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

Journal: Journal of Biomedical Optics

Article Title: Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy

doi: 10.1117/1.3290799

Figure Lengend Snippet: Dissection of the mouse cochlea. (a) Schematic of the cochlea after dissection. An opening overlying the basal turn of the cochlea (i.e., near the stapes and round window) is shown in this example. (b) Schematic of a cross section of one turn of the cochlea. (c) The entire temporal bone, which contains the cochlea, is shown submerged in extracellular solution after removal from the skull, as visualized through the dissecting microscope. The round window, oval window, and stapes can be seen. The scale bar is 1 mm. (d) The cochlea has been glued upright into a recording chamber, so that only the cochlea (white area to the left of the oval and round windows) is visible. Particular care needs to be taken to leave the round window, oval window, and stapes unencumbered by glue. The scale bar is 1 mm. (e) The apical turn of the cochlea has been opened. This permits visualization of the cochlear epithelium where the hair cells are located (curved dark area at tip of arrow). About one third to one half of a cochlea turn can be opened. The scale bar is 0.5 mm.

Article Snippet: Imaging setup Imaging was performed using a custom-built upright microscope (Fig. ) based on a moveable objective microscope (MOM, Sutter Instruments, Novato, California).

Techniques: Dissection, Microscopy

Journal: Journal of Biomedical Optics

Article Title: Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy

doi: 10.1117/1.3290799

Figure Lengend Snippet: Schematic of the microscope system.

Article Snippet: Imaging setup Imaging was performed using a custom-built upright microscope (Fig. ) based on a moveable objective microscope (MOM, Sutter Instruments, Novato, California).

Techniques: Microscopy

Journal: Journal of Biomedical Optics

Article Title: Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy

doi: 10.1117/1.3290799

Figure Lengend Snippet: Schematic diagram of the experiment setup. The size of the cochlea is exaggerated for clarity. In this diagram, the opening in the cochlea is shown overlying the basal turn. (b) Image of the preparation taken through the experimental microscope with the tip of the piezoactuator in contact with the stapes. The opening of the cochlea was over the apical turn in this example. The scale bar is 1 mm.

Article Snippet: Imaging setup Imaging was performed using a custom-built upright microscope (Fig. ) based on a moveable objective microscope (MOM, Sutter Instruments, Novato, California).

Techniques: Microscopy

Journal: Nature Communications

Article Title: Microfluidics for understanding model organisms

doi: 10.1038/s41467-022-30814-6

Figure Lengend Snippet: Novel microfabrication techniques combined with creativity enable the production of different microfluidic tools that, in turn, facilitate the development of novel experimental paradigms to be used with small multicellular model organisms. Microfluidics empower researchers with tremendous control over sample stimulation while also enabling precise manipulation of samples, and being compatible with high-resolution live imaging. The potential of these microtechnologies is accelerating as they are integrated with additional technologies, become more accessible, and are designed to be multifunctional. Figure created with BioRender.com.

Article Snippet: Fig. 3 Microfluidic systems can deliver stimulations with high precision and at throughputs not previously attainable while making once laborious experiments less demanding. a - i A 3D-printed microfluidic device compatible with a custom-built light-sheet microscope to stimulate zebrafish with precise flow vectors for brain-wide calcium imaging . a - ii A microfluidic device with an array of microposts for analysis of sleep behavior of C. elegans . b A microfluidic system that automatically aligned Drosophila embryos and precisely compressed them using pneumatically actuated deformable sidewalls with simultaneous live imaging . c A microfluidic system capable of trapping hundreds of C. elegans embryos quickly and enabling efficient reagent exchange . d - i A microfluidic system visualized the response of olfactory receptor neurons (ORNs) of Drosophila larva in response to controlled odorant exposure . d - ii A microfluidic device with integrated glass capillaries and a microneedle for chemical injection of Drosophila larvae . e A microfluidic device exposed Drosophila embryo to a thermal gradient along the anterior-posterior axis using two laminar flow streams with different temperatures .

