PMC:2940544 / 9933-14879 JSONTXT

{"project":"TEST0","denotations":[{"id":"20859526-212-220-568140","span":{"begin":610,"end":614},"obj":"[\"19636390\"]"},{"id":"20859526-103-111-568141","span":{"begin":720,"end":724},"obj":"[\"14587775\"]"},{"id":"20859526-124-132-568142","span":{"begin":741,"end":745},"obj":"[\"19636390\"]"},{"id":"20859526-126-134-568143","span":{"begin":1075,"end":1079},"obj":"[\"19495022\"]"},{"id":"20859526-104-112-568144","span":{"begin":2119,"end":2123},"obj":"[\"11240852\"]"},{"id":"20859526-87-95-568145","span":{"begin":2285,"end":2289},"obj":"[\"16442636\"]"},{"id":"20859526-180-188-568146","span":{"begin":2378,"end":2382},"obj":"[\"19636390\"]"},{"id":"20859526-125-133-568147","span":{"begin":4321,"end":4325},"obj":"[\"19129923\"]"}],"text":"Two-Photon Microscopy – Multibeam Methods\nA fundamentally different approach is described below: multibeam scanning. While some applications, such as deep tissue imaging require use of large amounts of power to compensate for scattering, many other applications use as little as 1/20th of the available laser power in order to keep single beam power below threshold for saturation and photodamage. To more efficiently utilize the available laser power in such circumstances we developed a multibeam approach in which the source laser is split into multiple beamlets using a diffractive element (Watson et al., 2009). These many beamlets are then simultaneously directed to different parts of the tissue (Sacconi et al., 2003, Watson et al., 2009). This allows use of the full power of the laser to either speed up imaging (similar to multibeam confocal imaging described above) or to increase sampling per frame and therefore signal-to-noise ratio.\nA particularly interesting though not yet practical approach takes this notion to its fullest conceptual extent (Oron et al., 2005). In this method, the laser simultaneously illuminates the entire field of view continuously. In a sense this is like “bathing” the sample in epifluorescent excitation but relies on two-photon excitation and on focusing the excitation pulse in time, therefore providing excellent Z-resolution. As might be imagined, the limitation of this method is average laser power – despite the high power of currently available lasers, this method demands so many photons per unit time that fluorescence intensities are very low and imaging is necessarily slow to allow sufficient integration. However in the future, with more powerful lasers this may be a promising approach.\n\nDiffractive optical elements and spatial light modulators\nOur initial approach used diffractive optical elements (DOEs) to spatially multiplex beamlets to improve imaging. Each of these beamlets has the same power as the single beam in a traditional optimized system. There exists a well-made and highly functional commercial variation on this technology (Nielsen et al., 2001) which uses multiple beam splitters rather than a DOE to create beamlets. This technology has been used to produce useful scientific observations (Kurtz et al., 2006) however it is over 100-fold more expensive than the method we describe (Watson et al., 2009) and does not clearly outperform our design.\nA DOE is an optical device – essentially a diffractive grating that allows splitting of a single beam from a coherent source (laser) into multiple beamlets using the effects of diffraction. In practice the DOE is usually a piece of transparent medium (glass, plastic, fused silica, etc.) with embossed pattern on its surface. A single laser beam is directed to the thin element and small variations in thickness of at different points in the element perturb the phase of electromagnetic waves to allow interference to create a specific final output pattern of beamlets, for instance a linear array as in Figure 1 (O'Shea, 2004).\nFigure 1 Multibeam two-photon excitation with diffractive optical elements (DOE) or spatial light modulators (SLM). Both the DOE and the SLM use diffraction to create an output pattern of multiple beamlets of light from a single input beam. (A) DOE is a static diffraction grating which creates a single particular output pattern, in this case a linear array of evenly spaced beamlets. These beamlets can be used to increase speed and/or increase sampling of imaging given that they allow for simultaneous exposure of multiple full power excitation points either in parallel or in series over portions of the field. (B) The SLM is dynamically controllable via computer and can create arbitrary patterns of output light to fit the spatial aspects of particular imaging fields (i.e., targeting particular neurites or cells). Furthermore, the SLM can have a new diffraction grating pattern thereby creating a new output pattern of beamlets every 16.7 ms (or even faster depending on type of the used SLM). SLMs allow scanless microscopy for both imaging as well as photostimulation experiments all at multiple spatial points simultaneously. Besides DOEs, which have an imprinted fixed pattern in them, we have used spatial light modulators (SLMs) (Nikolenko et al., 2008). SLMs are essentially DOEs made not of an etched medium, but instead a dynamic medium which can be controlled by a computer to create arbitrary diffraction patterns. This allows for a great deal of flexibility and customizability and we view it as particularly powerful for neuro-scientific experiments (Figures 1A vs 1B).\nWe utilize beam multiplexing approaches in three different ways: (1) Signal boost: increasing signal by oversampling, (2) Speed boost: increasing speed with parallel scanning, and (3) to create scanless dynamic SLM-based system; all of which will be described in detail in the following sections."}

