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Becker & Hickl flim spc 150 tcspc module

Plasma membrane viscosity in HeLa Kyoto cells with KillerRed during PDT. ( A ) Representative <t>FLIM</t> images of cells with both localizations of KillerRed. The bar is 40 µm, applicable to all images. ( B ) Quantification of viscosity of plasma membranes in HeLa Kyoto cells. Means ± SD, n = 100 cells for each time point. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Flim Spc 150 Tcspc Module, supplied by Becker & Hickl, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer"

Article Title: Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer

Journal: Biomedicines

doi: 10.3390/biomedicines12112550

Plasma membrane viscosity in HeLa Kyoto cells with KillerRed during PDT. ( A ) Representative FLIM images of cells with both localizations of KillerRed. The bar is 40 µm, applicable to all images. ( B ) Quantification of viscosity of plasma membranes in HeLa Kyoto cells. Means ± SD, n = 100 cells for each time point. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Figure Legend Snippet: Plasma membrane viscosity in HeLa Kyoto cells with KillerRed during PDT. ( A ) Representative FLIM images of cells with both localizations of KillerRed. The bar is 40 µm, applicable to all images. ( B ) Quantification of viscosity of plasma membranes in HeLa Kyoto cells. Means ± SD, n = 100 cells for each time point. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Techniques Used: Membrane, Viscosity, Control

Plasma membrane microviscosity in HeLa tumor spheroids after PDT with KillerRed localized in the nuclei (H2B) or within the plasma membrane (PM). ( A ) Schematic representation of the spheroid area (shown by the yellow square) imaged by FLIM. The spheroid had adhered to the glass bottom, and the images were acquired from a depth of ~30 μm. Higher-magnification image of the molecular rotor distribution in spheroid cell membranes indicated by the red squares. The scale bar is 80 μm. ( B ) FLIM images and live/dead (LD) assay of control and treated cells in spheroids. Bar = 80 μm. ( C ) Morphology of control and treated spheroids. The scale bar is 80 μm. ( D ) Quantification of membrane microviscosity of spheroid cells after PDT. Means ± SD, n = 4 spheroids, 60 cells in each. ( E ) Quantitative analysis of dead cells in control and treated cell populations, %. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Figure Legend Snippet: Plasma membrane microviscosity in HeLa tumor spheroids after PDT with KillerRed localized in the nuclei (H2B) or within the plasma membrane (PM). ( A ) Schematic representation of the spheroid area (shown by the yellow square) imaged by FLIM. The spheroid had adhered to the glass bottom, and the images were acquired from a depth of ~30 μm. Higher-magnification image of the molecular rotor distribution in spheroid cell membranes indicated by the red squares. The scale bar is 80 μm. ( B ) FLIM images and live/dead (LD) assay of control and treated cells in spheroids. Bar = 80 μm. ( C ) Morphology of control and treated spheroids. The scale bar is 80 μm. ( D ) Quantification of membrane microviscosity of spheroid cells after PDT. Means ± SD, n = 4 spheroids, 60 cells in each. ( E ) Quantitative analysis of dead cells in control and treated cell populations, %. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Techniques Used: Membrane, Control

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Journal: Biomedicines

Article Title: Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer

doi: 10.3390/biomedicines12112550

Figure Lengend Snippet: Plasma membrane viscosity in HeLa Kyoto cells with KillerRed during PDT. ( A ) Representative FLIM images of cells with both localizations of KillerRed. The bar is 40 µm, applicable to all images. ( B ) Quantification of viscosity of plasma membranes in HeLa Kyoto cells. Means ± SD, n = 100 cells for each time point. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Article Snippet: For viscous imaging, a LSM 880 laser scanning microscope (Carl Zeiss, Gottingen, Germany) equipped with a FLIM SPC 150 TCSPC module (Becker & Hickl GmbH, Berlin, Germany) and a Mai Tai HP femtosecond laser (80 MHz, 140 fs, Spectra Physics, Milpitas, CA, USA) were used.

