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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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Experimental setup and data analysis. (a) A microfluidics chip (CHIP) flow channel was positioned in the focus of a widefield epi-fluorescence microscope objective (OBJ). A circular illumination patch (IS) intersects flowing particles exciting fluorescence. The microscope features a picosecond pulsed laser (LASER), beam expander (BE), variable field aperture (AP), excitation filter (EX), tube lens (TL), dichroic mirror (DM), emission filter set (EM), and a detector (D), converting photons into electrical signals for TCSPC. (b) Train of detected photons is segmented into single-particle bursts. (c) Single-particle bursts are extracted by step-wise histogramming of photon arrival macro times into 20 μs time bins (RAW, blue line), median filtering (MED, black line), and applying minimum threshold (TH, dotted line). Bursts are characterized by burst duration (BD), photon count (PC), and mean count rate (MCR). (d) Fluorescence lifetime of each particle is obtained by fitting an exponential decay model into transients created by binning micro times of photons contained in each burst. (e) Burst parameters are plotted into 2D scatter plots used in flow cytometry analysis. 2.9% bursts result from the improper segmentation of two-particle events (inset). [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
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fig01: Experimental setup and data analysis. (a) A microfluidics chip (CHIP) flow channel was positioned in the focus of a widefield epi-fluorescence microscope objective (OBJ). A circular illumination patch (IS) intersects flowing particles exciting fluorescence. The microscope features a picosecond pulsed laser (LASER), beam expander (BE), variable field aperture (AP), excitation filter (EX), tube lens (TL), dichroic mirror (DM), emission filter set (EM), and a detector (D), converting photons into electrical signals for TCSPC. (b) Train of detected photons is segmented into single-particle bursts. (c) Single-particle bursts are extracted by step-wise histogramming of photon arrival macro times into 20 μs time bins (RAW, blue line), median filtering (MED, black line), and applying minimum threshold (TH, dotted line). Bursts are characterized by burst duration (BD), photon count (PC), and mean count rate (MCR). (d) Fluorescence lifetime of each particle is obtained by fitting an exponential decay model into transients created by binning micro times of photons contained in each burst. (e) Burst parameters are plotted into 2D scatter plots used in flow cytometry analysis. 2.9% bursts result from the improper segmentation of two-particle events (inset). [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Mentions: Measurements were performed on a modified automated microscope, developed in-house (61,67). The microscope (Fig. 1a) was equipped with a ×20 0.5 NA air objective (Nikon Instruments Ltd, Kingston Upon Thames, UK). A 473 nm pulsed diode laser (Becker & Hickl GmbH, Berlin, Germany) operated at 80 MHz was used for wide-field epifluorescent illumination of a circular area of 120 μm diameter. The filter set consisted of a 470 ± 11 nm single band-pass excitation filter (part FF01–470/22–25, Semrock, Rochester, NY), a FITC/TRITC dual band-pass dichroic mirror (part 59004, Chroma Inc, Olching, Germany), 520 ± 30 nm single band-pass excitation filter (part FF01–520/60-25, Semrock), and 488 nm long-pass filter (part LP02–488RE-25, Semrock). For dual-color detection, a 560 nm dichromatic mirror (part T560lpxr, Chroma) was added to split the emission light. The red part of the spectrum was further selected by 610 ± 37.5 nm emission filter (part HQ610/75, Chroma) and a 561 nm long-pass filter (part LP02–561RE-25, Semrock). The excitation source power was regulated by a series of absorptive ND filters in a motorized filter wheel (Thorlabs UK Ltd, Ely, UK). The filtered fluorescence light was relayed onto two hybrid photomultiplier detectors (part HPM-100-40, Becker & Hickl GmbH). The appropriate detector was connected to a TCSPC module (SPC-830, Becker & Hickl GmbH) and a control module (DCC-100, Becker & Hickl GmbH). In experiments requiring acquisition in two spectral channels, a detector router (HRT-41, Becker & Hickl GmbH) was connected between the two detectors and the TCSPC card. Microscope adjustment and acquisition were controlled by the Gray Institute Open Microscope control software (67), SPCM and DCC-100 programs (Becker & Hickl GmbH).


