Quasi-real-time fluorescence imaging with lifetime dependent contrast

J Biomed Opt. 2011 Aug;16(8):086001. doi: 10.1117/1.3609229.


Conventional fluorescence lifetime imaging requires complicated algorithms to extract lifetimes of fluorophores and acquisition of multiple data points at progressively longer delay times to characterize tissues. To address diminishing signal-to-noise ratios at these progressively longer time delays, we report a time-resolved fluorescence imaging method, normalized fluorescence yield imaging that does not require the extraction of lifetimes. The concept is to extract the "contrast" instead of the lifetime value of the fluorophores by using simple mathematical algorithms. This process converts differences in decay times directly to different intensities. The technique was verified experimentally using a gated iCCD camera and an ultraviolet light-emitting diode light source. It was shown that this method can distinguish between chemical dyes (Fluorescein and Rhodamine-B) and biomedical samples, such as powders of elastin and collagen. Good contrast was obtained between fluorophores that varied by less than 6% in lifetime. Additionally, it was shown that long gate times up to 16 ns achieve good contrast depending upon the samples to be studied. These results support the feasibility of time-resolved fluorescence imaging without lifetime extraction, which has a potential clinical role in noninvasive real-time imaging.

MeSH terms

  • Achilles Tendon / chemistry
  • Algorithms*
  • Animals
  • Calibration
  • Cattle
  • Collagen / chemistry
  • Elastin / chemistry
  • Equipment Design
  • Fluorescein / chemistry
  • Ligaments / chemistry
  • Microscopy, Fluorescence / instrumentation*
  • Microscopy, Fluorescence / methods*
  • Rhodamines / chemistry
  • Signal Processing, Computer-Assisted
  • Signal-To-Noise Ratio


  • Rhodamines
  • Collagen
  • Elastin
  • rhodamine B
  • Fluorescein