Journal: Communications Biology

Article Title: Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

doi: 10.1038/s42003-024-06702-8

Figure Lengend Snippet: a Schematic diagram of fluorescence lifetime-based multiplexing. Analog fluorescence signals (blue) are recorded, sampled (orange dots in ( b )), and processed via GPU computing to obtain their Fourier transform. c The geometrical unmixing process. d Confocal and lifetime images of three fluorescent solutions at 640 nm. Phosphate-buffered saline was utilized as the solvent for the three fluorescent solutions with the concentration 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{\mu g}{ml}$$\end{document} μ g m l . Phasor histogram corresponding to ( d ), ( e ) (5 × 5) median filter was applied before phasor plotting, and f individual contribution maps in the phasor domain for Alexa647, Atto647, and Attorho14. The blue lines in ( b ) and ( e ) represent the angle corresponding 1.5 ns. Scale bar: 1.5 mm.

Article Snippet: We utilized a custom-developed FLIM (Fluorescence Lifetime Imaging Microscopy) system specifically designed for our FLEX application (Intek Medi, iRT-FLIM-001, Supplementary Fig. ).

Techniques: Fluorescence, Multiplexing, Saline, Solvent, Concentration Assay

Journal: Communications Biology

Article Title: Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

doi: 10.1038/s42003-024-06702-8

Figure Lengend Snippet: a Fluorescence intensity images of 5 μm thin sections of FFPE tonsil tissue captured at four distinct excitation wavelengths (405, 450, 520, and 640 nm) and emission channels (415–450 nm, 510–550 nm, 550–610 nm, and 660–700 nm, respectively) before (left column) and after (right column) undergoing the autofluorescence elimination procedure. b Quantitative comparison of the autofluorescence elimination process across 3 distinct tissues. c Fluorescence lifetime image depicting AF555-CD3, both prior (top) and subsequent (bottom) to the elimination of autofluorescence. Insets in each image display the phasor histograms.

Article Snippet: We utilized a custom-developed FLIM (Fluorescence Lifetime Imaging Microscopy) system specifically designed for our FLEX application (Intek Medi, iRT-FLIM-001, Supplementary Fig. ).

Techniques: Fluorescence, Comparison

Journal: Communications Biology

Article Title: Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

doi: 10.1038/s42003-024-06702-8

Figure Lengend Snippet: a Intensity-weighted fluorescence lifetime images of individual tissue samples. the scale bar represents 1 mm. The average photon counts per image were over 1000 after the application of a median filter. b the means and standard deviations of fluorescence lifetimes corresponding to each image in Fig. 3a.Individual data points from each image were represented as colored dots.

Article Snippet: We utilized a custom-developed FLIM (Fluorescence Lifetime Imaging Microscopy) system specifically designed for our FLEX application (Intek Medi, iRT-FLIM-001, Supplementary Fig. ).

Techniques: Fluorescence

Journal: Communications Biology

Article Title: Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

doi: 10.1038/s42003-024-06702-8

Figure Lengend Snippet: a Graphical illustration of quality factor and phasor-domain crosstalk. b Quality factor variation with lifetime difference and photon count per pixel. c Monte Carlo simulations for quantifying crosstalk as a function of the quality factor, with the red dot indicating the mean from 1000 trials and error bars representing standard deviation. d Examination of antibody effects on fluorescence lifetime using seven different PE-antibodie conjugates (CD324, CD3, CD45, CD68, CD235, CD33, and CD15), showing a phasor standard deviation of 0.011, approximately 0.08 ns in fluorescence lifetime.the blue lines represent the angle corresponding 1 to 5 ns. e fluorescence lifetime images of tonsil tissues stained with three different fluorophores conjugated with two different antibodies. Scale bar: 1 mm ( f ) Violin plot corresponding to each image in Fig. 4e images.

Article Snippet: We utilized a custom-developed FLIM (Fluorescence Lifetime Imaging Microscopy) system specifically designed for our FLEX application (Intek Medi, iRT-FLIM-001, Supplementary Fig. ).

Techniques: Standard Deviation, Fluorescence, Staining

Journal: American Journal of Physiology - Cell Physiology

Article Title: Cigarette smoke activates CFTR through ROS-stimulated cAMP signaling in human bronchial epithelial cells

doi: 10.1152/ajpcell.00099.2017

Figure Lengend Snippet: An inhibitor of STIM1 translocation reduces the increase in intracellular [cAMP] ([cAMP]i) induced by CSE: evidence for store-operated cAMP signaling. CFBE cells expressing WT-CFTR were transfected with the exchange protein directly activated by cAMP (Epac)-based FRET sensor for cAMP (H134, Epac-SH134) or with a negative control construct mTurquoise2 (mT2). The lifetime of mT2 fluorescence was measured in the absence or presence of the FRET acceptor using fluorescence lifetime imaging microscopy (FLIM) to examine changes in intracellular [cAMP]. A: representative images of Epac-SH134-transfected cells under different conditions. Top left: basal conditions; top right: +20% CSE; bottom left: 20 µM STIM1 inhibitor ML9; bottom right: 20 µM STIM1 inhibitor ML9 + CSE. B: fluorescence lifetime of mT2 and H134 under different conditions. Means ± SE, n = no. of cells in two independent experiments, one-way ANOVA with Tukey’s multicomparison post test; effect of CSE: ***P < 0.001; effect of STIM1 inhibitor: †P < 0.01.

