Journal: Scientific Reports

Article Title: Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

doi: 10.1038/srep42730

Figure Lengend Snippet: ( a ) τ 1 of 3D Caco-2 luminal cysts before and after treatment with KCN (n = 6) or H 2 O 2 (n = 4). No significant impact is seen on the short fluorescence lifetime τ 1 of NADH. ( b ) τ 2 of 3D Caco-2 luminal cysts before and after treatment with KCN or H 2 O 2 . Treatment with KCN induces a significant decrease of τ 2 (n = 6, *p = 0.0004), whereas no significant impact was detected on τ 2 when treated with H 2 O 2 . ( c ) Changes are seen in the short fluorescence lifetime contribution α 1MEDF before and after treating 3D Caco-2 luminal cysts with KCN or H 2 O 2 : Treatment with KCN induces an increase in α 1MEDF (n = 6, *p = 0.0029), whereas H 2 O 2 treatment leads to a decrease in α 1MEDF (n = 4, *p = 0.0434). ( d–f ) False color-coded images of ( d) untreated, ( e) KCN-treated, and ( f ) H 2 O 2 -treated 3D Caco-2 luminal cyst models indicating the range of α 1MEDF from 65% (red) to 80% (blue).

Article Snippet: Fluorescence lifetime images of 3D Caco-2 models were acquired with a custom built 5D multiphoton FLIM microscope (JenLab GmbH, Jena, Germany).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

doi: 10.1038/srep42730

Figure Lengend Snippet: ( a ) EGF induces a significant decrease in the short fluorescence lifetime τ 1 of NADH (n = 4, *p = 0.002). (b ) EGF treatment induces a significant decrease in τ 2 (n = 4, *p = 0.0391). ( c ) Treatment of 3D Caco-2 models with EGF induces an increase in the short fluorescence lifetime contribution α 1MEDF (n = 4, *p = 0.0213). ( d,e ) False color-coded images of ( d ) untreated and ( e ) EGF-treated 3D Caco-2 luminal cyst models indicating the range of α 1MEDF from 65% (red) to 80% (blue).

Article Snippet: Fluorescence lifetime images of 3D Caco-2 models were acquired with a custom built 5D multiphoton FLIM microscope (JenLab GmbH, Jena, Germany).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

doi: 10.1038/srep42730

Figure Lengend Snippet: ( a ) Schematic of phasor plot properties showing the intersections of linear fitted line with universal circle representing lifetimes (e.g. τ 1 and τ 2 ) of contributing fluorophores for bi-exponential decays. Lifetime contributions β 1 and β 2 show distance of mean of phasor data from τ 1 and τ 2 . Shape of the phasor data is determined with calculation of 95% confidence ellipse. Relative lifetime contribution α i (i = 1, 2) is calculated using α i = β i /(β 1 + β 2 ). Elliptic ratio a/b , determined by semi-major axis a and semi-minor axis b gives further information about fluorescence properties. ( b ) Phasor plot of FLIM signal from coumarin 6. Green ellipse represents the 95% error ellipse of phasor approach calculations. Ratio between major and minor semi-axis is a/b = 1.13 and the subsequent fluorescence lifetime is τ coumarin = 2.48 ns. Calculations were made with harmonic number k = 4 and usage of first quarter of period.

Article Snippet: Fluorescence lifetime images of 3D Caco-2 models were acquired with a custom built 5D multiphoton FLIM microscope (JenLab GmbH, Jena, Germany).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

doi: 10.1038/srep42730

Figure Lengend Snippet: ( a ) τ 1 derived from phasor analysis before and after treatment with KCN (n = 6) or H 2 O 2 (n = 4). KCN treatment induces a significant reduction in τ 1 (n = 6, *p = 0.0007). ( b ) KCN treatment of 3D Caco-2 luminal cysts induces a significant decrease of τ 2 (n = 6, *p = 0.0003). ( c ) 3D Caco-2 luminal cysts treated with KCN displayed a significant increase in the short fluorescence lifetime contribution α 1phasor (n = 6, *p = 0.0169), whereas H 2 O 2 leads to a decrease in α 1phasor (n = 4, *p = 0.0113). ( d ) Phasor plot of 3D Caco-2 models before and after treatment with KCN shows 95% confidence ellipses and fitted linear functions on the universal circle. ( e ) Phasor plot of 3D Caco-2 models before and after H 2 O 2 treatment reveals 95% confidence ellipses and fitted linear functions. ( f–h ) EGF treatment of 3D Caco-2 luminal cysts shows no significant changes in phasor analysis-derived ( f ) short fluorescence lifetime τ 1 , ( g ) long fluorescence lifetime τ 2 and ( h ) the short fluorescence lifetime contribution α 1phasor of NADH (n = 4). ( i ) Phasor plot of untreated and EGF-treated 3D Caco-2 models, showing 95% confidence ellipses and fitted linear functions on the universal circle. All phasor calculations were made with harmonic number k = 2 and usage of first half the period.

Article Snippet: Fluorescence lifetime images of 3D Caco-2 models were acquired with a custom built 5D multiphoton FLIM microscope (JenLab GmbH, Jena, Germany).

Techniques: Derivative Assay, Fluorescence