Separation of spectrally overlapping fluorophores using intra-exposure excitation modulation

Abstract

Multicolor fluorescence imaging is an excellent method for the simultaneous visualization of multiple structures, although it is limited by the available spectral window. More labels can be measured by distinguishing these on properties, such as their fluorescence dynamics, but usually these dynamics must be directly resolvable by the instrument. We propose an approach to distinguish emitters over a much broader range of light-induced dynamics by combining fast modulation of the light source with the detection of the time-integrated fluorescence. We demonstrate our method by distinguishing four spectrally overlapping photochromic fluorophores within Escherichia coli bacteria, showing that we can accurately classify all four probes by acquiring just two to four fluorescence images. Our strategy expands the range of probes and processes that can be used for fluorescence multiplexing.

Figure 1

Concept of the strategy. (A) Spectrally indistinguishable photochromic labels with different on- and off-switching efficiencies are exposed to quickly varying illumination patterns. (B) The camera detector measures the integrated fluorescence over each pattern, leading to a distinct intensity profile for each of the labels.

Figure 2

Characterization of photoswitching dynamics of four green fluorescent proteins expressed in E.coli. (A) Normalized fluorescence intensity decays of two cycles. (B) Normalized fluorescence intensity traces of FPs when exposed to the irradiation sequence shown in Fig. 1A (shaded region represents the standard deviations).

Figure 3

Separation of four green FPs expressed in E. coli. (A) Fluorescence image of transformed bacteria. (B) False color image of separated proteins. (C) Visualization of the bacterial fluorescence intensities observed in the first three fluorescence images, normalized to the intensity of the fourth image. The different colors show bacteria expressing only the indicated label and a mixture of such bacteria. (n_EGFP= 8516, n_ffDronpa = 10,801, n_ffDronpaF = 14,898, n_ffDronpa2F = 6249, and n_Mixture = 9378). (D) Confusion matrices showing the accuracy of the separation algorithm using four, three, and two fluorescence images (expressed as percentage).