Journal: PLoS Biology

Article Title: Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

doi: 10.1371/journal.pbio.3000965

Figure Lengend Snippet: ( a ) Mutations of NIR-GECO2 and NIR-GECO2G relative to NIRGECO1. The different mutations between NIR-GECO2 and NIR-GECO2G are highlighted in green. ( b ) Relative fluorescence intensity (mean ± SEM) of NIR-GECO1, NIR-GECO2, NIR-GECO2G, and miRFP720 in neurons ( n = 160, 120, 219, and 84 neurons, respectively, from 2 cultures). Fluorescence was normalized by co-expression of GFP via self-cleavable 2A peptide. ( c – e ) Comparison of NIR-GECO variants, as a function of stimulus strength (the same color code is used in panels c – e ). ( c ) ΔF/F 0 ; ( d ) rise time; ( e ) half decay time. Values are shown as mean ± SD ( n = 10 wells from 3 cultures). The underlying data for ( b ) to ( e ) can be found in . GFP, green fluorescent protein; NIR, near-infrared; SD, standard deviation; SEM, standard error of the mean.

Article Snippet: Plasmids pAAV-CAG-NIR-GECO2 (plasmid no. 159603), pAAV-NIR-GECO2G (plasmid no. 159605), and pSF11-wNIR-GECO2-T2A-HO1(plasmid no.159606) are available via Addgene according to the terms of the Uniform Biological Material Transfer Agreement.

Techniques: Fluorescence, Expressing, Comparison, Standard Deviation

Journal: PLoS Biology

Article Title: Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

doi: 10.1371/journal.pbio.3000965

Figure Lengend Snippet: ( a – c ) Fluorescence traces of NIR-GECO2G, NIR-GECO2, and NIR-GECO1 in response to 100 ms ( a ), 500 ms ( b ), and 1 s ( c ) blue light activation (470 nm at a power of 1.9 mW/mm 2 ) in HeLa cells with co-expression of Opto-CRAC. Opto-CRAC is composed of the STIM1-CT and LOV2 domain. The fusion of STIM1-CT to the LOV2 domain allows photo-controllable exposure of the active site of SRIMI-CT, which is able to interact with ORAI1 and trigger Ca 2+ entries across the plasma membrane . Black, green, and dark blue lines represent averaged data for NIR-GECO2G ( n = 32 cells), NIR-GECO2 ( n = 25 cells), and NIR-GECO1 ( n = 23 cells), respectively. The same color code is used in panels a–c . Shaded areas represent the SD. ( d ) Quantitative -ΔF/F 0 (mean ± SD) for NIR-GECO2G, NIR-GECO2, and NIR-GECO1 in a–c . The underlying data for a – d can be found in . NIR, near-infrared; SD, standard deviation.

Article Snippet: Plasmids pAAV-CAG-NIR-GECO2 (plasmid no. 159603), pAAV-NIR-GECO2G (plasmid no. 159605), and pSF11-wNIR-GECO2-T2A-HO1(plasmid no.159606) are available via Addgene according to the terms of the Uniform Biological Material Transfer Agreement.

Techniques: Fluorescence, Activation Assay, Expressing, Clinical Proteomics, Membrane, Standard Deviation

Journal: PLoS Biology

Article Title: Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

doi: 10.1371/journal.pbio.3000965

Figure Lengend Snippet: ( a ) Left, fluorescent image of neurons expressing NLS-jGCaMP7s (λ ex = 488-nm laser light, λ em = 527/50 nm). Right, fluorescent image of neurons expressing NIR-GECO2-T2A-HO1 (λ ex = 640-nm laser light, λ em = 685/40 nm). Representative of more than 3 worms, both under tag-168 promoter. Scale bar, 50 μm. ( b ) Fluorescence traces of NLS-jGCaMP7s (top) and NLS-NIR-GECO2 (bottom) in response to the stimulation of microfluidic containing 200 mM NaCl. Solid lines represent averaged data from 3 neurons. Shaded areas are shown as SD. Triangles on the top of the traces indicate the time points of stimulation (20 seconds for each stimulation). ( c ) Quantitative fluorescence changes of NLS-jGCaMP7s and NLS-NIR-GECO2 in b ( n = 36 spikes from 3 neurons). ( d ) Fluorescence image of the 4 C . elegans expressing NIR-GECO2-T2A-HO1 in AVA neurons (under flp-18 promoter) and CoChR-GFP in ASH neurons (under sra-6 promoter). The merged image is shown. Imaging conditions: NIR-GECO2, λ ex = 640-nm laser light, λ em = 685/40; GFP, λ ex = 488-nm laser light, λ ex = 527/50 nm. ( e ) Individual traces of NIR-GECO2 fluorescence in an AVA neuron under blue light illumination (20 mW/mm 2 , λ ex = 488-nm laser light, 100 ms; blue bars). The underlying data for b , c , and e can be found in . NIR, near-infrared; SD, standard deviation.

Article Snippet: Plasmids pAAV-CAG-NIR-GECO2 (plasmid no. 159603), pAAV-NIR-GECO2G (plasmid no. 159605), and pSF11-wNIR-GECO2-T2A-HO1(plasmid no.159606) are available via Addgene according to the terms of the Uniform Biological Material Transfer Agreement.

Techniques: Expressing, Fluorescence, Imaging, Standard Deviation

Journal: PLoS Biology

Article Title: Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

doi: 10.1371/journal.pbio.3000965

Figure Lengend Snippet: ( a ) Fluorescence intensity of NLS-jGCaMP7s and NIR-GECO2 in C . elegans neurons at resting state. Fluorescence was normalized to the same excitation intensity ( n = 132 ROIs from 5 worms; data are shown as mean ± SD) ( b ) SBR of NLS-jGCaMP7s and NIR-GECO2 in neurons of C . elegans at resting state ( n = 132 ROIs from 5 worms; data are shown as mean ± SD). SBR was obtained via dividing the fluorescence intensity from neurons by the averaged autofluorescence from the intestine area. ( c ) SNR of NLS-jGCaMP7s and NLS-NIR-GECO2 quantified from spontaneously spiking neurons (n = 78 ROIs from 4 worms; data are shown as mean ± SD). SNR was calculated by dividing the fluorescence change associated with a spike by the SD of the baseline fluorescence over the 2-second period immediately before the spike. ( d ) The ratio of SBR NIR-GECO2 to SBR NLS-jGCaMP7s at different imaging depths ( n = 5 worms; data are shown as mean ± SD). ( e ) The ratio of SNR NLS-NIR-GECO2 to SNR NLS-jGCaMP7s at different imaging depths ( n = 4 worms; data are shown as mean ± SD). NIR-GECO2 (without NLS) and NLS-jGCaMP7s were used for the experiments in a , b , and d ; NLS-NIR-GECO2 and NLS-jGCaMP7s were used for the experiments in c and e . The underlying data for a – e can be found in . ROI, region of interest; SBR, signal-to-background ratio; SD, standard deviation; SNR, signal-to-noise ratio.

Article Snippet: Plasmids pAAV-CAG-NIR-GECO2 (plasmid no. 159603), pAAV-NIR-GECO2G (plasmid no. 159605), and pSF11-wNIR-GECO2-T2A-HO1(plasmid no.159606) are available via Addgene according to the terms of the Uniform Biological Material Transfer Agreement.

Techniques: Fluorescence, Imaging, Standard Deviation