Techniques: Membrane, Viscosity, Control

Journal: Biomedicines

Article Title: Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer

doi: 10.3390/biomedicines12112550

Figure Lengend Snippet: Plasma membrane microviscosity in HeLa tumor spheroids after PDT with KillerRed localized in the nuclei (H2B) or within the plasma membrane (PM). ( A ) Schematic representation of the spheroid area (shown by the yellow square) imaged by FLIM. The spheroid had adhered to the glass bottom, and the images were acquired from a depth of ~30 μm. Higher-magnification image of the molecular rotor distribution in spheroid cell membranes indicated by the red squares. The scale bar is 80 μm. ( B ) FLIM images and live/dead (LD) assay of control and treated cells in spheroids. Bar = 80 μm. ( C ) Morphology of control and treated spheroids. The scale bar is 80 μm. ( D ) Quantification of membrane microviscosity of spheroid cells after PDT. Means ± SD, n = 4 spheroids, 60 cells in each. ( E ) Quantitative analysis of dead cells in control and treated cell populations, %. * p < 0.05 with control; # p < 0.05 with KillerRed-H2B. CNT KR: control with different localization of KillerRed. H2B: cells with nuclear localization of KillerRed. PM: cells with membrane localization of KillerRed.

Techniques: Membrane, Control

TCSPC FLIM setup. TCSPC FLIM requires a pulsed excitation source. In our system, a multiphoton laser (repetition rate = 78 MHz, T = 12.82 ns, pulse width < 150 fs) is used. The pulsed laser is coupled to the Bio-Rad Radiance 2100 scanning system that is attached to a Nikon TE300 microscope. Thus, spatial information of a specimen is obtained by an XY raster scanning mechanism, and is also available in Z for optical sections. Photons emitted from the specimen pass through the emission (EM) filter and are detected by a fast detector (timing jitter: 25–300 ps). A photomultiplier tube (PMT) with a response time of ~150 ps is placed in the side port of the microscope (nondescanned detection) and is connected to the TCSPC device. The TCSPC device contains PC plug-in boards (commercially available) that function as the time-to-amplitude converter, time-to-digital converter, discriminator and multichannel analyzer. The TCSPC device synchronizes the detector to the excitation pulse and records the arrival time and spatial information for the detected photons. A reference (REF) signal is acquired from a glass cover slip (GC) reflecting 4% of the excitation light, and this reference beam (RB) is passed to a photodiode (PD) that is connected to the TCSPC device. Given a time period (a few seconds or minutes) of accumulating emitted photons for thousands or millions of excitation pulses, a ‘photon counts’ histogram is built for each pixel of an image. The fluorescence lifetime at each pixel can be estimated by fitting the corresponding photon counts (decay) data into a single- or multiexponential model.

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: TCSPC FLIM setup. TCSPC FLIM requires a pulsed excitation source. In our system, a multiphoton laser (repetition rate = 78 MHz, T = 12.82 ns, pulse width < 150 fs) is used. The pulsed laser is coupled to the Bio-Rad Radiance 2100 scanning system that is attached to a Nikon TE300 microscope. Thus, spatial information of a specimen is obtained by an XY raster scanning mechanism, and is also available in Z for optical sections. Photons emitted from the specimen pass through the emission (EM) filter and are detected by a fast detector (timing jitter: 25–300 ps). A photomultiplier tube (PMT) with a response time of ~150 ps is placed in the side port of the microscope (nondescanned detection) and is connected to the TCSPC device. The TCSPC device contains PC plug-in boards (commercially available) that function as the time-to-amplitude converter, time-to-digital converter, discriminator and multichannel analyzer. The TCSPC device synchronizes the detector to the excitation pulse and records the arrival time and spatial information for the detected photons. A reference (REF) signal is acquired from a glass cover slip (GC) reflecting 4% of the excitation light, and this reference beam (RB) is passed to a photodiode (PD) that is connected to the TCSPC device. Given a time period (a few seconds or minutes) of accumulating emitted photons for thousands or millions of excitation pulses, a ‘photon counts’ histogram is built for each pixel of an image. The fluorescence lifetime at each pixel can be estimated by fitting the corresponding photon counts (decay) data into a single- or multiexponential model.

