Related Experiment Video
Updated: May 20, 2026
06:01
Fluorescence Lifetime Macro Imager for Biomedical Applications
Published on: April 7, 2023
Fluorescence lifetime imaging microscopy using near-infrared contrast agents.
R Nothdurft1, P Sarder, S Bloch
1Washington University, School of Medicine, Department of Radiology, Scott Avenue, St. Louis, Missouri 63110, USA.
Journal of Microscopy
|July 14, 2012
Summary
Near-infrared fluorescence lifetime imaging microscopy (FLIM) now images live cells using probes emitting beyond 700 nm. This technique distinguishes cell populations and estimates dye concentrations, bridging in vitro and in vivo imaging.
Area of Science:
- Biophotonics
- Cellular Imaging
- Fluorescence Microscopy
Background:
- Single-photon fluorescence lifetime imaging microscopy (FLIM) is limited to visible spectrum emissions.
- Near-infrared (NIR) probes offer advantages for deep tissue penetration and reduced autofluorescence.
Purpose of the Study:
- To explore the feasibility of NIR (emission >700 nm) fluorescent probes for live-cell FLIM.
- To establish a method for distinguishing cell populations and estimating dye concentrations using NIR FLIM.
Main Methods:
- Utilized a confocal microscope with a 785 nm laser, red-enhanced photomultiplier tube, and time-correlated single photon counting.
- Applied NIR fluorescent dyes (cypate and DTTCI) to label live cells.
- Acquired and analyzed fluorescence lifetime distributions.
Main Results:
- Demonstrated successful lifetime measurements of NIR dyes (cypate, DTTCI) in live cells.
- Showcased NIR FLIM's ability to distinguish cell populations based on distinct dye lifetimes.
- Enabled estimation of relative dye concentrations within complex cellular microenvironments.
Conclusions:
- NIR FLIM is a viable technique for live-cell imaging beyond the visible spectrum.
- This method provides a bridge for translating in vitro fluorophore characterization to in vivo small animal imaging.
- NIR FLIM offers new possibilities for cellular analysis and in vivo applications.
Related Concept Videos
01:05Total Internal Reflection Fluorescence Microscopy
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
01:37Super-resolution Fluorescence Microscopy
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.