Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

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.

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy

“…Exposure time ( τ ), defined as the amount of time that a photodetector is subjected to the light for image formation, is the key factor for the maximum frame acquisition rate. For simultaneous multicolor imaging, the maximum frame acquisition rate equals 1/( τ ) [ 11 , 13 , 18 ]. Since a multicolor image is formed by the sequential acquisition of emitted fluorescence light from three different spectrum bands using the round-robin method, the maximum frame rate is computed as 1/(3 τ ).…”

“…In numerous application domains ranging from single-cell visualization to rapid diagnosis, multicolor fluorescence microscopy is recognized as a key emerging technology [11][12][13][14][15][16][17][18][19][20][21][22][23]. The working principle of this microscopy technique is based on performing imaging using spectrally-different conventional fluorescent dyes [11], endogenous fluorophores [22], fluorescent proteins [24], or quantum dots [25]. Relatively narrow excitation and emission wavelength ranges enable image acquisition from different spectrum bands with multiple fluorophores.…”

“…However, performing imaging by positioning the filters either manually or using a motorized drive creates a delay and limits the image acquisition rate. An appealing solution for stationary components is to use a multi-band emission filter [ 11 , 15 ]. Since the delay associated with the spectrum band change is removed, the acquisition is limited by the frame rate of the imaging sensor.…”

“…To form focused multicolor fluorescence images using widefield microscopy, individual image acquisition from different focal planes is required. Since micro-structures in the previously used samples have almost the same height, defocus aberration correction is not addressed for widefield multicolor imaging [ 11 , 18 , 31 , 32 ]. In this study, our multicolor fluorescence microscope is specially developed for defocus aberration correction by spectrally-different image acquisition from multiple focal planes within the simplified tumor model.…”

Investigating dye performance and crosstalk in fluorescence enabled bioimaging using a model system

“…Several solutions have been proposed to deal with cross-talk. [ 38 – 45 ] As the model system gives direct access to data from systems with and without co-localization, the amount of crosstalk can be readily determined.…”