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Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening.

Nedbal J, Visitkul V, Ortiz-Zapater E, Weitsman G, Chana P, Matthews DR, Ng T, Ameer-Beg SM - Cytometry A (2014)

Bottom Line: The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell.The maximum safe throughput of the instrument reaches 3,000 particles per minute.This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Studies, King's College London, United Kingdom; Randall Division of Cell and Molecular Biophysics, King's College London, United Kingdom.

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Two-color fluorescence lifetime flow cytometry. (a) Cholesterol present in cellular membranes increases their lipid order. Di-4-ANEPPDHQ is a membrane-specific dye undergoing spectral and (inset) fluorescence lifetime changes with varying lipid order. (b) Cells treated with cholesterol or cholesterol-depleting cyclodextrin increase or decrease their membrane lipid order, respectively. Di-4-ANEPPDHQ fluorescence lifetime was measured in spectral bands centered on 520 nm and 610 nm. Fluorescent lifetime progressively increased in both spectral channels with higher liquid order. (c) The same cell populations analyzed on a flow cytometer exhibit a blue shift in the emission spectrum toward shorter wavelengths (585 nm → 530 nm) with increasing membrane lipid order. (d) Cells were treated with Opti-MEM® solutions of saturated cholesterol/cyclodextrin (ch100), ch100 diluted in one-to-one volume ratio (ch50), Opti-MEM only as a control (ctrl) and 3.4 mg ml−1 (cd50) or 6.8 mg ml−1 (cd100) solution of methyl-β-cyclodextrin. The graph presents fractional intensities (Eq. (11)) for fluorescence lifetime components Y of 0.2 ns and 2.95 ns measured at wavelengths X of 520 nm and 610 nm for each of the five cell samples. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
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fig02: Two-color fluorescence lifetime flow cytometry. (a) Cholesterol present in cellular membranes increases their lipid order. Di-4-ANEPPDHQ is a membrane-specific dye undergoing spectral and (inset) fluorescence lifetime changes with varying lipid order. (b) Cells treated with cholesterol or cholesterol-depleting cyclodextrin increase or decrease their membrane lipid order, respectively. Di-4-ANEPPDHQ fluorescence lifetime was measured in spectral bands centered on 520 nm and 610 nm. Fluorescent lifetime progressively increased in both spectral channels with higher liquid order. (c) The same cell populations analyzed on a flow cytometer exhibit a blue shift in the emission spectrum toward shorter wavelengths (585 nm → 530 nm) with increasing membrane lipid order. (d) Cells were treated with Opti-MEM® solutions of saturated cholesterol/cyclodextrin (ch100), ch100 diluted in one-to-one volume ratio (ch50), Opti-MEM only as a control (ctrl) and 3.4 mg ml−1 (cd50) or 6.8 mg ml−1 (cd100) solution of methyl-β-cyclodextrin. The graph presents fractional intensities (Eq. (11)) for fluorescence lifetime components Y of 0.2 ns and 2.95 ns measured at wavelengths X of 520 nm and 610 nm for each of the five cell samples. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Mentions: Multi-spectral detection capability, enabling concurrent examination of multiple biomarkers, is essential in modern flow cytometry. We added a second hybrid photomultiplier, additional spectral filters and a TCSPC router to the instrument to enable dual-color fluorescence lifetime flow cytometry. The dual-spectral channel functionality was demonstrated on cells stained with di-4-ANEPPDHQ. The fluorescence properties of di-4-ANEPPDHQ change with the degree of cellular membrane lipid packing (73,74). The dye adopts two photophysically distinct states throughout the membrane consisting of lipid ordered and lipid disordered phases (Fig. 2a). Owing to the significant overlap in the spectral components (74), at least two fluorescence lifetimes were expected across the emission band associated with these two states. Compared to the lipid ordered phase, the dye in the disordered phase should exhibit a red shift in its emission spectrum (74) and a dominant shorter fluorescence lifetime component (73,75) (Fig. 2a, inset). In accordance with the limited total number of photons emitted in a burst (typically < 10,000), the data were fitted with a mono-exponential decay, which closely resembles the average fluorescence lifetime (Fig. 2b). The proportional representation of the two assumed photophysical states of the dye was obtained through a bi-exponential global analysis of the fluorescence decays at both emission wavelengths (Fig. 2d) (69,76,77).


Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening.

