Bright far-red fluorescent protein for whole-body imaging

Abstract

For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.

A monomeric red fluorescent protein with low cytotoxicity

“…Notably, after expression of mRuby and mCherry in HeLa cells for 4 days, we observed punctate fluorescent labelling that resembled lysosomal localization, as previously described by Miyawaki et al 12 This observation confirms our previous data for mCherry, although it should be mentioned here that in our previous work the punctate labelling was defined as 'aggregates' 6 . In contrast, we did not observe lysosomal localization of mKate2 and FusionRed after 4 days of expression under the same conditions ( Supplementary Fig.…”

“…3 and Supplementary movies [1][2][3][4][5][6][7][8]. Owing to the lack of a statistical methodology to make direct comparisons of fluorescent protein performance in fusions, the practice is utilized only to provide one or several representative images that demonstrate the ability of the reporter to properly perform in each of the constructs.…”

Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: Comparison of transgenic strains and relevance for fetal cell microchimerism

“…FCM and immunohistochemistry) to continue to understand this phenomenon. These experiments also include the assessment of bright, far-red fluorescent proteins, such as the far-red mutant TurboFP635 (14), and DsRed mouse strains that are currently commercially available, such as B6.Cg-Tg(ACTB-Bgeo,-DsRed*MST)1Nagy/J. These new and highly innovative vectors may allow for improved sensitivity and specificity due to better tissue penetration, increased signal to noise ratios through the reduction of autofluorescence.…”

Solid state yellow and orange lasers for flow cytometry

“…useful for exciting the shorter wavelength FPs, proteins with longer excitation wavelengths such as mCherry (k EX 5 587 nm, k EM 5 610 nm), mPlum (k EX 5 592 nm, k EM 5 645 nm), and the new bright red FP mKate (k EX 5 588 nm, k EM 5 635 nm) optimally require longer wavelength laser sources (12,15). This is shown in Figure 5 for HcRed (k EX 5 590 nm, k EM 5 649 nm) and the newly reported dimerized version of mKate, Katushka (k EX 5 588 nm, k EM 5 635 nm) (15), where 559, 580, 592, and 633 nm power-matched sources were used to excite bacteria expressing the relatively dim protein HcRed (left column), and the far brighter Katushka (right column).…”