Article Snippet: TCSPC FLIM system BH SPC-150 module (Becker & Hickl) BH PMC-100-0 photomultiplier tube (PMT; Becker & Hickl) BH SPCM software v8.91 (Becker & Hickl) BH SPCImage software v2.9.2.2989 (Becker & Hickl) Biorad Radiance 2100 confocal/multiphoton imaging system (Carl Zeiss) Coherent Verdi pumped Mira 900 tunable mode-locked ultrafast pulsed laser (Coherent) Nikon TE300 inverted epifluorescence microscope (Nikon) Nikon ×60/1.2 NA water immersion (Nikon) Emission filter: HQ 480/40 nm—2p (Chroma Technology, cat. no. NC030887) LaserSharp 2000 software (Carl Zeiss) Glass cover slips (Fisherbrand, 12-545-102 25CIR.-1) Custom-built steel chamber (Note: A commercially available alternative is Invitrogen Attofluor cell chamber, Invitrogen, cat. no. A-7816; details are available at the Invitrogen website) FD FLIM system ISS ALBA FastFLIM system (ISS) ISS VistaVision software v4.0.102 (ISS) Two avalanche photodiodes (APDs; Micro Photon Devices, PDM series) Nichia 5 mW 440 nm pulsed diode laser (Nichia, NDHB510APA) Olympus IX71 inverted epifluorescence microscope (Olympus America) Olympus ×60/1.2 NA water immersion (Olympus America) Semrock FF495-Di02-25-D beam splitter (Semrock, cat. no. 208529) Emission filter, 530/43 nm (Chroma Technology, cat. no. 509302) Emission filter, ET 480/40 nm (Chroma Technology, cat. no. 224544) Chambered cover glass (Thermo Scientific, cat. no. 155379, 155409)

Techniques: Microscopy, Fluorescence

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: Frequency-domain (FD) FLIM setup. FD FLIM uses a modulated light source to excite the specimen. The basic principle of the FD FLIM method is illustrated, showing the phase delay (Φ) and modulation ratio (m = (B/A)/(b/a)) of the emission (Em, dashed sinusoidal curve) relative to the excitation (Ex, solid sinusoidal curve), which are used to estimate the fluorescence lifetime. For cyan fluorescent proteins, a 440-nm pulsed-diode laser is directly modulated by the ISS FastFLIM module at the fundamental frequency of 20 MHz with several sinusoidal harmonics. The modulated laser is coupled to the ISS scanning system attached to an Olympus IX71 microscope to scan specimens. Photons emitted from the specimen travel through the scanning system (descanned detection) and are then routed by a beam splitter (BS) through emission (EM) filters to two identical avalanche photodiodes (APD) detectors. The phase delays and modulation ratios of the emission relative to the excitation are measured at up to seven modulation frequencies (ω = 20–140 MHz) for each XY raster scanning location, and are also available in Z by changing focus. The excitation profile is obtained through a calibration procedure in which a reference fluorophore of known lifetime is used.

Techniques: Fluorescence, Microscopy

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: Photobleaching affects the accuracy of FLIM measurements. Live cells that only express the donor (here, Cerulean-bZip) are used to establish a suitable excitation power level for the TCSPC FLIM-FRET measurements. The amount of photobleaching can be evaluated by monitoring the intensity changes in selected cell regions over time. (a) Shown is a typical photobleaching level at the excitation power level used in the TCSPC FLIM-FRET study (~1.2 mW at the specimen plane). (b) The increased photobleaching when 250% (2.5-fold) of the excitation power level (~ 3 mW at the specimen plane) is used. (c) The effect of the increased photobleaching on the FLIM measurements is shown, demonstrating that the decay profile for Cerulean-bZip is markedly shifted by photobleaching. The mean lifetime obtained from the FLIM data acquired at the high laser power level (~1.3 ns) is much shorter than the normal value (~2.75 ns).

Techniques:

TCSPC FLIM data representation of the FRET standard. (a) The representative Coumarin 6, C5A (donor-alone control) and C5V (FRET standard) raw decay data and corresponding fitting curves are shown. Fittings were carried out using the measured instrument response function (IRF) with a full width at half maximum of ~300 ps. The fluorescent lifetime decay kinetics for Cerulean in the C5A and C5V constructs was determined through the comparisons of fitting the decay data into both single- and double-exponential decay models. There was little difference observed between the single- and double-exponential fits of the C5A decay data, confirmed by the calculated χ2s—1.08 (single) versus 1.07 (double). However, the C5V FRET standard was better represented by the double-exponential fit (χ2 = 1.07) compared with the single-exponential fit (χ2 = 2.38). The results clearly show the quenched state of Cerulean (the donor) in the presence of Venus (the acceptor) resulting from FRET. (b) The lifetime distributions in representative Coumarin 6, C5A or C5V lifetime (overlaid with intensity) images are shown (the apparent lifetime for C5V, see equation (2)). Scale bar, 10 μm.