Techniques: Control, Imaging

Journal: Nature Communications

Article Title: Microfluidics for understanding model organisms

doi: 10.1038/s41467-022-30814-6

Figure Lengend Snippet: a-i A microfluidic device with a shape memory alloy actuator immobilized zebrafish and examined the hydrodynamic flow resultant from tail beating . a - ii A worm chamber for visualization and periodic immobilization of C. elegans . b A microfluidic device coupled with a live detection system designed to gently immobilize, orient, and inject zebrafish larvae . c - i A microfluidic system to sort fruit fly samples at high throughput and enrichment ratio . c - ii A non-invasive zebrafish larvae sorting system based on microfluidics that utilized light and acoustics to corral individual samples . All panels are cropped and adapted versions of the originals. Panel c - ii was reproduced from Mani and Chen , with the permission of AIP Publishing.

Article Snippet: Fig. 3 Microfluidic systems can deliver stimulations with high precision and at throughputs not previously attainable while making once laborious experiments less demanding. a - i A 3D-printed microfluidic device compatible with a custom-built light-sheet microscope to stimulate zebrafish with precise flow vectors for brain-wide calcium imaging . a - ii A microfluidic device with an array of microposts for analysis of sleep behavior of C. elegans . b A microfluidic system that automatically aligned Drosophila embryos and precisely compressed them using pneumatically actuated deformable sidewalls with simultaneous live imaging . c A microfluidic system capable of trapping hundreds of C. elegans embryos quickly and enabling efficient reagent exchange . d - i A microfluidic system visualized the response of olfactory receptor neurons (ORNs) of Drosophila larva in response to controlled odorant exposure . d - ii A microfluidic device with integrated glass capillaries and a microneedle for chemical injection of Drosophila larvae . e A microfluidic device exposed Drosophila embryo to a thermal gradient along the anterior-posterior axis using two laminar flow streams with different temperatures .

Techniques: High Throughput Screening Assay

Journal: Nature Communications

Article Title: Microfluidics for understanding model organisms

doi: 10.1038/s41467-022-30814-6

Figure Lengend Snippet: a - i A 3D-printed microfluidic device compatible with a custom-built light-sheet microscope to stimulate zebrafish with precise flow vectors for brain-wide calcium imaging . a - ii A microfluidic device with an array of microposts for analysis of sleep behavior of C. elegans . b A microfluidic system that automatically aligned Drosophila embryos and precisely compressed them using pneumatically actuated deformable sidewalls with simultaneous live imaging . c A microfluidic system capable of trapping hundreds of C. elegans embryos quickly and enabling efficient reagent exchange . d - i A microfluidic system visualized the response of olfactory receptor neurons (ORNs) of Drosophila larva in response to controlled odorant exposure . d - ii A microfluidic device with integrated glass capillaries and a microneedle for chemical injection of Drosophila larvae . e A microfluidic device exposed Drosophila embryo to a thermal gradient along the anterior-posterior axis using two laminar flow streams with different temperatures . All panels are cropped and adapted versions of the originals. Panel c is adapted with permission from Charles et al. , copyright 2020 American Chemical Society.

Article Snippet: Fig. 3 Microfluidic systems can deliver stimulations with high precision and at throughputs not previously attainable while making once laborious experiments less demanding. a - i A 3D-printed microfluidic device compatible with a custom-built light-sheet microscope to stimulate zebrafish with precise flow vectors for brain-wide calcium imaging . a - ii A microfluidic device with an array of microposts for analysis of sleep behavior of C. elegans . b A microfluidic system that automatically aligned Drosophila embryos and precisely compressed them using pneumatically actuated deformable sidewalls with simultaneous live imaging . c A microfluidic system capable of trapping hundreds of C. elegans embryos quickly and enabling efficient reagent exchange . d - i A microfluidic system visualized the response of olfactory receptor neurons (ORNs) of Drosophila larva in response to controlled odorant exposure . d - ii A microfluidic device with integrated glass capillaries and a microneedle for chemical injection of Drosophila larvae . e A microfluidic device exposed Drosophila embryo to a thermal gradient along the anterior-posterior axis using two laminar flow streams with different temperatures .

Techniques: Microscopy, Imaging, Injection