Browsing by Subject "Fluorescence lifetime imaging microscopy (FLIM)"

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    Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells
    (Wiley, 2012) Day, Richard N.; Davidson, Michael W.; Cellular and Integrative Physiology, School of Medicine
    The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.
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    Instant FLIM enables 4D in vivo lifetime imaging of intact and injured zebrafish and mouse brains
    (Optica, 2021) Zhang, Yide; Guldner, Ian H.; Nichols, Evan L.; Benirschke, David; Smith, Cody J.; Zhang, Siyuan; Howard, Scott S.; Medicine, School of Medicine
    Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, most existing FLIM techniques are slow to acquire and process lifetime images, difficult to implement, and expensive. Here, we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 μm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains up to 12 hours.