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: TCSPC FLIM data representation of the FRET standard. (a) The representative Coumarin 6, C5A (donor-alone control) and C5V (FRET standard) raw decay data and corresponding fitting curves are shown. Fittings were carried out using the measured instrument response function (IRF) with a full width at half maximum of ~300 ps. The fluorescent lifetime decay kinetics for Cerulean in the C5A and C5V constructs was determined through the comparisons of fitting the decay data into both single- and double-exponential decay models. There was little difference observed between the single- and double-exponential fits of the C5A decay data, confirmed by the calculated χ2s—1.08 (single) versus 1.07 (double). However, the C5V FRET standard was better represented by the double-exponential fit (χ2 = 1.07) compared with the single-exponential fit (χ2 = 2.38). The results clearly show the quenched state of Cerulean (the donor) in the presence of Venus (the acceptor) resulting from FRET. (b) The lifetime distributions in representative Coumarin 6, C5A or C5V lifetime (overlaid with intensity) images are shown (the apparent lifetime for C5V, see equation (2)). Scale bar, 10 μm.

Techniques: Construct

FD FLIM data representation of the FRET standard. (a) The representative HPTS, Coumarin 6, C5A (donor-alone control) and C5V (FRET standard) data measured at the fundamental modulation frequency (20 MHz) are displayed in the phasor plot. Both HPTS and Coumarin 6 are expected to have a single lifetime component, and this is clearly shown by the phasor plot, in which the lifetime distributions are centered on the universal semicircle of the phasor plot. This is also observed for the lifetime distribution for the cell expressing the C5A fusion protein. (b) However, the lifetime distribution for the cell expressing the C5V fusion protein falls inside the semicircle, and is better fitted with a biexponential model than a monoexponential model, as shown. As HPTS (~5.3 ns) has a much longer lifetime than Coumarin 6 (~2.5 ns), the two populations in the phasor plot are well separated. The phasor plot clearly demonstrates the quenching of the donor (Cerulean) in the C5V protein compared with the unquenched donor in C5A. Scale bar, 10 μm.

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: FD FLIM data representation of the FRET standard. (a) The representative HPTS, Coumarin 6, C5A (donor-alone control) and C5V (FRET standard) data measured at the fundamental modulation frequency (20 MHz) are displayed in the phasor plot. Both HPTS and Coumarin 6 are expected to have a single lifetime component, and this is clearly shown by the phasor plot, in which the lifetime distributions are centered on the universal semicircle of the phasor plot. This is also observed for the lifetime distribution for the cell expressing the C5A fusion protein. (b) However, the lifetime distribution for the cell expressing the C5V fusion protein falls inside the semicircle, and is better fitted with a biexponential model than a monoexponential model, as shown. As HPTS (~5.3 ns) has a much longer lifetime than Coumarin 6 (~2.5 ns), the two populations in the phasor plot are well separated. The phasor plot clearly demonstrates the quenching of the donor (Cerulean) in the C5V protein compared with the unquenched donor in C5A. Scale bar, 10 μm.

Techniques: Expressing

Localization of dimerized C/EBPα-bZip in living cell nucleus using TCSPC FLIM-FRET microscopy. C/EBPα-bZip was tagged with either Cerulean (C) or Venus (V). The fluorescent lifetime decay kinetics for the C–bZip (FRET donor) in the absence and the presence of V–bZip (FRET acceptor) was determined by fitting the measured decay data into a single or double-exponential decay model, respectively, with the measured instrument response function (IRF). Use of a threshold allowed fitting to the pixels in regions of centromeric heterochromatin of the cell nucleus. The comparison between the representative measured decay data points, the fitting curves and the lifetime (overlaid with intensity) images of the two cases clearly shows that the C in cells expressing both C–bZip and V–bZip decayed faster (or has a shorter lifetime) than that in cells expressing C–bZip alone, indicating that the C attached to bZip was quenched by the V attached to bZip because of FRET. Scale bar, 10 μm.