Nedbal J, Visitkul V, Ortiz-Zapater E, Weitsman G, Chana P, Matthews DR, Ng T, Ameer-Beg SM - Cytometry A (2014)

Two-color fluorescence lifetime flow cytometry. (a) Cholesterol present in cellular membranes increases their lipid order. Di-4-ANEPPDHQ is a membrane-specific dye undergoing spectral and (inset) fluorescence lifetime changes with varying lipid order. (b) Cells treated with cholesterol or cholesterol-depleting cyclodextrin increase or decrease their membrane lipid order, respectively. Di-4-ANEPPDHQ fluorescence lifetime was measured in spectral bands centered on 520 nm and 610 nm. Fluorescent lifetime progressively increased in both spectral channels with higher liquid order. (c) The same cell populations analyzed on a flow cytometer exhibit a blue shift in the emission spectrum toward shorter wavelengths (585 nm → 530 nm) with increasing membrane lipid order. (d) Cells were treated with Opti-MEM® solutions of saturated cholesterol/cyclodextrin (ch100), ch100 diluted in one-to-one volume ratio (ch50), Opti-MEM only as a control (ctrl) and 3.4 mg ml−1 (cd50) or 6.8 mg ml−1 (cd100) solution of methyl-β-cyclodextrin. The graph presents fractional intensities (Eq. (11)) for fluorescence lifetime components Y of 0.2 ns and 2.95 ns measured at wavelengths X of 520 nm and 610 nm for each of the five cell samples. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
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Related In: Results  -  Collection

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fig02: Two-color fluorescence lifetime flow cytometry. (a) Cholesterol present in cellular membranes increases their lipid order. Di-4-ANEPPDHQ is a membrane-specific dye undergoing spectral and (inset) fluorescence lifetime changes with varying lipid order. (b) Cells treated with cholesterol or cholesterol-depleting cyclodextrin increase or decrease their membrane lipid order, respectively. Di-4-ANEPPDHQ fluorescence lifetime was measured in spectral bands centered on 520 nm and 610 nm. Fluorescent lifetime progressively increased in both spectral channels with higher liquid order. (c) The same cell populations analyzed on a flow cytometer exhibit a blue shift in the emission spectrum toward shorter wavelengths (585 nm → 530 nm) with increasing membrane lipid order. (d) Cells were treated with Opti-MEM® solutions of saturated cholesterol/cyclodextrin (ch100), ch100 diluted in one-to-one volume ratio (ch50), Opti-MEM only as a control (ctrl) and 3.4 mg ml−1 (cd50) or 6.8 mg ml−1 (cd100) solution of methyl-β-cyclodextrin. The graph presents fractional intensities (Eq. (11)) for fluorescence lifetime components Y of 0.2 ns and 2.95 ns measured at wavelengths X of 520 nm and 610 nm for each of the five cell samples. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
Mentions: Multi-spectral detection capability, enabling concurrent examination of multiple biomarkers, is essential in modern flow cytometry. We added a second hybrid photomultiplier, additional spectral filters and a TCSPC router to the instrument to enable dual-color fluorescence lifetime flow cytometry. The dual-spectral channel functionality was demonstrated on cells stained with di-4-ANEPPDHQ. The fluorescence properties of di-4-ANEPPDHQ change with the degree of cellular membrane lipid packing (73,74). The dye adopts two photophysically distinct states throughout the membrane consisting of lipid ordered and lipid disordered phases (Fig. 2a). Owing to the significant overlap in the spectral components (74), at least two fluorescence lifetimes were expected across the emission band associated with these two states. Compared to the lipid ordered phase, the dye in the disordered phase should exhibit a red shift in its emission spectrum (74) and a dominant shorter fluorescence lifetime component (73,75) (Fig. 2a, inset). In accordance with the limited total number of photons emitted in a burst (typically < 10,000), the data were fitted with a mono-exponential decay, which closely resembles the average fluorescence lifetime (Fig. 2b). The proportional representation of the two assumed photophysical states of the dye was obtained through a bi-exponential global analysis of the fluorescence decays at both emission wavelengths (Fig. 2d) (69,76,77).

Bottom Line: The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell.The maximum safe throughput of the instrument reaches 3,000 particles per minute.This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Studies, King's College London, United Kingdom; Randall Division of Cell and Molecular Biophysics, King's College London, United Kingdom.

Show MeSH
Related in: MedlinePlus