Performance of the red-shifted fluorescent proteins in deep-tissue molecular imaging applications

Abstract

The discovery of new fluorescent proteins (FPs) that emit in the far-red part of the spectrum, where light absorption from tissue is significantly lower than in the visible, offers the possibility for noninvasive biological interrogation at the entire organ or small animal level in vivo. The performance of FPs in deep-tissue imaging depends not only on their optical characteristics, but also on the wavelength-dependent tissue absorption and the depth of the fluorescence activity. In order to determine the optimal choice of FP and illumination wavelength we compared the performance of five of the most promising FPs, i.e. tdTomato, mCherry, mRaspberry, mPlum, and Katushka. We experimentally measured the signal strength through mice and employed theoretical predictions to obtain an understanding on the performance of different illumination scenarios, especially as it pertains to tomographic imaging. It was found that the appropriate combination of red-shifted proteins and illumination wavelengths can improve detection sensitivity in small animals by at least 2 orders of magnitude compared to Green FP (GFP). It is also shown that the steep attenuation change of the hemoglobin spectrum around the 600nm range may significantly affect the detection sensitivity and necessitates the careful selection of illumination wavelengths for optimal imaging performance.

Recombinant Canine Distemper Virus Strain Snyder Hill Expressing Green or Red Fluorescent Proteins Causes Meningoencephalitis in the Ferret

“…It is important to investigate the utility of red fluorescent proteins for pathogenesis studies due to the longer wavelength of emitted light, which significantly enhances tissue penetrance (13). Moreover, expression of the fluorescent proteins from an additional transcription unit (ATU) rather than as fusion proteins has the significant advantage that the entire cytoplasm of the infected cells is flooded with the reporter proteins.…”

Performance of computer vision in vivo flow cytometry with low fluorescence contrast

“…For example, comparison of extinction coefficient and emission quantum yield data suggests that cells labeled with the Turbo-FP650 red fluorescent protein (RFP; Evrogen, Moscow, Russia) [44][45][46] would be about three times less-brightly labeled than with vybrant-DiD. Likewise, the use of a green fluorophore would reduce the contrast by approximately a factor of 10 due to increased autofluorescence, [47][48][49][50] which (according to the analysis here) would not significantly degrade CV-IVFC sensitivity.…”

Preclinical whole body time domain fluorescence lifetime multiplexing of fluorescent proteins

“…The results demonstrate that TD lifetime multiplexing of FPs and tissue AF can enable the in vivo detection of metastatic lesions at earlier stages than CW imaging alone, without the need for cumbersome background subtraction techniques that can depend on experimental scaling parameters. 9, 18 We also demonstrate that TD FL multiplexing allows for the accurate discrimination of up to three subcutaneously placed, spectrally overlapping red shifted FPs in mice, purely based on their distinct lifetimes. The techniques presented here have the potential to positively impact studies necessitating in vivo visualization of multiple genetically encoded tags.…”