Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabd2102
DOI: 10.1126/sciadv.abd2102


  • Fig. 1 Principle of operation for the PM method and the parallelized PM method.

    (A) Sinusoidal excitation light and the corresponding fluorescence. (B) Phase delay between excitation light and fluorescence. (C) Parallelized PM method with spatially frequency-multiplexed excitation.

  • Fig. 2 Principle of operation for f-DCM.

    (A) Dual-comb optical beating between dual OFCs. (B) 2D spectral mapping of dual OFCs. RF multiplexing of excitation light in (C) the frequency domain and (D) the image domain. RF multiplexing of fluorescence in (E) the image domain and (F) the frequency domain.

  • Fig. 3 Experimental setup and sample.

    (A) Schematic drawing of the experimental setup. OFC1 and OFC2, dual OFCs; PPLNs, chirped periodically poled lithium niobate crystals; BS1 and BS2, beam splitters; L1 and L2, lenses; DM, dichroic mirror; OL, objective lens; BPF1 and BPF2, optical band-pass filters; and PMT1 and PMT2 PMTs. Inset shows optical spectra of OFC1 and OFC2 after converting into green light. a.u., arbitrary units. (B) Schematic drawing of the sample.

  • Fig. 4 Scan-less imaging of fluorescence intensity.

    (A) Temporal waveform and (B) amplitude spectrum of fluorescent RF comb modes. (C) Fluorescence intensity image reconstructed from the amplitude spectrum of fluorescent RF comb modes.

  • Fig. 5 Fluorescence intensity image with respect to the number of accumulated signals.

    (A) 100, (B) 1000, (C) 10,000, and (D) 100,000. (E) Relation between number of accumulated signals and image SNR.

  • Fig. 6 Scan-less bimodal imaging of fluorescence lifetime and fluorescence intensity.

    (A) Temporal waveform, (B) amplitude spectrum, and (C) phase spectrum of fluorescent RF comb modes. (D) Fluorescence intensity image, (E) fluorescence phase delay image, and (F) fluorescence lifetime image.

  • Fig. 7 Fluorescence lifetime images of samples with different fluorescent lifetimes.

    (A) Rhodamine 6G aqueous solution, (B) rhodamine B aqueous solution, (C) rhodamine B methanol solution, and (D) rhodamine B ethanol solution. (E) Comparison of fluorescence lifetime between literature values and measured values.


  • Table 1 Characteristics of prepared fluorescent solutions (37, 38).

    Molar concentration
    lifetime (ns)
    Rhodamine 6G
    aqueous solution
    Rhodamine B aqueous
    Rhodamine B
    methanol solution
    Rhodamine B ethanol

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