saiedeh saghafi - Academia.edu (original) (raw)

Papers by saiedeh saghafi

Research paper thumbnail of <title>Beam propagation analysis in unstable laser resonators (ULR): low to high magnification</title>

19th Congress of the International Commission for Optics: Optics for the Quality of Life, 2003

ABSTRACT

Research paper thumbnail of Chemical Clearing of Brains

Handbook of Neurophotonics, 2020

Research paper thumbnail of Ultramicroscopy of Nerve Fibers and Neurons: Fine-Tuning the Light Sheets

Research paper thumbnail of Chemical Clearing of GFP-Expressing Neural Tissues

Research paper thumbnail of 3D histopathology of human tumours by fast clearing and ultramicroscopy

Scientific Reports, 2020

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malign... more Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a–c is fully reversible, allowing the morphological correlation of one and the same histological structures, onc...

Research paper thumbnail of Breaking the diffraction limit of light sheets allows fast isotropic imaging of large samples by ultramicroscopy

Proceedings for Annual Meeting of The Japanese Pharmacological Society, 2018

By breaking the diffraction limit of light sheets of low numerical aperture (NA) we were able to ... more By breaking the diffraction limit of light sheets of low numerical aperture (NA) we were able to generate extremely long thin sheets of light with a thickness in the one micron range and a vastly increased Rayleigh range. We measured the thickness of the light sheets with different methods including standard point spread function measurement with beads. By using these light sheets in our ultramicroscope fast 3D imaging of whole mouse brains with objectives with a large field of view was possible. Due to the extremely low divergence of the light sheets mouse brains could be reconstructed from a single stack of optical sections with isotropic resolution. The light sheets used were essentially non-Gaussian generated by new optics we developed. Compared to a Gaussian light sheet of the same NA our new light sheet is much thinner. Thus the diffraction limit which holds also for low NA Gaussian light sheets was significantly surpassed. These optics will allow the application of ultramicroscopy to ever increasing samples beyond the whole mouse brain range. Besides mouse brains we imaged also cleared whole adult drosophilae. We were able to get good transparency for all developmental stages of the insect from larvae to adult animals with fully preserved GFP signal. We showed that also multiview imaging of cleared adult drosophilae is possible and and allows isotropic resolution with standard ultramicroscopy for such kind of specimens. With the new light sheet and a new ultrafast clearing also large human samples could be quickly imaged.

Research paper thumbnail of Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Journal of Biophotonics, 2018

This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Research paper thumbnail of A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity

Science Advances, 2020

A tailored protocol combines pigment removal with clearing, facilitating labeling and whole-body ... more A tailored protocol combines pigment removal with clearing, facilitating labeling and whole-body imaging across phylogeny.

Research paper thumbnail of Effect of salinity and radiation on proline accumulation in seeds of canola (Brassica napus L.)

Plant, Soil and Environment, 2010

Since laser beam may affect plant traits, it was used to enhance accumulation of proline in rapes... more Since laser beam may affect plant traits, it was used to enhance accumulation of proline in rapeseed and therefore to improve its tolerance to the salinity stress. This investigation was performed to study the effect of NaCl concentration in irrigated water (0, 100, 200 and 300 mmol NaCl) on proline accumulation of Canola (Brassica napus L.) after laser irradiation (Red, Infra-red and Nd:YAG) at two exposure treatments. In each exposure, seeds were irradiated for three minutes once or twice by the laser set. Free proline content in leaves increased significantly by increasing of NaCl concentration. Also proline content significantly increased with irradiation by laser beam. The Red laser irradiation used once and the Nd:YAG laser used twice had the greatest effect on the proline content whereas the Infrared laser had a low effect. Double application of irradiation induced a significantly higher amount of proline in the leaves compared to only one application. This is the first repor...

Research paper thumbnail of High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

Journal of Biophotonics, 2019

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts ben... more Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep‐tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)‐expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP‐expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity‐dependent expression pattern of tdTomato.

