Ultramicroscopy: development and outlook

Dodt, Hans‐Ulrich; Saghafi, Saiedeh; Becker, Klaus; Jährling, Nina; Hahn, Christian; Pende, Marko; Wanis, Martina; Niendorf, Axel

doi:10.1117/1.nph.2.4.041407

Cited by 27 publications

(18 citation statements)

References 28 publications

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“…The samples were dehydrated in an ascending aqueous concentration series of tetrahydrofuran (THF, Sigma‐Aldrich, Austria, 50%, 80%, 96%, 100%, 1 to 2 hours per step, last step overnight). Afterward, they were immersed in dibenzyl ether (DBE) for refractive index matching until they became transparent (3‐6 weeks) .…”

Section: Methodsmentioning

confidence: 99%

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Saghafi

Haghi-Danaloo

Becker

et al. 2018

Journal of Biophotonics

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Based on the modal analysis method, we developed a model that describes the output beam of a diode-pumped solid state (DPSS) laser emitting a multimode beam. Measuring the output beam profile in the near field and at the constructed far field the individual modes, their respective contributions, and their optical parameters are determined. Using this information, the beam is optically reshaped into a quasi-Gaussian beam by the interference and superposition of the various modes. This process is controlled by a mode modulator unit that includes different meso-aspheric elements and a soft-aperture. The converted beam is guided into a second optical unit comprising achromatic-aspheric elements to produce a thin light sheet for ultramicroscopy. We found that this light sheet is markedly thinner and exhibits less side shoulders compared with a light sheet directly generated from the output of a DPSS multimode laser.

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Section: Methodsmentioning

confidence: 99%

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Saghafi

Haghi-Danaloo

Becker

et al. 2018

Journal of Biophotonics

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“…Incubation was done at room temperature with a period of 1 day each. Finally, transparency was achieved by refractive‐index matching in dibenzyl ether (Becker et al, ; Dodt et al, ). Figure presents images of human tumour specimen taken during cancer surgery that obtained by the optimized UM via various objectives equipped with the modulator.…”

Section: Imaging Of Biological Samples Cleared By a Methods Using Dbe mentioning

confidence: 99%

“…For further improvement of the image quality, we have tried various techniques for optimizing the sample transparency, which is the subject of a further article. Furthermore, we have equipped our objectives with a custom‐made modulator unit to overcome the usual problems that researchers encounter due to refractive index mismatch while collecting the signals through microscope objectives as shown in Figure (Dodt et al, ). The details are given in the patent application DE102013012182A1, and WO2015010783A8.…”

Section: Beam Shaping In Light Sheet Ultramicroscopy‐ummentioning

confidence: 99%

Outlook on optimizing ultramicroscopy imaging technique through optical characterization

Saghafi¹,

Becker²,

Hahn³

et al. 2016

Microscopy Res & Technique

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Here, we present an optically optimized system for static ultramicroscopy imaging technique. The unit for generating an ultra-thin light sheet employs aspheric and meso-optical elements (meso-aspheric system). An analytical as well as an experimental comparison between the light sheet produced by the standard system (using a rectangular slit aperture and one cylindrical lens) and the one produced by our latest optimized system, which converts a symmetrical Gaussian beam into an ultra-thin light sheet is presented. Using the new light sheet in combination with our objective equipped with a modulator unit to compensate the refractive index mismatch between air and mediums with indices of 1.45-1.56, we present high resolution images of various biological samples that were chemically cleared using different methods. They demonstrate a marked improvement in quality, contrast and resolution.

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“…Researchers commonly use air objectives, introducing very large spherical aberration that leads to a dramatic reduction in the detected signal and in the optical sectioning capabilities of LSM. 37 A promising approach to recover diffraction-limited performances is the use of specialized optical elements for the correction of air objectives, as described by Dodt et al 38 If these devices would become commercially available, they could boost the use of organic solvents for clearing.…”

Section: Endogenous Fluorescencementioning

confidence: 99%

Clearing of fixed tissue: a review from a microscopist’s perspective

Costantini²,

et al. 2016

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Abstract. Chemical clearing of fixed tissues is becoming a key instrument for the three-dimensional reconstruction of macroscopic tissue portions, including entire organs. Indeed, the growing interest in this field has both triggered and been stimulated by recent advances in high-throughput microscopy and data analysis methods, which allowed imaging and management of large samples. The strong entanglement between clearing methods and imaging technology is often overlooked, as typical classification of the former is based only on the chemicals used. Here, we review the recent literature in the field, proposing a taxonomy of clearing techniques based on their mating with the major high-throughput microscopies. We hope that this application-oriented classification can help researchers to find the protocol best suited to their experiment among the many present in the literature.

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Ultramicroscopy: development and outlook

Cited by 27 publications

References 28 publications

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Outlook on optimizing ultramicroscopy imaging technique through optical characterization

Clearing of fixed tissue: a review from a microscopist’s perspective

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