Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source

Grant, David M.; Elson, Daniel S.; Schimpf, Damian N.; Dunsby, Christopher; Requejo-Isidro, José; Auksorius, Egidijus; Munro, Ian; Neil, Mark A. A.; French, Paul M. W.; Nye, Edwin R.; Stamp, Gordon; Courtney, Patrick

doi:10.1364/ol.30.003353

Cited by 44 publications

(31 citation statements)

References 13 publications

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“…This instrument bears a resemblance to a number of multifocal multiphoton configurations, whose major advantage is that they increase the image acquisition rate over that of point-scanning systems (12,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Most use widefield detection for parallelized detection of the multiple focal spots and this is important for 2P-MSIM for two critical reasons.…”

Section: Msim Excitation Was Originally Implemented With a Digital MImentioning

confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

112

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Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 μm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.multiphoton | superresolution F luorescence microscopy is an invaluable tool for biologists.Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200-300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 μm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.

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Section: Msim Excitation Was Originally Implemented With a Digital MImentioning

confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

112

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“…The technique is almost 100 years old though it is still used in various modern imaging instruments such as the optical confocal microscope [12]. The Nipkow disk used in our terahertz imaging system is made of aluminium, consisting of a series of 24 apertures with a diameter of 2 mm for scanning the object image, as shown in we can obtain a 50 mm x 50 mm imaging area with an axial (Y-axis ) resolution of 2 mm/pixel.…”

Section: Terahertz Single Pixel Imaging Based On a Nipkow Diskmentioning

confidence: 99%

Terahertz single pixel imaging based on a Nipkow disk

Grant

Saha

et al. 2012

Opt. Lett.

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“…When spatial resolution is needed, such as with microscopy, different considerations come into play depending on the kind of microscope used. One major difference between the laser scanning confocal microscope and the camera-based microscopes is that in the former the detector works in the serial mode, although there are some recent scanning instruments using multiple foci in conjunction with a camera to collect the image (Grant et al, 2005;van Munster et al, 2007). For FLIM instruments operating in the serial mode, a bottleneck in the rate of data acquisition is caused by the recovery time of the TAC element that is common to the TCSPC-based instruments.…”

Section: Introductionmentioning

confidence: 99%

A novel fluorescence lifetime imaging system that optimizes photon efficiency

Colyer

Lee

Gratton

2007

Microscopy Res & Technique

171

149

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Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost digital frequency domain FLIM (DFD-FLIM) system, which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. On the basis of the mathematical model, we present an error analysis that shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination. Microsc. Res. Tech. 71:201-213, 2008. V V C 2007 Wiley-Liss, Inc. INTRODUCTIONWe have developed a physical implementation and statistical model of a new method for fluorescence lifetime imaging (FLIM) data collection and analysis. Our approach falls in the category of the ''frequency domain'' approach to lifetime acquisition, yet uses a detector operating in the photon counting mode. This digital frequency domain (DFD) method overcomes the problems of duty cycle, modulation of the detector gain and expensive radio frequency synthesizers used in the classical analog frequency domain approach. In our approach we implemented all the operations performed in a frequency-domain lifetime instrument in a digital form using a single field programmable chip. Since all operations including the generation of the light modulation frequency, the generation of the cross-correlation sampling frequency and the assignment of the time of arrival of a photon to a bin are digital, there are no calibrations or adjustments to be performed. The mathematical model presented below fully accounts for all the elements of the DFD method. In addition, the mathematical model reproduces, as a limiting case, the principle of the time-correlated single photon counting (TCSPC) approach. Therefore, on a common statistical basis we can compare the two approaches and derive some general conclusions about the relative precision of the two methods. We found that with proper system design the two methods can be made to have comparable precision. More importantly, the mathematical model was used to maximize the precision of the DFD implementation and to determine which parameters are crucial to reach optimal performance. In terms of precision of the lifetime measurement, we were able to fully quantify the effect of the instrument response including the jitter of the detection system.Fluorescence lifetime is a fundamental spectroscopic quantity that allows quantitative analysis through several approaches, including the identification of molecu-

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Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source

Cited by 44 publications

References 13 publications

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Terahertz single pixel imaging based on a Nipkow disk

A novel fluorescence lifetime imaging system that optimizes photon efficiency

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