Research paper thumbnail of High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster

Nature Communications, 2018

The fruit fly, Drosophila melanogaster, is an important experimental model to address central que... more The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique we developed, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope set-up utilising an improved light-sheet generator allows us to visualise long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applyin...

Research paper thumbnail of Trichobilharzia regenti (Schistosomatidae): 3D imaging techniques in characterization of larval migration through the CNS of vertebrates

Micron, 2016

Migration of parasitic worms through the host tissues, which may occasionally result in fatal dam... more Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches.

Research paper thumbnail of Ultramicroscopy: development and outlook

Neurophotonics, 2015

We present an overview of the ultramicroscopy technique we developed. Starting from developments ... more We present an overview of the ultramicroscopy technique we developed. Starting from developments 100 years ago, we designed a light sheet microscope and a chemical clearing to image complete mouse brains. Fluorescence of green fluorescent protein (GFP)-labeled neurons in mouse brains could be preserved with our 3DISCO clearing and high-resolution three-dimensional (3-D) recordings were obtained. Ultramicroscopy was also used to image whole mouse embryos and flies. We improved the optical sectioning of our light sheet microscope by generating longer and thinner light sheets with aspheric optics. To obtain high-resolution images, we corrected available air microscope objectives for clearing solutions with high refractive index. We discuss how eventually super resolution could be realized in light sheet microscopy by applying stimulated emission depletion technology. Also the imaging of brain function by recording of mouse brains expressing cfos-GFP is discussed. Finally, we show the first 3-D recordings of human breast cancer with light sheet microscopy as application in medical diagnostics.

Research paper thumbnail of Recent developments in light sheet ultramicroscopy imaging techniques

Advanced Microscopy Techniques IV; and Neurophotonics II, 2015

ABSTRACT Ultramicroscopy-UM allows 3D-vizualization of biological specimens with μm-resolution. T... more ABSTRACT Ultramicroscopy-UM allows 3D-vizualization of biological specimens with μm-resolution. The spatial intensity distribution and thickness of the light sheet illuminating the specimen have the foremost impact on the quality of image in UM. In this paper, we investigate and compare different designs for generating light sheet and their optical characteristics

Research paper thumbnail of Ultramicroscopy: light-sheet-based microscopy for imaging centimeter-sized objects with micrometer resolution

Cold Spring Harbor protocols, 2013

Ultramicroscopy (UM) is a powerful imaging technique that achieves precise and accurate three-dim... more Ultramicroscopy (UM) is a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. It was developed for specimens in the size range of ∼1-15 mm, such as whole mouse brains, mouse embryos, mouse organs, and Drosophila melanogaster. In UM, the specimen is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. UM is closely related to a growing family of comparable microscopy approaches based on light sheet illumination developed in recent years. This article presents an overview of light-sheet-based microscopy and describes the underlying physics of light sheet generation. The assembly of an "ultramicroscope" for investigating fixed chemically cleared tissue is described in detail, and the functions of the essential components, such as mechanics, camera, and objectives, are discussed. Finally, practical applications of UM...

Research paper thumbnail of Immunostaining, dehydration, and clearing of mouse embryos for ultramicroscopy

Cold Spring Harbor protocols, 2013

This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful ima... more This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. In combination with fluorescein isothiocyanate (FITC) immunostaining, UM allows visualization of somatic motor and sensorial nerve fibers in whole mouse embryos. Even the fine branches of the sensomotoric fibers can be visualized over a distance of up to several millimeters. In this protocol, mouse embryos are fixed and immunostained in preparation for UM. Because UM requires the excitation light sheet to travel throughout the entire horizontal width of the specimen, specimens usually have to be rendered transparent before microscope inspection. Here, the embryos are dehydrated in ethan...

Research paper thumbnail of Dehydration and clearing of whole mouse brains and dissected hippocampi for ultramicroscopy

Cold Spring Harbor protocols, 2013

This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultram... more This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, mouse brains and hippocampi are carefully dissected and dehydrated, and then cleared in a solution of benzyl benzoate and benzyl alcohol.

