Optical refocusing three-dimensional wide-field fluorescence lifetime imaging microscopy

Wu, Qiang; Guo, Shangyu; Ma, Yinxing; Gao, F.; Yang, Chengliang; Yang, Ming; Yu, Xiaoyang; Zhang, Xinzheng; Rupp, Romano A.; Xu, Jingjun

doi:10.1364/oe.20.000960

Cited by 7 publications

(5 citation statements)

References 19 publications

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“…Most FLIM systems use laser scanning-based microscopes, that is, confocal [4,5] and multiphoton [1,6,7] with single-channel detectors, such as photomultiplier tube or avalanche photodiode. Meanwhile, widefield type FLIM systems could provide higher imaging speed than scanning systems [8][9][10], which is particularly useful for studies with critical requirements in speed. Frequency domain technique is commonly used in widefield FLIM experiments.…”

Section: Introductionmentioning

confidence: 99%

Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Chen

Kagawa

et al. 2019

Journal of Biophotonics

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Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD‐FLIM system based on a complementary metal‐oxide semiconductor camera equipped with two‐tap true correlated double sampling lock‐in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31‐harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean‐Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD‐FLIM system is a valuable tool in the biomedical field.

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Section: Introductionmentioning

confidence: 99%

Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Chen

Kagawa

et al. 2019

Journal of Biophotonics

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“…However, typical laser scanning systems are slower in data acquisition and the overall setup needed for FLIM is expensive. Instead, widefield FLIM systems could provide higher imaging speed because of massive parallel frame acquisition (Clegg et al, ; Holub, et al, ; Wu et al, ), which is particularly useful for kinetic studies of lifetime changes occurring in a large area. Widefield FLIM can be implemented in two functionally equivalent ways: the frequency domain or the time domain (Dong et al, ).…”

Section: Introductionmentioning

confidence: 99%

Modulated CMOS camera for fluorescence lifetime microscopy

Chen

Holst²,

Gratton

2015

Microscopy Res & Technique

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Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The non-uniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition.

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“…The first category requires moving the sample or the objective lens relative to the other using translational stages or hydraulic, pneumatic or mechanical scanners [2][3][4]. The other category does not require any mechanical movement and utilizes moving and shaping of focus and/or wavefront by exploiting chromatic aberrations [5,6], using adaptive phase compensation techniques [7][8][9][10], or utilizing a variable focal length lens [11][12][13]. These "optical axial scanning" mechanisms can enable relatively faster axial scanning that is less susceptible to motion artifacts in smaller package endoscopes.…”

Section: Introductionmentioning

confidence: 99%

Optical axial scanning in confocal microscopy using an electrically tunable lens

Jabbour

Malik

Olsovsky

et al. 2014

Biomed. Opt. Express

116

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This paper presents the use and characterization of an electrically focus tunable lens to perform axial scanning in a confocal microscope. Lateral and axial resolution are characterized over a >250 µm axial scan range. Confocal microscopy using optical axial scanning is demonstrated in epithelial tissue and compared to traditional stage scanning. By enabling rapid axial scanning, minimizing motion artifacts, and reducing mechanical complexity, this technique has potential to enhance in vivo threedimensional imaging in confocal endomicroscopy.

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Optical refocusing three-dimensional wide-field fluorescence lifetime imaging microscopy

Cited by 7 publications

References 19 publications

Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Modulated CMOS camera for fluorescence lifetime microscopy

Optical axial scanning in confocal microscopy using an electrically tunable lens

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