Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Abstract

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators

“…5500 M −1 cm −1 at 365 nm, L5 is brighter at that wavelength than the Lumi4(Tb) complex (brightness at 365 nm, 5200 M −1 cm −1 ) that we have used successfully for time-gated luminescence microscopy. 4, 6 …”

Lanthanide Fluorobenzoates as Bio‐Probes: a Quest for the Optimal Ligand Fluorination Degree

“…Ag rowing interest for lanthanidel uminescence is accelerated by the increased demand for luminescent materials for ab road range of applicationst hat include telecommunications, [1][2][3] lighting, electroluminescent devices, [3,4] bio-sensors, [5][6][7][8][9] and bio-imagingp robes. [5,10,11] Their rebirtha nd increasing importance in biology and materials research has been highlighted by numerous reviews.…”

NIR‐to‐NIR Two‐Photon Scanning Laser Microscopy Imaging of Single Nanoparticles Doped by YbIII Complexes

“…[1] Traditionally,t he fluorescent probest hat are used for such applicationsa re organic dyes, although they present inevitable limitations such as short lifetimes, small Stokes shift and low photostability.T oo vercome these problems-and depending on the targeted application-metal complexes such as those involving Ln III cations have been widelys tudied due to their uniquelys harp" atom-like" line shapes and long-lived emission. [5] These Eu-or Tb-luminescent bioprobes found actually important commercial or fundamental applications forincellulo and in vivo imaging [6] or sensing [7] or as bio-conjugated tag acting as donor for time-resolved FRET-based applications. [5] These Eu-or Tb-luminescent bioprobes found actually important commercial or fundamental applications forincellulo and in vivo imaging [6] or sensing [7] or as bio-conjugated tag acting as donor for time-resolved FRET-based applications.…”

“…[2] To date, among the series of Ln III ions, europium and terbium complexes have been used preferentially for these purposes because of their optimized luminescence brightness (defineda st he product of the molar absorption coefficient and the quantum yield efficiency) [3,4] and their ms range lifetime enabling time-gated detection. [5] These Eu-or Tb-luminescent bioprobes found actually important commercial or fundamental applications forincellulo and in vivo imaging [6] or sensing [7] or as bio-conjugated tag acting as donor for time-resolved FRET-based applications. [8] All these experimentsa re performed with aV is-to-Vis configuration with both excitation and detection in the UV/Vis spectralrange [250-650nm].However,t hese wavelengthsa re strongly absorbed or scattered by tissues and may cause important biological photo-damage or induces trong parasitic auto-fluorescence.…”