Research paper thumbnail of Dehydration and clearing of adult Drosophila for ultramicroscopy

Cold Spring Harbor protocols, 2013

This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imagi... more This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, Drosophila melanogaster adults are fixed, dehydrated in ethanol, and then cleared in a solution of benzyl alcohol and benzyl benzoate.

Research paper thumbnail of Recent development in light Ultramicroscopy using aspherical optical elements

Optical Systems Design 2012, 2012

ABSTRACT Here we present a new light sheet generator unit for Ultramicroscopy (UM) employing a co... more ABSTRACT Here we present a new light sheet generator unit for Ultramicroscopy (UM) employing a combination of optical lenses with aspherical surface structure. UM allows 3D-vizualization of chemically transparent biological specimens with μm-resolution. Improving optical characteristics parameters of light sheet such as the uniformity factor of spatial intensity distribution along the line of focus, the thickness of light sheet, and chromatic aberrations are the most important criteria in this design. Since we do not use any hard edge aperture there is no truncation of the beam and laser energy is used more efficiently. Due to these improvements, a marked enhancement in presenting fine details of biological specimens such as Drosophila melanogaster, entire mouse brain, and hippocampus are achieved.

Research paper thumbnail of Alteration of optical and morphological properties of polycarbonate illuminated by visible/IR laser beams

Journal of the European Optical Society: Rapid Publications, 2010

In this paper we investigate the effects of visible to near infrared (NIR) laser illumination on ... more In this paper we investigate the effects of visible to near infrared (NIR) laser illumination on the optical transmission (OT) and morphological (MC) alterations of thin, curved surfaces of polycarbonate (PC). The second harmonic of Nd:YAG laser (532 nm) and two diode lasers (665 and 980 nm) were used as illuminating sources. The morphological changes of the PC surfaces are determined using atomic force microscopy (AFM), demonstrating the appreciable changes caused by shorter wavelengths (higher energy). When analyzing the OT spectra of PC thin films, a measurable decrease in the OT of the PC samples which were illuminated by 532, 665 and 980 nm, in particular 532 nm, for energy densities greater than 25 J/cm 2 can be seen.

Research paper thumbnail of <title>Beam propagation analysis in unstable laser resonators (ULR): low to high magnification</title>

19th Congress of the International Commission for Optics: Optics for the Quality of Life, 2003

ABSTRACT

Research paper thumbnail of Chemical Clearing of Brains

Handbook of Neurophotonics, 2020

Research paper thumbnail of Ultramicroscopy of Nerve Fibers and Neurons: Fine-Tuning the Light Sheets

Research paper thumbnail of Chemical Clearing of GFP-Expressing Neural Tissues

Research paper thumbnail of 3D histopathology of human tumours by fast clearing and ultramicroscopy

Scientific Reports, 2020

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malign... more Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a–c is fully reversible, allowing the morphological correlation of one and the same histological structures, onc...

Research paper thumbnail of Breaking the diffraction limit of light sheets allows fast isotropic imaging of large samples by ultramicroscopy

Proceedings for Annual Meeting of The Japanese Pharmacological Society, 2018

By breaking the diffraction limit of light sheets of low numerical aperture (NA) we were able to ... more By breaking the diffraction limit of light sheets of low numerical aperture (NA) we were able to generate extremely long thin sheets of light with a thickness in the one micron range and a vastly increased Rayleigh range. We measured the thickness of the light sheets with different methods including standard point spread function measurement with beads. By using these light sheets in our ultramicroscope fast 3D imaging of whole mouse brains with objectives with a large field of view was possible. Due to the extremely low divergence of the light sheets mouse brains could be reconstructed from a single stack of optical sections with isotropic resolution. The light sheets used were essentially non-Gaussian generated by new optics we developed. Compared to a Gaussian light sheet of the same NA our new light sheet is much thinner. Thus the diffraction limit which holds also for low NA Gaussian light sheets was significantly surpassed. These optics will allow the application of ultramicroscopy to ever increasing samples beyond the whole mouse brain range. Besides mouse brains we imaged also cleared whole adult drosophilae. We were able to get good transparency for all developmental stages of the insect from larvae to adult animals with fully preserved GFP signal. We showed that also multiview imaging of cleared adult drosophilae is possible and and allows isotropic resolution with standard ultramicroscopy for such kind of specimens. With the new light sheet and a new ultrafast clearing also large human samples could be quickly imaged.

