly improve the separation quality between multiple labels and tissue AF. We demonstrate this approach in highly autofluorescent human lung tissue, where the fluorescence signals from specific stainings are sometimes weaker than tissue AF. We use dual color Pulsed Interlevaed Excitation (PIE) in conjunction with a spectral FLIM (sFLIM) detection system featuring eight separate TCSPC timing channels and analyze the data by applying a unique pattern matching technique.
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