Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

Abstract

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

Physicochemical properties of potential porphyrin photosensitizers for photodynamic therapy

“…Endogenous chromophores already present in tissue can hinder the observation of emission related solely to the desired photosensitizer due to similar emission wavelengths of the endogenous chromophores. However porphyrins fluorescence lifetimes are significantly longer than fluorescence lifetimes of endogenous components [36] and time-resolved evaluation of observed light establishes a reliable method to distinguish between photosensitizers and endogenous chromophores [36][37][38].…”

Azadioxatriangulenium: Synthesis and Photophysical Properties of Reactive Dyes for Bioconjugation

“…[10] In either application, the long fluorescence lifetime enables efficient suppression of autofluorescence and clear identification of probe signal. Similarly, the long fluorescence lifetime allows for lifetime based sensing of biorelevant species and determination of material related properties.…”

Red Emitting Coumarins: Insights of Photophysical Properties with DFT Methods

“…The red emitting dyes are important in OLEDs to complement their blue [9,10] and green [11,12] dye counterparts in fabrication of displays. In biological applications, red emitting dyes stand high and apart due to their ability to produce emission signals discrete from the autofluorescence [13][14][15] of biomolecules (195-600 nm), low energy excitation and emission in biological window [16,17]. The optics used for the red region is simpler, as the scattering effect for the red region is minimal and the sources such as dye lasers in the red region are readily available [18].…”