Research paper thumbnail of Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet

Journal of Biophotonics, 2018

This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Research paper thumbnail of A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity

Science Advances, 2020

A tailored protocol combines pigment removal with clearing, facilitating labeling and whole-body ... more A tailored protocol combines pigment removal with clearing, facilitating labeling and whole-body imaging across phylogeny.

Research paper thumbnail of Effect of salinity and radiation on proline accumulation in seeds of canola (Brassica napus L.)

Plant, Soil and Environment, 2010

Since laser beam may affect plant traits, it was used to enhance accumulation of proline in rapes... more Since laser beam may affect plant traits, it was used to enhance accumulation of proline in rapeseed and therefore to improve its tolerance to the salinity stress. This investigation was performed to study the effect of NaCl concentration in irrigated water (0, 100, 200 and 300 mmol NaCl) on proline accumulation of Canola (Brassica napus L.) after laser irradiation (Red, Infra-red and Nd:YAG) at two exposure treatments. In each exposure, seeds were irradiated for three minutes once or twice by the laser set. Free proline content in leaves increased significantly by increasing of NaCl concentration. Also proline content significantly increased with irradiation by laser beam. The Red laser irradiation used once and the Nd:YAG laser used twice had the greatest effect on the proline content whereas the Infrared laser had a low effect. Double application of irradiation induced a significantly higher amount of proline in the leaves compared to only one application. This is the first repor...

Research paper thumbnail of High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

Journal of Biophotonics, 2019

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts ben... more Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep‐tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)‐expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP‐expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity‐dependent expression pattern of tdTomato.

Research paper thumbnail of High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster

Nature Communications, 2018

The fruit fly, Drosophila melanogaster, is an important experimental model to address central que... more The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique we developed, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope set-up utilising an improved light-sheet generator allows us to visualise long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applyin...

Research paper thumbnail of Trichobilharzia regenti (Schistosomatidae): 3D imaging techniques in characterization of larval migration through the CNS of vertebrates

Micron, 2016

Migration of parasitic worms through the host tissues, which may occasionally result in fatal dam... more Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches.

Research paper thumbnail of Ultramicroscopy: development and outlook

Neurophotonics, 2015

We present an overview of the ultramicroscopy technique we developed. Starting from developments ... more We present an overview of the ultramicroscopy technique we developed. Starting from developments 100 years ago, we designed a light sheet microscope and a chemical clearing to image complete mouse brains. Fluorescence of green fluorescent protein (GFP)-labeled neurons in mouse brains could be preserved with our 3DISCO clearing and high-resolution three-dimensional (3-D) recordings were obtained. Ultramicroscopy was also used to image whole mouse embryos and flies. We improved the optical sectioning of our light sheet microscope by generating longer and thinner light sheets with aspheric optics. To obtain high-resolution images, we corrected available air microscope objectives for clearing solutions with high refractive index. We discuss how eventually super resolution could be realized in light sheet microscopy by applying stimulated emission depletion technology. Also the imaging of brain function by recording of mouse brains expressing cfos-GFP is discussed. Finally, we show the first 3-D recordings of human breast cancer with light sheet microscopy as application in medical diagnostics.