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)

Experimental setup and data analysis. (a) A microfluidics chip (CHIP) flow channel was positioned in the focus of a widefield epi-fluorescence microscope objective (OBJ). A circular illumination patch (IS) intersects flowing particles exciting fluorescence. The microscope features a picosecond pulsed laser (LASER), beam expander (BE), variable field aperture (AP), excitation filter (EX), tube lens (TL), dichroic mirror (DM), emission filter set (EM), and a detector (D), converting photons into electrical signals for TCSPC. (b) Train of detected photons is segmented into single-particle bursts. (c) Single-particle bursts are extracted by step-wise histogramming of photon arrival macro times into 20 μs time bins (RAW, blue line), median filtering (MED, black line), and applying minimum threshold (TH, dotted line). Bursts are characterized by burst duration (BD), photon count (PC), and mean count rate (MCR). (d) Fluorescence lifetime of each particle is obtained by fitting an exponential decay model into transients created by binning micro times of photons contained in each burst. (e) Burst parameters are plotted into 2D scatter plots used in flow cytometry analysis. 2.9% bursts result from the improper segmentation of two-particle events (inset). [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
© Copyright Policy
Related In: Results  -  Collection

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fig01: Experimental setup and data analysis. (a) A microfluidics chip (CHIP) flow channel was positioned in the focus of a widefield epi-fluorescence microscope objective (OBJ). A circular illumination patch (IS) intersects flowing particles exciting fluorescence. The microscope features a picosecond pulsed laser (LASER), beam expander (BE), variable field aperture (AP), excitation filter (EX), tube lens (TL), dichroic mirror (DM), emission filter set (EM), and a detector (D), converting photons into electrical signals for TCSPC. (b) Train of detected photons is segmented into single-particle bursts. (c) Single-particle bursts are extracted by step-wise histogramming of photon arrival macro times into 20 μs time bins (RAW, blue line), median filtering (MED, black line), and applying minimum threshold (TH, dotted line). Bursts are characterized by burst duration (BD), photon count (PC), and mean count rate (MCR). (d) Fluorescence lifetime of each particle is obtained by fitting an exponential decay model into transients created by binning micro times of photons contained in each burst. (e) Burst parameters are plotted into 2D scatter plots used in flow cytometry analysis. 2.9% bursts result from the improper segmentation of two-particle events (inset). [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
Mentions: Measurements were performed on a modified automated microscope, developed in-house (61,67). The microscope (Fig. 1a) was equipped with a ×20 0.5 NA air objective (Nikon Instruments Ltd, Kingston Upon Thames, UK). A 473 nm pulsed diode laser (Becker & Hickl GmbH, Berlin, Germany) operated at 80 MHz was used for wide-field epifluorescent illumination of a circular area of 120 μm diameter. The filter set consisted of a 470 ± 11 nm single band-pass excitation filter (part FF01–470/22–25, Semrock, Rochester, NY), a FITC/TRITC dual band-pass dichroic mirror (part 59004, Chroma Inc, Olching, Germany), 520 ± 30 nm single band-pass excitation filter (part FF01–520/60-25, Semrock), and 488 nm long-pass filter (part LP02–488RE-25, Semrock). For dual-color detection, a 560 nm dichromatic mirror (part T560lpxr, Chroma) was added to split the emission light. The red part of the spectrum was further selected by 610 ± 37.5 nm emission filter (part HQ610/75, Chroma) and a 561 nm long-pass filter (part LP02–561RE-25, Semrock). The excitation source power was regulated by a series of absorptive ND filters in a motorized filter wheel (Thorlabs UK Ltd, Ely, UK). The filtered fluorescence light was relayed onto two hybrid photomultiplier detectors (part HPM-100-40, Becker & Hickl GmbH). The appropriate detector was connected to a TCSPC module (SPC-830, Becker & Hickl GmbH) and a control module (DCC-100, Becker & Hickl GmbH). In experiments requiring acquisition in two spectral channels, a detector router (HRT-41, Becker & Hickl GmbH) was connected between the two detectors and the TCSPC card. Microscope adjustment and acquisition were controlled by the Gray Institute Open Microscope control software (67), SPCM and DCC-100 programs (Becker & Hickl GmbH).

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