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Published on: September 16, 2014
Time-resolved microscopy for imaging lanthanide luminescence in living cells
Nivriti Gahlaut1, Lawrence W Miller
1Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, Chicago, IL 60607, USA.
Summary
Time-resolved luminescence microscopy overcomes autofluorescence for imaging lanthanide probes in live cells. This new system achieves high-quality images with fast acquisition times, enabling sensitive, quantitative cellular analysis.
Area of Science:
- Biophotonics
- Cellular Imaging
- Spectroscopy
Background:
- Time-resolved luminescence (TRL) microscopy effectively images long-lifetime probes, suppressing short-lifetime autofluorescence in biological samples.
- Conventional fluorescent probes emit more photons but lack the specificity and long lifetimes of lanthanide complexes.
- Low photon emission from lanthanide complexes hinders rapid, high-quality imaging at biologically relevant concentrations.
Purpose of the Study:
- To develop and characterize a novel TRL microscope for enhanced live-cell imaging.
- To evaluate the instrument's performance in background rejection, photon collection, contrast, and signal-to-noise ratio (SNR).
- To demonstrate the feasibility of quantitative imaging of lanthanide luminescence in living cells.
Main Methods:
- A TRL microscope was constructed using a pulsed LED (365 nm) for epi-illumination and an intensified charge-coupled device (ICCD) camera for gated detection.
- Europium chelate-impregnated microspheres (200 nm and 40 nm) were used for instrument performance evaluation.
- A terbium complex (Lumi4-Tb®) was introduced into cultured cells via osmotic lysis of pinocytic vesicles for live-cell imaging.
Main Results:
- Complete autofluorescence suppression was achieved for 200 nm microspheres within the ICCD's time resolution (66.7 ms).
- Approximately 40 nm microspheres were detected within 1-second acquisition times.
- Live cells loaded with terbium complex were imaged in as little as 333 ms, with improved contrast and SNR at longer exposure times and frame summing.
Conclusions:
- The developed TRL microscope provides high sensitivity and precision for imaging lanthanide luminescence.
- The system enables high-resolution, quantitative imaging of lanthanide probes in living cells under physiological conditions.
- This advancement facilitates detailed studies of cellular processes using lanthanide-based probes.
