Measuring BODIPY Fluorescence with UV Vis Spectroscopy

BODIPY is an organic fluorophore with impressive fluorescent quantum yield, small stokes shift and impressive chemical and photostability. These are often used in biological labelling and as an organic fluorescent dye.

To fabricate the samples, we dissolved BODIPY-Br at varying concentrations in a solution of polystyrene (a transparent polymer) in the solvent toluene. We then coated this onto quartz-coated glass substrates using an Ossila Spin Coater to form 180 nm thin films (see our complete guide to spin coating for more information on how to fabricate thin films through spin coating).

Absorbance and fluorescence spectra for the molecular dye BODIPY-Br (a bromine-substituted boron dipyrromethene derivative) are shown below. These spectra of BODIPY fluorescence were measured using the Ossila Optical Spectrometer. Here, we see that optical spectroscopy can be incredibly useful tool for identify and explain the behaviour and properties of small molecules at varying concentrations.

As expected, the absorbance of the films increases as the relative dye concentration is increased. This is because the more absorbent material in the film, the more light is absorbed.

absorbance spectra of BODIPY at various concentrations — Absorbance spectra of BODIPY-Br/polystyrene thin films with varying dye concentration

Fluorescence Spectra

Perhaps unexpectedly, the peak fluorescence intensity of the films does not increase linearly with dye concentration. In fact, the 50% film has the lowest peak intensity, while the 10% film has the greatest.

This reduction in fluorescence intensity at higher concentrations is due to intermolecular interactions caused by the aggregation of the BODIPY-Br molecules (see Fluorescence Quenching and Non-Radiative Relaxation). At low concentrations, the fluorescent molecules are spatially separated, and no intermolecular interactions are possible. As the relative concentration of the molecules is increased, the spacing between adjacent molecules is reduced and intermolecular interactions become significant. This reduces the total fluorescence from the sample.

fluorescence spectra of BODIPY at various concentrations — Fluorescence spectra of BODIPY-Br/polystyrene thin films with varying dye concentration

Normalised Fluorescence Spectra

In addition to the reduction in the fluorescence intensity, the spectral shape also changes. The most obvious change is shown in the figure below, which shows the normalised fluorescence spectra. As the concentration of BODIPY-Br increases, the shoulder around 600 nm emerges and increases in intensity. This is also an effect of aggregation and in the case of BODIPY-Br is due to the formation of excimers (see Exciplex and Excimer Absorption and Emission).

normalised spectra of BODIPY at various concentrations — Normalised fluorescence spectra of BODIPY-Br/polystyrene thin films with varying dye concentration. The red arrow shows the emergence of the shoulder around 600 nm that corresponds to excimer emission at higher concentrations.

Zooming into the main peak shows how the position of the peak redshifts with increasing concentration. This is further evidence of aggregation resulting in excimer formation.

Reduced fluorescence spectra of BODIPY at various concentrations — Close-up of main fluorescence peak, illustrating its increasing redshift as the BODIPY-Br/polystyrene concentration is increased

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Contributing Authors

Written by

Kirsty McGhee

PhD Student Collaborator