Abstract
We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization.
Highlights
Epi-fluorescence microscopy is a very simple method that can identify the locations of various organelles of single cells using staining of a selectively targeted organelle [1,2,3,4,5]
For a better understanding of single cells, each organelle in the cells should be stained by individual fluorescent dyes having different emission spectra, and the stained organelles should be imaged with individual filter sets
We demonstrated epi-fluorescence microscopy for multicolor imaging by employing a digital lock-in technique known as four-bucket detection
Summary
Epi-fluorescence microscopy is a very simple method that can identify the locations of various organelles of single cells using staining of a selectively targeted organelle [1,2,3,4,5]. For a better understanding of single cells, each organelle in the cells should be stained by individual fluorescent dyes having different emission spectra, and the stained organelles should be imaged with individual filter sets. Several kinds of epi-fluorescence microscopy have been developed for multicolor imaging using different filter sets (i.e., full-multiband filter set, multiband Pinkel filter set, multiband Sedat filter set). Configurations using the multiband Pinkel filter and Sedat filter sets can achieve higher quality images than the full-multiband filter set, but these methods are time-consuming because filters need to be exchanged with a motorized wheel in order to obtain a multicolor image
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