Investigating dye performance and crosstalk in fluorescence enabled bioimaging using a model system

Abstract

Detailed imaging of biological structures, often smaller than the diffraction limit, is possible in fluorescence microscopy due to the molecular size and photophysical properties of fluorescent probes. Advances in hardware and multiple providers of high-end bioimaging makes comparing images between studies and between research groups very difficult. Therefore, we suggest a model system to benchmark instrumentation, methods and staining procedures. The system we introduce is based on doped zeolites in stained polyvinyl alcohol (PVA) films: a highly accessible model system which has the properties needed to act as a benchmark in bioimaging experiments. Rather than comparing molecular probes and imaging methods in complicated biological systems, we demonstrate that the model system can emulate this complexity and can be used to probe the effect of concentration, brightness, and cross-talk of fluorophores on the detected fluorescence signal. The described model system comprises of lanthanide (III) ion doped Linde Type A zeolites dispersed in a PVA film stained with fluorophores. We tested: F18, MitoTracker Red and ATTO647N. This model system allowed comparing performance of the fluorophores in experimental conditions. Importantly, we here report considerable cross-talk of the dyes when exchanging excitation and emission settings. Additionally, bleaching was quantified. The proposed model makes it possible to test and benchmark staining procedures before these dyes are applied to more complex biological systems.

Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

“…The microscope was used to record spectrally resolved images of a model system comprised of zeolites doped with lanthanide(III) ions in a polyvinyl alcohol (PVA) film on a glass cover slip. The model system is described in detail elsewhere [ 14 ]. In each image, the data recorded in each pixel were treated according to the scheme outlined in Fig 1 .…”

“…The methods here described are reiterated and adapted from ref. [ 14 ]. Fluorescein F18 was from (Sigma-Aldrich, St. Louis, MO, US), MitoTracker Red (MT, CMXRos) from Molecular Probes (ThermoFisher Scientific, Waltham, MA) and ATTO647N from ATTO-TEC GmbH (Siegen, Germany).…”

“…Prior to use, the microscope slides were cleaned by pyrolysis at 450°C for a minimum of 1 hour. The samples used in this manuscript are the same as in ref [ 14 ].…”

“…We recently introduced a model system that we suggest as a benchmark for developing new microscopy methods [ 14 ]. In our model system we use lanthanide ions as the signal that is to be discriminated from a background simulated by emission from bright organic fluorophores, see Methods and Materials for details.…”

Best Practices for Technical Reproducibility Assessment of Multiplex Immunofluorescence

“…The second aspect to consider is crosstalk, which is an additional signal from the non-target fluorophore captured by the microscopic system (60, ( Arppe et al, 2017 ). There are commonly recommended practices to cut back this effect; for example, crosstalk is often considerably reduced by choosing fluorophores whose excitation and emission spectra match those of the corresponding channels but minimally overlap those of non-corresponding ones.…”