Article Snippet: Fluorescence lifetime images were acquired using a Zeiss LSM-710-fluorescence lifetime imaging microscopy (FLIM) (PicoQuant) microscope at ×20 magnification.

Techniques: Translocation Assay, Expressing, Transfection, Negative Control, Construct, Fluorescence, Imaging, Microscopy

Journal: Fertility and sterility

Article Title: Metabolic imaging using Fluorescence Lifetime Imaging Microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes

doi: 10.1016/j.fertnstert.2018.07.022

Figure Lengend Snippet: Comparative assessments of Clpp+/+ and Clpp−/− oocytes. A) Brightfield images show gross morphological features of oocytes, while NADH FLIM intensity images reflect the mitochondrial distribution. B) Obvious defects in Clpp−/− oocytes were not observed with brightfield, but fluorescence imaging revealed aberrant mitochondrial distributions in many oocytes. C) Metabolic imaging of Clpp+/+ (n=55) and Clpp−/− oocytes (n=52) detected highly significant differences in five of the eight parameters measured. Parameters are plotted in order of decreasing separation, with asterisks indicating the following p-values: (*: p<10−11) and (**: p<10−26) D) If the three most sensitive metabolic parameters (NADH long lifetime, NADH fraction engaged, and FAD long lifetime) are represented in a 3D plot, we can fit a plane that perfectly separates the two data sets. E) Clpp+/+ oocytes (n=46) and Clpp−/− oocytes (n=43) were lysed to obtain individual mtDNA copy number measurements. T-tests on mtDNA measurements showed only a marginally significant difference with a p-value of 0.042. Error bars represent standard errors.

Article Snippet: Disclosures: Tim Sanchez and Dan Needleman are co-founders and stake holders at LuminOva, Inc. Summary sentence: Mitochondrial dysfunction caused by reproductive aging (mild) and CLPP-deficiency (severe) is accurately detected by Fluorescence Lifetime Imaging Microscopy (FLIM).

Techniques: Fluorescence, Imaging

Journal: Fertility and sterility

Article Title: Metabolic imaging using Fluorescence Lifetime Imaging Microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes

doi: 10.1016/j.fertnstert.2018.07.022

Figure Lengend Snippet: Comparative assessments of oocytes from old (12-month) and young (3-month) mice. A,B) Neither brightfield images nor FLIM intensity images revealed obvious differences in morphology or mitochondrial distribution. 20um bar. C) Conversely, metabolic imaging measurements on the same oocytes effectively differentiated young (n=35) and old (n=29) groups, with four of the eight parameters showing significant differences. Parameters are plotted in order of decreasing separation, with asterisks indicating the following p-values: (*:p<0.02) and (**: p<10−3). P-values were not as low as with Clpp, the more severe case of metabolic dysfunction. D) If the three most sensitive metabolic parameters (NADH intensity NADH short lifetime, and NADH long lifetime) are represented in a 3D plot, we can draw a plane that effectively separates the two data sets; however, there is more overlap between the distributions than in the more extreme case of Clpp. E) mtDNA copy number measurements were taken on young (n=35) and old (n=29) oocytes, and no significant difference between the two groups was observed (p=0.14). Error bars represent standard errors.

Article Snippet: Disclosures: Tim Sanchez and Dan Needleman are co-founders and stake holders at LuminOva, Inc. Summary sentence: Mitochondrial dysfunction caused by reproductive aging (mild) and CLPP-deficiency (severe) is accurately detected by Fluorescence Lifetime Imaging Microscopy (FLIM).

Techniques: Imaging

Journal: Fertility and sterility

Article Title: Metabolic imaging using Fluorescence Lifetime Imaging Microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes

doi: 10.1016/j.fertnstert.2018.07.022

Figure Lengend Snippet: Evaluation of safety for varying photodoses of FLIM illumination. Top: Reactive oxygen species levels were measured via HC-DCFDA fluorescence (custom units). Significant differences between illuminated and non-illuminated embryos were not observed for any of the photodoses studied. Embryos exposed to 30mM H2O2 were measured as a positive control. Error standard error bars represent variation between individual embryo measurements. Bottom: Embryos were cultured on the microscope, and blastocyst development rates of illuminated embryos were compared to non-illuminated embryos in the same dish. Embryos cultured in a standard incubator were used as a control. FLIM illumination did not have any significant impact on blastocyst development rates. Standard error bars represent variation between experiment batches.

Article Snippet: Disclosures: Tim Sanchez and Dan Needleman are co-founders and stake holders at LuminOva, Inc. Summary sentence: Mitochondrial dysfunction caused by reproductive aging (mild) and CLPP-deficiency (severe) is accurately detected by Fluorescence Lifetime Imaging Microscopy (FLIM).

Techniques: Fluorescence, Positive Control, Cell Culture, Microscopy, Control