Multiplexed imaging in live cells using pulsed interleaved excitation spectral FLIM.
Opt Express
; 32(3): 3290-3307, 2024 Jan 29.
Article
in En
| MEDLINE
| ID: mdl-38297554
ABSTRACT
Multiplexed fluorescence detection has become increasingly important in the fields of biosensing and bioimaging. Although a variety of excitation/detection optical designs and fluorescence unmixing schemes have been proposed to allow for multiplexed imaging, rapid and reliable differentiation and quantification of multiple fluorescent species at each imaging pixel is still challenging. Here we present a pulsed interleaved excitation spectral fluorescence lifetime microscopic (PIE-sFLIM) system that can simultaneously image six fluorescent tags in live cells in a single hyperspectral snapshot. Using an alternating pulsed laser excitation scheme at two different wavelengths and a synchronized 16-channel time-resolved spectral detector, our PIE-sFLIM system can effectively excite multiple fluorophores and collect their emission over a broad spectrum for analysis. Combining our system with the advanced live-cell labeling techniques and the lifetime/spectral phasor analysis, our PIE-sFLIM approach can well unmix the fluorescence of six fluorophores acquired in a single measurement, thus improving the imaging speed in live-specimen investigation.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Diagnostic Imaging
/
Fluorescence Resonance Energy Transfer
Type of study:
Diagnostic_studies
Language:
En
Journal:
Opt Express
/
Opt. express
/
Optics express
Journal subject:
OFTALMOLOGIA
Year:
2024
Document type:
Article
Country of publication:
Estados Unidos