{"project":"0_colil","denotations":[{"id":"20859526-19636390-568140","span":{"begin":610,"end":614},"obj":"19636390"},{"id":"20859526-14587775-568141","span":{"begin":720,"end":724},"obj":"14587775"},{"id":"20859526-19636390-568142","span":{"begin":741,"end":745},"obj":"19636390"},{"id":"20859526-19495022-568143","span":{"begin":1075,"end":1079},"obj":"19495022"},{"id":"20859526-11240852-568144","span":{"begin":2119,"end":2123},"obj":"11240852"},{"id":"20859526-16442636-568145","span":{"begin":2285,"end":2289},"obj":"16442636"},{"id":"20859526-19636390-568146","span":{"begin":2378,"end":2382},"obj":"19636390"},{"id":"20859526-19129923-568147","span":{"begin":4321,"end":4325},"obj":"19129923"}],"text":"Two-Photon Microscopy – Multibeam Methods\nA fundamentally different approach is described below: multibeam scanning. While some applications, such as deep tissue imaging require use of large amounts of power to compensate for scattering, many other applications use as little as 1/20th of the available laser power in order to keep single beam power below threshold for saturation and photodamage. To more efficiently utilize the available laser power in such circumstances we developed a multibeam approach in which the source laser is split into multiple beamlets using a diffractive element (Watson et al., 2009). These many beamlets are then simultaneously directed to different parts of the tissue (Sacconi et al., 2003, Watson et al., 2009). This allows use of the full power of the laser to either speed up imaging (similar to multibeam confocal imaging described above) or to increase sampling per frame and therefore signal-to-noise ratio.\nA particularly interesting though not yet practical approach takes this notion to its fullest conceptual extent (Oron et al., 2005). In this method, the laser simultaneously illuminates the entire field of view continuously. In a sense this is like “bathing” the sample in epifluorescent excitation but relies on two-photon excitation and on focusing the excitation pulse in time, therefore providing excellent Z-resolution. As might be imagined, the limitation of this method is average laser power – despite the high power of currently available lasers, this method demands so many photons per unit time that fluorescence intensities are very low and imaging is necessarily slow to allow sufficient integration. However in the future, with more powerful lasers this may be a promising approach.\n\nDiffractive optical elements and spatial light modulators\nOur initial approach used diffractive optical elements (DOEs) to spatially multiplex beamlets to improve imaging. Each of these beamlets has the same power as the single beam in a traditional optimized system. There exists a well-made and highly functional commercial variation on this technology (Nielsen et al., 2001) which uses multiple beam splitters rather than a DOE to create beamlets. This technology has been used to produce useful scientific observations (Kurtz et al., 2006) however it is over 100-fold more expensive than the method we describe (Watson et al., 2009) and does not clearly outperform our design.\nA DOE is an optical device – essentially a diffractive grating that allows splitting of a single beam from a coherent source (laser) into multiple beamlets using the effects of diffraction. In practice the DOE is usually a piece of transparent medium (glass, plastic, fused silica, etc.) with embossed pattern on its surface. A single laser beam is directed to the thin element and small variations in thickness of at different points in the element perturb the phase of electromagnetic waves to allow interference to create a specific final output pattern of beamlets, for instance a linear array as in Figure 1 (O'Shea, 2004).\nFigure 1 Multibeam two-photon excitation with diffractive optical elements (DOE) or spatial light modulators (SLM). Both the DOE and the SLM use diffraction to create an output pattern of multiple beamlets of light from a single input beam. (A) DOE is a static diffraction grating which creates a single particular output pattern, in this case a linear array of evenly spaced beamlets. These beamlets can be used to increase speed and/or increase sampling of imaging given that they allow for simultaneous exposure of multiple full power excitation points either in parallel or in series over portions of the field. (B) The SLM is dynamically controllable via computer and can create arbitrary patterns of output light to fit the spatial aspects of particular imaging fields (i.e., targeting particular neurites or cells). Furthermore, the SLM can have a new diffraction grating pattern thereby creating a new output pattern of beamlets every 16.7 ms (or even faster depending on type of the used SLM). SLMs allow scanless microscopy for both imaging as well as photostimulation experiments all at multiple spatial points simultaneously. Besides DOEs, which have an imprinted fixed pattern in them, we have used spatial light modulators (SLMs) (Nikolenko et al., 2008). SLMs are essentially DOEs made not of an etched medium, but instead a dynamic medium which can be controlled by a computer to create arbitrary diffraction patterns. This allows for a great deal of flexibility and customizability and we view it as particularly powerful for neuro-scientific experiments (Figures 1A vs 1B).