Figure 1.

(a) Schematics of the frequency-encoded multicolor confocal fluorescence setup with color-blind detection. Up to four modulated wavelengths excite the sample at a time using an AOTF. Fluorescence generated by the sample is collected by the same microscope objective as used for excitation and relayed directly to the detector through a set of notch filters. An optional polarizing beam splitter and second detector can be placed on the detection side to enable polarization sensitive measurements. (b) The fluorescence incident on the detector is converted into a digital data stream, and an FFT is performed on a pixel-by-pixel basis to convert the photon data stream into the frequency domain, where the amplitudes of the signal at each modulation frequency are measured and plotted as pixel intensities in individual images for each excitation source. (c–g) Representative demodulated confocal images of a sample containing four colors of spectrally overlapping fluorescent nanospheres are shown (absorption spectra shown in Fig. S2), recorded at (c) 470 nm, (d) 514 nm, (e) 561 nm, and (f) 640 nm. (g) A composite overlay of the individual images is shown, with arrows highlighting individual nanospheres of each color. Images were acquired simultaneously with a 2-ms pixel dwell time, maximal values of 72, 60, 119, and 108 photons in a pixel, respectively. Scale bars, 2 μm. To see this figure in color, go online.