Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system

Periasamy, Ammasi; Wodnicki, Pawel; Wang, Xue F.; Kwon, Seongwook; Gordon, Gerald W.; Herman, Brian

doi:10.1063/1.1147139

Cited by 89 publications

(53 citation statements)

References 25 publications

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“…Conventionally, nondairy coffee creamer can be used to record the IRF of a FLIM system for visible light excitation. 33 In this study, where infrared light excitation was used, we measured the IRF of the FLIM system through collecting the second-harmonic generation ͑SHG͒ signals emitted from urea crystals. 34 SHG is an ultrafast nonlinear process that delivers a signal at one-half the excitation wavelength.…”

Section: Measuring the Instrument Response Function ͑Irf͒ Of The Flimmentioning

confidence: 99%

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

et al. 2009

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Abstract. Orange fluorescent proteins ͑FPs͒ are attractive candidates as Förster resonance energy transfer ͑FRET͒ partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 ͑mKO2͒ FP as a FRET acceptor for monomeric teal FP ͑mTFP͒ as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency ͑E % ͒ of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red/far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair.

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Section: Measuring the Instrument Response Function ͑Irf͒ Of The Flimmentioning

confidence: 99%

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

et al. 2009

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“…In addition, LSFM has been combined with SR techniques such as STORM [48,[80][81][82], STED [92], and structured illumination [69,75,93]. The versatility of LSFM also allows combining it with more established techniques, such as FLIM [94][95][96], FRET [96], hyperspectral fluorescence [97], Raman [98][99][100][101], and OPT [102,103]. The combined advantages of LSFM are thus shared with multimodal approaches and allow extracting structural, functional, chemical, and molecular information from the sample, enabling a comprehensive interpretation of the biological process of study.…”

Section: 1g Lsfm Allows Multimodal Imaging Of Biological Samplesmentioning

confidence: 99%

Light-sheet microscopy: a tutorial

et al. 2018

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This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.

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“…The fluorescence lifetime is then calculated for each pixel, and an image is constructed. [7][8][9][10][11] This approach reduces the time for data acquisition to construct the image (less than 1 min), since the laser irradiates a wide area of vision. In this case, the temporal resolution depends on the pulse width of the laser and the gate width of the CCD camera.…”

Section: Introductionmentioning

confidence: 99%

“…In the first approach, the fluorescence lifetime is measured by means of time-correlated single-photon counting (TCSPC), and the laser beam is scanned for measuring the cell image. [1][2][3][4][5][6][7] A fluorescence lifetime image can be obtained with a temporal resolution of several tens of picoseconds, when a highly-repetitive laser having a short pulse width is used. Unfortunately, the time required to measure the lifetime image is relatively long, e.g., 30 min, and is dependent on the area of the image.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Lifetime Imaging Microscope Consisting of a Compact Picosecond Dye Laser and a Gated Charge-Coupled Device Camera for Applications to Living Cells

et al. 2006

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An inverted microscope was combined with a compact dye laser with a pulse width of < 190 ps and an intensified chargecoupled device (ICCD) camera with a minimum gate width of 200 ps. The resulting fluorescence lifetime imaging microscope, which has a temporal resolution of 340 ps, was used to measure the fluorescence lifetime of polymer microspherers. The results indicated a fluorescence lifetime of 0.9 ns. The present analytical instrument was also employed in an evaluation of biological cells after labeling them with SYTO 13, a fluorescent dye.

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Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system

Cited by 89 publications

References 25 publications

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

Light-sheet microscopy: a tutorial

Fluorescence Lifetime Imaging Microscope Consisting of a Compact Picosecond Dye Laser and a Gated Charge-Coupled Device Camera for Applications to Living Cells

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