High efficiency beam splitter for multifocal multiphoton microscopy

Nielsen, Tim; Fricke, Marc; Hellweg, Dirk; Andresen, Peter L.

doi:10.1046/j.1365-2818.2001.00852.x

Cited by 135 publications

(116 citation statements)

References 9 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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“…Devices, which allow a faster scanning of the sample, e.g. acusto-optical deflectors, are good alternatives to the standard galvanometer scanners, but apart of their complicated and sometimes unstable instrumental implementation, they still have the disadvantage that due to photodamage only 10% of the full laser power can be used for biological investigations 20,21 . Techniques based on a multi-beam scanning combined with synchronous fluorescence detection, i.e.…”

Section: Fluorescence Imaging Techniquesmentioning

confidence: 99%

Fluorescence lifetime imaging in biosciences: technologies and applications

Niesner

Gericke

2008

Front. Phys. China

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The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.

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“…Pulsed near-infrared laser light from a tunable Ti:sapphire laser (see Table 1) was directed to a mirror-based beam multiplexer (Nielsen et al, 2001). The principle of the beam multiplexer is to combine a central 50%-beamsplitter mirror with high-reflectivity mirrors to both of its sides (see Fig.…”

Section: Principle Of Multiline Tplsmmentioning

confidence: 99%

“…As we show later, multiline TPLSM can also be used with point-scanning detection, although at a much lower temporal resolution than with imaging detection (see later this section and Section 3 for a critical evaluation of both detection schemes). Nielsen et al (2001). A linear array of in this case eight laser beams is generated by repeated separation at a 50%-beamsplitter mirror (BS) and reflection at high-reflectivity mirrors (HR).…”

Section: Emission-light Detection By Imaging In Multiline Tplsmmentioning

confidence: 99%

“…The principle of multiline TPLSM builds on a mirror-based beam multiplexer, which divides a single laser beam into multiple beams (Nielsen et al, 2001). In TPLSM, where excitation is intrinsically confined to the tiny focal volume of a laser, this beam multiplexer can be used to generate several of such excitation spots in the focal plane of the sample.…”

Section: Introductionmentioning

confidence: 99%

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Application of multiline two-photon microscopy to functional in vivo imaging

Kurtz

Fricke

Kalb

et al. 2006

Journal of Neuroscience Methods

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High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and 'spine'-like structures. Calcium dynamics depended on branch size, but 'spines' did not systematically differ from their 'parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode.

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High efficiency beam splitter for multifocal multiphoton microscopy

Cited by 135 publications

References 9 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

Fluorescence lifetime imaging in biosciences: technologies and applications

Application of multiline two-photon microscopy to functional in vivo imaging

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