Research paper thumbnail of Recent developments in light sheet ultramicroscopy imaging techniques

Advanced Microscopy Techniques IV; and Neurophotonics II, 2015

ABSTRACT Ultramicroscopy-UM allows 3D-vizualization of biological specimens with μm-resolution. T... more ABSTRACT Ultramicroscopy-UM allows 3D-vizualization of biological specimens with μm-resolution. The spatial intensity distribution and thickness of the light sheet illuminating the specimen have the foremost impact on the quality of image in UM. In this paper, we investigate and compare different designs for generating light sheet and their optical characteristics

Research paper thumbnail of Ultramicroscopy: light-sheet-based microscopy for imaging centimeter-sized objects with micrometer resolution

Cold Spring Harbor protocols, 2013

Ultramicroscopy (UM) is a powerful imaging technique that achieves precise and accurate three-dim... more Ultramicroscopy (UM) is a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. It was developed for specimens in the size range of ∼1-15 mm, such as whole mouse brains, mouse embryos, mouse organs, and Drosophila melanogaster. In UM, the specimen is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. UM is closely related to a growing family of comparable microscopy approaches based on light sheet illumination developed in recent years. This article presents an overview of light-sheet-based microscopy and describes the underlying physics of light sheet generation. The assembly of an "ultramicroscope" for investigating fixed chemically cleared tissue is described in detail, and the functions of the essential components, such as mechanics, camera, and objectives, are discussed. Finally, practical applications of UM...

Research paper thumbnail of Immunostaining, dehydration, and clearing of mouse embryos for ultramicroscopy

Cold Spring Harbor protocols, 2013

This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful ima... more This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. In combination with fluorescein isothiocyanate (FITC) immunostaining, UM allows visualization of somatic motor and sensorial nerve fibers in whole mouse embryos. Even the fine branches of the sensomotoric fibers can be visualized over a distance of up to several millimeters. In this protocol, mouse embryos are fixed and immunostained in preparation for UM. Because UM requires the excitation light sheet to travel throughout the entire horizontal width of the specimen, specimens usually have to be rendered transparent before microscope inspection. Here, the embryos are dehydrated in ethan...

Research paper thumbnail of Dehydration and clearing of whole mouse brains and dissected hippocampi for ultramicroscopy

Cold Spring Harbor protocols, 2013

This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultram... more This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, mouse brains and hippocampi are carefully dissected and dehydrated, and then cleared in a solution of benzyl benzoate and benzyl alcohol.

Research paper thumbnail of Dehydration and clearing of adult Drosophila for ultramicroscopy

Cold Spring Harbor protocols, 2013

This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imagi... more This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, Drosophila melanogaster adults are fixed, dehydrated in ethanol, and then cleared in a solution of benzyl alcohol and benzyl benzoate.

Research paper thumbnail of Recent development in light Ultramicroscopy using aspherical optical elements

Optical Systems Design 2012, 2012

ABSTRACT Here we present a new light sheet generator unit for Ultramicroscopy (UM) employing a co... more ABSTRACT Here we present a new light sheet generator unit for Ultramicroscopy (UM) employing a combination of optical lenses with aspherical surface structure. UM allows 3D-vizualization of chemically transparent biological specimens with μm-resolution. Improving optical characteristics parameters of light sheet such as the uniformity factor of spatial intensity distribution along the line of focus, the thickness of light sheet, and chromatic aberrations are the most important criteria in this design. Since we do not use any hard edge aperture there is no truncation of the beam and laser energy is used more efficiently. Due to these improvements, a marked enhancement in presenting fine details of biological specimens such as Drosophila melanogaster, entire mouse brain, and hippocampus are achieved.

Research paper thumbnail of Alteration of optical and morphological properties of polycarbonate illuminated by visible/IR laser beams

Journal of the European Optical Society: Rapid Publications, 2010

In this paper we investigate the effects of visible to near infrared (NIR) laser illumination on ... more In this paper we investigate the effects of visible to near infrared (NIR) laser illumination on the optical transmission (OT) and morphological (MC) alterations of thin, curved surfaces of polycarbonate (PC). The second harmonic of Nd:YAG laser (532 nm) and two diode lasers (665 and 980 nm) were used as illuminating sources. The morphological changes of the PC surfaces are determined using atomic force microscopy (AFM), demonstrating the appreciable changes caused by shorter wavelengths (higher energy). When analyzing the OT spectra of PC thin films, a measurable decrease in the OT of the PC samples which were illuminated by 532, 665 and 980 nm, in particular 532 nm, for energy densities greater than 25 J/cm 2 can be seen.