Comparison of the performance of the non-fitting strategies as a function of the total number of photons for three distinct fractions of interacting donor <i>f_D</i>: 0.25 (A), 0.5 (B) and 0.75 (C).

<p>The polar approach is indicated in black, the moments method in blue and the <i>mf_D</i> in red. For all methods, we have reported the estimated <i>f_D</i> and the estimated <i>mf_D</i> value in the left part of the figure. The estimated donor lifetime in presence of the acceptor <i>τ_F,</i> and the estimated mean lifetime <<i>τ</i>> are reported in the right part. In all cases, medians are indicated with markers and error bars correspond to the interquartile ranges of 4096 simulated histograms whose parameters are: <i>τ_F</i> = 1.5 ns, <i>τ_D</i> = 2.5 ns and <i>N_ch</i> = 64 channels (TCSPC simulations) and the simulated values are indicated in dotted lines.</p

Importance of the initial conditions for the standard fitting method for three distinct total numbers of photons: 200 (in light gray), 1000 (in dark gray) and 100000 (in black).

<p>We have considered three fractions of interacting donor <i>f_D</i>: 0.25 (A), 0.5 (B) and 0.75 (C). The minimal fractions of interacting donor are plotted in the left part and the lifetimes of the donor in presence of the acceptor in the right part. For each condition, the dotted lines represent the simulated values and the markers with error bars represent the medians and interquartile ranges of 4096 simulated TCSPC histograms (with <i>τ_F</i> = 1.5 ns, <i>τ_D</i> = 2.5 ns and <i>N_ch</i> = 64 channels).</p

Performance of the non-fitting methods (polar approach in black, moments method in blue and <i>mf_D</i> in red) as a function of the number of temporal channels for <i>N</i> = 1000 photons (time gated simulations).

<p>We have considered three fractions of interacting donor <i>f_D</i>: 0.25 (A), 0.5 (B) and 0.75 (C). <i>τ_F</i> and <<i>τ></i> are reported in the right part whereas <i>f_D^P, f_D^M</i> and <i>mf_D</i> are indicated in the left. For each condition, the dotted lines represent the simulated values and the markers with error bars represent the corresponding medians and interquartile ranges of 4096 simulated histograms with parameters: <i>τ_F</i> = 1.5 ns and <i>τ_D</i> = 2.5 ns.</p

Performance of the non-fitting methods (polar approach in black, moments method in blue and <i>mf_D</i> in red) as a function of the number of temporal channels for <i>N</i> = 200 photons acquired with time gated system.

<p>Three fractions of interacting donor <i>f_D</i> are considered: 0.25 (A), 0.5 (B) and 0.75 (C). We have indicated the fraction of interacting donor and the <i>mf_D</i> in the left part; the donor lifetime in presence of the acceptor <i>τ_F</i> and the mean lifetime <<i>τ></i> are in the right part. In all graphs, the dotted lines represent the simulated values and the markers with error bars represent the corresponding medians and interquartile ranges of 4096 simulated histograms whose parameters are: <i>τ_F</i> = 1.5 ns and <i>τ_D</i> = 2.5 ns.</p

Theoretical fractions of interacting donor calculated with the polar approach <i>f_D^P</i> or the moments method <i>f_D^M</i> (in black) and theoretical <i>mf_D</i> values (in red) as a function of the lifetime of the donor in presence of the acceptor <i>τ_F</i> (for a donor lifetime of 2.5 ns).

<p>Three distinct <i>f_D</i> values were considered in (A). We have also plotted the means in (B) and the standard deviations in (C) of <i>f_D^P</i> in black (deduced from Eqs. 15 and 16), those of <i>f_D^M</i> in blue (deduced from Eqs. 18 and 19) and those of <i>mf_D</i> in red (deduced from Eqs. 13 and 14) as a function of the total number of photons <i>N</i>. The following FRET parameters were used: <i>τ_F</i> = 1.5 ns, <i>τ_D</i> = 2.5n s and <i>f_D</i> = 0.25, 0.5 and 0.75.</p

Fast-FLIM scheme.

<p>The supercontinuum laser is collimated out of the fiber, spectrally filtered (473–491 nm) and injected into the Yokogawa spinning disk system where shaping optics extend the beam. The micro-lenses disk creates a multitude of beams focused in the pinholes of the coupled disk conjugated with the sample plane. The emitted fluorescence is selected by a dichroic mirror DM (transmission peak at 488 nm) and an emission filter (500–550 nm), and converted into electrons with the photocathode of the intensifier. Each laser pulse triggers the photocathode so that it runs as an ultra-fast shutter (time gate of 2.25 ns at 80 MHz). The electrons are amplified by a micro channel plate and converted back into light with a phosphorescent screen. The photons are finally acquired with a CCD camera (with binning 3×3) and the fluorescent images are saved. A home-made MetaMorph user program called Flimager (MFQ, IGDR) was developed for both controlling the complete system (delay generator, CCD camera and microscope), and calculating the FLIM images on-line.</p

Experimental FLIM measurements with our fast-FLIM prototype for quantifying G protein activation in living cells.

