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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points
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Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.

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    Programmable lifetime imaging (PLI) accelerates fluorescence lifetime measurements by using a digital micromirror device for object-oriented data acquisition. This novel method significantly reduces imaging time for biological assays compared to traditional techniques.

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    Area of Science:

    • Biophotonics
    • Spectroscopy
    • Microscopy

    Background:

    • Time-correlated single-photon counting (TCSPC) is crucial for lifetime spectroscopy in biological assays.
    • Traditional laser scanning fluorescence lifetime imaging (FLIM) is slow due to point-by-point scanning.
    • Wide-field TCSPC methods offer speed but compromise temporal resolution and are limited by photon counting rates.

    Purpose of the Study:

    • To introduce Programmable Lifetime Imaging (PLI) for faster and accurate fluorescence lifetime determination.
    • To combine wide-field imaging with advanced TCSPC detectors and a digital micromirror device (DMD).
    • To enable object-oriented lifetime analysis in biological assays.

    Main Methods:

    • PLI utilizes total internal reflection excitation and a DMD to segment fluorescence from individual objects.
    • Fluorescent emission is projected onto the DMD for sequential acquisition and lifetime determination.
    • An automated PLI implementation uses camera feedback for targeted measurement of emitting objects.

    Main Results:

    • PLI demonstrates sensitivity in fixed cell assays through manual segmentation.
    • Automated PLI effectively segments fluorescence from objects of interest.
    • PLI reduces fluorescence lifetime data acquisition time by over an order of magnitude compared to laser scanning FLIM.

    Conclusions:

    • PLI offers a significant advancement in fluorescence lifetime imaging speed and efficiency.
    • The object-oriented approach minimizes data acquisition to relevant areas, maximizing efficiency.
    • PLI provides accurate lifetime determination with substantially reduced acquisition times for biological assays.