Journal: Micromachines

Article Title: Multimodal Nonlinear Microscopy for Therapy Monitoring of Cold Atmospheric Plasma Treatment

doi: 10.3390/mi10090564

Figure Lengend Snippet: Photographs of kINPen-device ® ( a ) and endoscopic plasma device ( b ) during operation; ( c ) Sketch of treatment of a thin tissue section using the kINPen-device ® as shown in ( a ). The plasma covers the whole area of the tissue sections under investigation, approximately 3 × 3 mm² in size. ( d ) The plasma jet of the endoscopic plasma device shown in panel ( b ) is moved across the area of the specimen for treatment of the full section. ( e ) Scheme of the setup used for multimodal nonlinear microscopy combining coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon fluorescence lifetime imaging (2P-FLIM). The picosecond (ps) pulse trains of the Ti: sapphire laser/optical parametric oscillator (OPO) system (1) is coupled into the laser scanning microscope (2). The laser light is focused onto the sample by a microscope objective (5) for image acquisition by scanning the specimen (6). Two-photon excited autofluorescence (TPEF) signals are collected by the objective (5) and reflected to the 2P-FLIM detector (4) by a 600 nm short pass dichroic mirror (3). The TP-FLIM signal is filtered from residual laser light by a 650 nm short pass filter and a 458/64 nm bandpass filter (both Semrock, Rochester, MN, USA). The CARS and SHG signals from the sample are collected in a forward direction by a condenser (7), split by a 514 nm long pass dichroic mirror and detected by photomultiplier tube (PMT) modules (9 SHG, 10 CARS) after spectral filtering as described in .

Article Snippet: The fluorescence signal is collected by the microscope objective and filtered from the laser light by the dichroic mirror (see e number 3) and two filters (short pass 650 nm, bandpass 458/64 nm, Semrock, USA) before detection by the FLIM module (see e number 4, Becker & Hickl, Berlin, Germany).

Techniques: Microscopy, Fluorescence, Imaging, Laser-Scanning Microscopy

Journal: Micromachines

Article Title: Multimodal Nonlinear Microscopy for Therapy Monitoring of Cold Atmospheric Plasma Treatment

doi: 10.3390/mi10090564

Figure Lengend Snippet: Results from two-photon time correlated single photon counting (TCSPC) fluorescence measurements ( a ) The relative change of the total fluorescence (two-photon excitation at 672.5 nm and 832.2 nm, corresponding to single photon excitation at 336 and 416 nm, fluorescence emission at 426–490 nm) after CAP treatment is plotted for all samples normalized to the total fluorescence of the control (=1.0), i.e., all measurements and experimental conditions (treatment time, plasma source, gas composition, specimen), the two plasma devices kINPen MED ® (using Ar and an Ar–oxygen gas mixture for operation and treatment times from 10–70 s) and the endoscopic plasma device (using Ne and a Ne–oxygen gas mixture for operation and treatment times of 10 and 60 s), treatment times of 10 s (in combination with different CAP devices and gas mixtures), longer treatment times (20 s, 60 s and 70 s) and different gas mixtures. Here, Ar and Ar–oxygen gas mixtures were used with the kINPen-device only, Ne and Ne–oxygen gas mixtures were used with the endoscopic plasma device only. ( b ) Change of the median fluorescence lifetime for the parameters of ( a ) for all samples under investigation and all 12 experimental conditions using a monoexponential decay function and plotting the median lifetime of the whole FLIM image. The experimental conditions are in detail: 1,2: Skin, 10 s kINPen, Ar gas; 3: Mucosa lesion, 10 s kINPen, Ar gas; 4: Mucosa, 10 s kINPen, Ar gas; 5,6: Skin, 10 s kINPen, Ar–oxygen gas mixture; 7: Skin, 20 s kINPen, Ar–oxygen gas mixture; 8: Skin, 70 s kINPen, Ar gas; 9: Skin, 10 s endoscopic plasma source, Ne gas; 10,11: Skin, 1 min endoscopic plasma source, Ne gas; 12: Skin, 1 min endoscopic plasma source, Ne–oxygen gas. In eight cases a lifetime increase was observed, while in four cases the fluorescence lifetime was reduced.

Article Snippet: The fluorescence signal is collected by the microscope objective and filtered from the laser light by the dichroic mirror (see e number 3) and two filters (short pass 650 nm, bandpass 458/64 nm, Semrock, USA) before detection by the FLIM module (see e number 4, Becker & Hickl, Berlin, Germany).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Real-time Brain Tumor imaging with endogenous fluorophores: a diagnosis proof-of-concept study on fresh human samples

doi: 10.1038/s41598-018-33134-2

Figure Lengend Snippet: ( a ) 3D scatter plot of three quantitative tissue indicators: the redox ratio, fluorescence lifetime, and SHG signal averaged for each tissue subgroup with a Gaussian ellipsoid fit, ( b ) box plot of the average lifetimes, ( c ) bar graph of the redox ratios for each tissue type with the errors corresponding to the standard deviations across all measurements, and ( d ) overlaid TPEF and SHG intensity images.

Article Snippet: The microscope integrated a FLIM module from PicoQuant (GmbH, Berlin, Germany), in order to acquire fluorescence lifetime imaging.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Multiplexed in vivo imaging with fluorescence lifetime modulating tags

doi: 10.1101/2024.04.12.589181

Figure Lengend Snippet: ( a ) Principle of fluorescence lifetime multiplexing of FAST variants. FAST:fluorogen assemblies with similar spectral properties but different lifetime signatures can be distinguished analyzing their lifetimes. ( b-e ) Intensity-weighted average lifetime distributions of shortT550, midT550 and longT550 in HEK293T cells ( b ), HeLa cells ( c ) and U2OS cells ( d ). N cells from two biological replicates were analyzed (the number N of analyzed cells is given in ( j )). Each cell is color-coded according to the biological replicate it came from. The solid circles correspond to the mean of each biological replicate. The black line represents the mean ± SD of the two biological replicates. ( e ) Means of intensity-weighted average fluorescence lifetimes of shortT550, midT550 and longT550 in the three cell lines. ( f-i ) Intensity-weighted average lifetime distributions of shortT560, midT560 and longT560 in HEK293T cells ( f ), HeLa cells ( g ) and U2OS cells ( h ). N cells from two biological replicates were analyzed (the number N of analyzed cells is given in ( j )). Each cell is color-coded according to the biological replicate it came from. The solid circles correspond to the mean of each biological replicate. The black line represents the mean ± SD of the two biological replicates. ( i ) Means of intensity-weighted average fluorescence lifetimes of shortT560, midT560 and longT560 in the three cell lines. ( j ) Number of cells for each set of experiments. ( k,l ) Photophysical properties of FAST variants with HBR-2,5DM ( k ) and HBR-3,5DM ( l ). Each dot corresponds to one FAST:fluorogen assembly according to their radiative and non-radiative decay constant values, and is scaled to the FQY of the assembly.

Article Snippet: FLIM images were acquired on a Zeiss LSM 980 confocal system coupled with Becker and Hickl FLIM module and equipped with a 20 ξ dry (NA 0.8) objective or a 63 ξ oil immersion (NA 1.4) objective.

Techniques: Fluorescence, Multiplexing