<p>The polar plots of a 3T3 cell co-expressing Rac-GFP and PBD-mCherry (with <i>N_mean</i> = 850 photons) and of a cell co-expressing Rac-GFP+mCherry (with <i>N_mean</i> = 830 photons) as a reference (negative control) are shown in (A). The shift between the two spots is the proof of lifetime modification which is also clearly visible in the mean lifetime images <<i>τ</i>> (scale bar: 10 µm). We have plotted in (B) the temporal variations of the mean lifetime for each cell. We have also calculated <i>f_D^P</i>, <i>f_D^M</i> and <i>mf_D</i> of the cell co-expressing Rac-GFP and PBD-mCherry and the images are reported respectively in (C1), (C2) and (C3). The evolution of each parameter calculated in the white region of interest is also plotted in (C4). We finally show the images of <i>τ_F^P</i> and <i>τ_F^M</i> in (D1) and (D2) and the corresponding lifetime distributions in (D3).</p

Experimental FLIM measurements on fluorescent solutions with our fast-FLIM prototype.

<p>Lifetime images of Rd6G alone, AO alone and of three mixtures of Rd6G and AO with theoretical fractions of interacting donor of 0.17, 0.44 and 0.65 were acquired with <i>N</i>≈1500 photons. The polar plot and the mean lifetime images of each solution are represented in (A). All spots corresponding to the mixtures in the polar plot are well localized on a line connecting pure Rh6G and pure AO. We have also calculated <i>f_D^P</i>, <i>f_D^M</i> and <i>mf_D</i> for the three mixtures and the corresponding images are indicated in (B). We show in (C) the images of donor lifetime in presence of the acceptor estimated with the polar approach and the moment method. We have also performed FLIM acquisitions with different numbers of detected photons for each fraction of interacting donor: 0.17 (D), 0.44 (E) and 0.65 (F); the corresponding plots of <i>f_D^P</i>, <i>f_D^M</i>, <i>mf_D, τ_F^P</i> and <i>τ_F^M</i> as a function of <i>N</i> are reported in the right part. For all graphs, markers with error bars represent the medians and interquartile ranges of FLIM images. The theoretical <i>f_D</i> and <i>τ_F</i> are indicated in dotted lines.</p

Myofibril Changes in the Copepod <i>Pseudodiaptomus marinus</i> Exposed to Haline and Thermal Stresses

<div><p>Copepods are small crustaceans capable to survive in various aquatic environments. Their responses to changes in different external factors such as salinity and temperature can be observed at different integration levels from copepod genes to copepod communities. Until now, no thorough observation of the temperature or salinity effect stresses on copepods has been done by optical microscopy. In this study, we used autofluorescence to visualize these effects on the morphology of the calanoid copepod <i>Pseudodiaptomus marinus</i> maintained during several generations in the laboratory at favorable and stable conditions of salinity (30 psu) and temperature (18°C). Four different stress experiments were conducted: at a sharp decrease in temperature (18 to 4°C), a moderate decrease in salinity (from 30 to 15 psu), a major decrease in salinity (from 30 to 0 psu), and finally a combined stress with a decrease in both temperature and salinity (from 18°C and 30 psu to 4°C and 0 psu). After these stresses, images acquired by confocal laser scanning microscopy (CLSM) revealed changes in copepod cuticle and muscle structure. Low salinity and/or temperature stresses affected both the detection of fluorescence emitted by muscle sarcomeres and the distance between them. In the remaining paper we will use the term sarcomeres to describe the elements located within sarcomeres and emitted autofluorescence signals. Quantitative study showed an increase in the average distance between two consecutive sarcomeres from 2.06 +/- 0.11 μm to 2.44 +/- 0.42 μm and 2.88 +/- 0.45μm after the exposure to major haline stress (18°C, 0 psu) and the combined stress (4°C, 0 psu), respectively. These stresses also caused cuticle cracks which often occurred at the same location, suggesting the cuticle as a sensitive area for osmoregulation. Our results suggest the use of cuticular and muscle autofluorescence as new biomarkers of stress detectable in formalin-preserved <i>P</i>. <i>marinus</i> individuals. Our label-free method can be easily applied to a large number of other copepod species or invertebrates with striated musculature.</p></div

Myofibril Changes in the Copepod <i>Pseudodiaptomus marinus</i> Exposed to Haline and Thermal Stresses - Fig 4

<p>a) percentage of muscle fibers exhibiting stripes. b) Average distances between stripes in each muscle region and each stress. C, control (18°C, 30psu); H15, minor haline stress (18°C, 0psu, 12 min); H0, major haline stress (18°C, 15psu, 210 min); M, mixed stress (4°C, 0psu, less than one minute); T, thermal stress (4°C, 30psu, 75 minutes). Vertical bars correspond to standard errors.</p