See also Figure S2 and Table 2.Adjust Brightness and Contrast Tutorial Adjust Brightness and Contrast Tutorial Mean NIR fluorescence intensity was normalized to mean green fluorescence intensity of EGFP co-expressed via T2A peptide upstream of NIR FPs (to account for transfection efficiency) and to mean fluorescence intensity of mock transfected cells in the 660/20 nm channel (for miRFP670 and smURFP) or in the 730/45 nm channel (for miRFP703 and mIFP) (to account for cellular autofluorescence). (b) Quantification of the data presented in (a). Fluorescence distribution of cells co-expressing NIR FPs with catalytically inactive HO1 (H25A/D140F) mutant as a control is shown with dashed lines. ![]() The supplements were added for 24 h period. (a) Fluorescence intensity distribution of live HeLa cells transfected with smURFP, miRFP670, mIFP and miRFP703 co-expressed with HO1 downstream of IRES2 element (in EGFP-T2A-NIRFP-IRES2-HO1 constructs) with or without 250 μM 5-ALA and 40 μM FeSO 4 was analyzed using flow cytometry. Biliverdin dimethyl ester (BVMe 2) can be produced using organic synthesis by esterification of carboxylic groups of BV (blue rings in BV and BVMe 2 structures). BV can be further converted into bilirubin with biliverdin reductase. Molecular oxygen enter to the reaction resulted in carbon monoxide (CO) production, and the iron is released from the BV molecule. In this reaction NADPH is used as the reducing agent. Heme can be further converted to biliverdin IXα (BV) by heme oxygenase (HO), which cleaves the heme ring at the α-methene bridge (red ring in both heme and BV structures). PPIX is loaded by Fe 2+ ions with help of ferrochelatase, which results in a formation of heme. Formed protoporphyrinogen IX is further oxidized to protoporphyrin IX (PPIX). Decarboxylation of uroporphyrinogen III results in a formation of coproporphyrinogen III, which is converted by coproporphyrinogen III oxidase, located in the mitochondrial membrane. There it is merging in a chain of several enzymatic reactions through the formation of porphobilinogen (simple pyrrole), hydroxymethylbilane (linear tetrapyrrole) and uroporphyrinogen III (cyclic tetrapyrrole). All rights reserved.īiosynthesis of a heme starts with the synthesis of 5-aminolevulinic acid (5-ALA) in mitochondria, and then 5-ALA is transferred to the cytosol. ![]() ![]() This feature makes them easy to use as GFP-like proteins for spectral multiplexing with FPs of visible range.ĥ-ALA IFP biliverdin firefly luciferase heme oxygenase iRFP in vivo imaging near infrared phytochrome smURFP.Ĭopyright © 2017 Elsevier Ltd. mIFP showed intermediate, while monomeric miRFP670 and miRFP703 exhibited high binding efficiency of endogenous biliverdin chromophore. Exogenously added chromophore substantially increased smURFP brightness but only slightly enhanced brightness of other NIR FPs. Either co-expression of heme oxygenase or incubation of cells with heme precursor weakly affected NIR fluorescence, however, in the latter case elevated cellular autofluorescence. For instance, in common HeLa cells miRFP703, mIFP, and smURFP were 2-, 9-, and 53-fold dimmer than miRFP670. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher's choice rather difficult.
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