High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method

Park, K.S.; Choi, Woo June; Eom, Joo Beom; Chang, Ki Soo; Lee, Byeong Ha

doi:10.1016/j.optcom.2015.07.003

Cited by 2 publications

(2 citation statements)

References 7 publications

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“…Similar to lock-in amplification widely applied in electronics, OLID has been demonstrated to be a powerful tool for contrast-enhanced imaging 37 , 38 . OLID utilizes the photoswitchable property of fluorescent probes, including fluorescent dyes, proteins, and inorganic probes 39 – 42 . The fluorescent probes exhibit a non-linear response to the optical modulation, most of which behave switchable between fluorescent and dark states.…”

Section: Resultsmentioning

confidence: 99%

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells

et al. 2022

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The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.

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Section: Resultsmentioning

confidence: 99%

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells

et al. 2022

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“…The proposed method does not use emission filters for discriminating each fluorophore, and thus, the fluorescence sensitivity is intrinsically higher than that when the narrow bandpass filter is used to reduce crosstalk. In our previous research, we demonstrated that applying the four-bucket technique to fluorescence imaging improves the signal-tobackground ratio of the fluorescence image [19]. The current transmission rate of the individual filters has been improving.…”

Section: Discussionmentioning

confidence: 98%

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

Park

Kim

Lee

et al. 2016

Biomed. Opt. Express

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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.

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High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method

Cited by 2 publications

References 7 publications

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells

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

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