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: Localization of dimerized C/EBPα-bZip in living cell nucleus using TCSPC FLIM-FRET microscopy. C/EBPα-bZip was tagged with either Cerulean (C) or Venus (V). The fluorescent lifetime decay kinetics for the C–bZip (FRET donor) in the absence and the presence of V–bZip (FRET acceptor) was determined by fitting the measured decay data into a single or double-exponential decay model, respectively, with the measured instrument response function (IRF). Use of a threshold allowed fitting to the pixels in regions of centromeric heterochromatin of the cell nucleus. The comparison between the representative measured decay data points, the fitting curves and the lifetime (overlaid with intensity) images of the two cases clearly shows that the C in cells expressing both C–bZip and V–bZip decayed faster (or has a shorter lifetime) than that in cells expressing C–bZip alone, indicating that the C attached to bZip was quenched by the V attached to bZip because of FRET. Scale bar, 10 μm.

Techniques: Microscopy, Expressing

Investigation of the dimerization of C/EBPα-bZip in living cell nucleus using FD FLIM-FRET microscopy. bZip was tagged with either Cerulean (C) or Venus (V). (a) The intensity and lifetime images of representative cells, which only express C–bZip (donor-alone control) and that co-express C–bZip (FRET donor) and V–bZip (FRET acceptor), are compared. The FD FLIM data acquired at the fundamental modulation frequency (20 MHz) is displayed on the phasor plot. The comparison demonstrates a shorter lifetime of Cerulean in the cell that expresses both C–bZip and V–bZip. The lifetimes of C–bZip in the absence and the presence of V–bZip were estimated by single- and double-exponential fittings, respectively. (b) The average donor lifetime, obtained from ten cells that expressed only C–bZip, was 3.15 ns (indicated by the black dot). The apparent lifetimes for 80 regions of interest (ROIs) identified in ten cells coexpressing C–bZip and V–bZip were then determined, and the range was from 2.5 to 3.05 ns, resulting in a variety of energy transfer efficiencies (E) calculated on the basis of equation (3). To investigate how the quenched C–bZip lifetimes were influenced by the acceptor-to-donor ratio, we roughly determined the ratio using the intensities obtained in the acceptor and donor channels for each ROI. With all 80 ROIs, the lifetime (blue dots with a dark blue trend line: R = 0.35) or E (red triangles with a dark red trend line: R = 0.19) shows a negative or positive dependency on the acceptor-to-donor ratio, respectively (scale bar, 10 μm).

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: Investigation of the dimerization of C/EBPα-bZip in living cell nucleus using FD FLIM-FRET microscopy. bZip was tagged with either Cerulean (C) or Venus (V). (a) The intensity and lifetime images of representative cells, which only express C–bZip (donor-alone control) and that co-express C–bZip (FRET donor) and V–bZip (FRET acceptor), are compared. The FD FLIM data acquired at the fundamental modulation frequency (20 MHz) is displayed on the phasor plot. The comparison demonstrates a shorter lifetime of Cerulean in the cell that expresses both C–bZip and V–bZip. The lifetimes of C–bZip in the absence and the presence of V–bZip were estimated by single- and double-exponential fittings, respectively. (b) The average donor lifetime, obtained from ten cells that expressed only C–bZip, was 3.15 ns (indicated by the black dot). The apparent lifetimes for 80 regions of interest (ROIs) identified in ten cells coexpressing C–bZip and V–bZip were then determined, and the range was from 2.5 to 3.05 ns, resulting in a variety of energy transfer efficiencies (E) calculated on the basis of equation (3). To investigate how the quenched C–bZip lifetimes were influenced by the acceptor-to-donor ratio, we roughly determined the ratio using the intensities obtained in the acceptor and donor channels for each ROI. With all 80 ROIs, the lifetime (blue dots with a dark blue trend line: R = 0.35) or E (red triangles with a dark red trend line: R = 0.19) shows a negative or positive dependency on the acceptor-to-donor ratio, respectively (scale bar, 10 μm).

Techniques: Microscopy

Journal: Nature protocols

Article Title: Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

doi: 10.1038/nprot.2011.364

Figure Lengend Snippet: Troubleshooting table.

Techniques: Inverted Microscopy, Fluorescence, Over Expression, Expressing, Transfection, Plasmid Preparation, Concentration Assay, Isolation

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1) Product Images from "Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer"

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