\nWe utilize beam multiplexing approaches in three different ways: (1) Signal boost: increasing signal by oversampling, (2) Speed boost: increasing speed with parallel scanning, and (3) to create scanless dynamic SLM-based system; all of which will be described in detail in the following sections."}

{"project":"2_test","denotations":[{"id":"20859526-19636390-38464482","span":{"begin":610,"end":614},"obj":"19636390"},{"id":"20859526-14587775-38464483","span":{"begin":720,"end":724},"obj":"14587775"},{"id":"20859526-19636390-38464484","span":{"begin":741,"end":745},"obj":"19636390"},{"id":"20859526-19495022-38464485","span":{"begin":1075,"end":1079},"obj":"19495022"},{"id":"20859526-11240852-38464486","span":{"begin":2119,"end":2123},"obj":"11240852"},{"id":"20859526-16442636-38464487","span":{"begin":2285,"end":2289},"obj":"16442636"},{"id":"20859526-19636390-38464488","span":{"begin":2378,"end":2382},"obj":"19636390"},{"id":"20859526-19129923-38464489","span":{"begin":4321,"end":4325},"obj":"19129923"}],"text":"Two-Photon Microscopy – Multibeam Methods\nA fundamentally different approach is described below: multibeam scanning. While some applications, such as deep tissue imaging require use of large amounts of power to compensate for scattering, many other applications use as little as 1/20th of the available laser power in order to keep single beam power below threshold for saturation and photodamage. To more efficiently utilize the available laser power in such circumstances we developed a multibeam approach in which the source laser is split into multiple beamlets using a diffractive element (Watson et al., 2009). These many beamlets are then simultaneously directed to different parts of the tissue (Sacconi et al., 2003, Watson et al., 2009). This allows use of the full power of the laser to either speed up imaging (similar to multibeam confocal imaging described above) or to increase sampling per frame and therefore signal-to-noise ratio.\nA particularly interesting though not yet practical approach takes this notion to its fullest conceptual extent (Oron et al., 2005). In this method, the laser simultaneously illuminates the entire field of view continuously. In a sense this is like “bathing” the sample in epifluorescent excitation but relies on two-photon excitation and on focusing the excitation pulse in time, therefore providing excellent Z-resolution. As might be imagined, the limitation of this method is average laser power – despite the high power of currently available lasers, this method demands so many photons per unit time that fluorescence intensities are very low and imaging is necessarily slow to allow sufficient integration. However in the future, with more powerful lasers this may be a promising approach.\n\nDiffractive optical elements and spatial light modulators\nOur initial approach used diffractive optical elements (DOEs) to spatially multiplex beamlets to improve imaging. Each of these beamlets has the same power as the single beam in a traditional optimized system. There exists a well-made and highly functional commercial variation on this technology (Nielsen et al., 2001) which uses multiple beam splitters rather than a DOE to create beamlets. This technology has been used to produce useful scientific observations (Kurtz et al., 2006) however it is over 100-fold more expensive than the method we describe (Watson et al., 2009) and does not clearly outperform our design.\nA DOE is an optical device – essentially a diffractive grating that allows splitting of a single beam from a coherent source (laser) into multiple beamlets using the effects of diffraction. In practice the DOE is usually a piece of transparent medium (glass, plastic, fused silica, etc.) with embossed pattern on its surface. A single laser beam is directed to the thin element and small variations in thickness of at different points in the element perturb the phase of electromagnetic waves to allow interference to create a specific final output pattern of beamlets, for instance a linear array as in Figure 1 (O'Shea, 2004).\nFigure 1 Multibeam two-photon excitation with diffractive optical elements (DOE) or spatial light modulators (SLM). Both the DOE and the SLM use diffraction to create an output pattern of multiple beamlets of light from a single input beam. (A) DOE is a static diffraction grating which creates a single particular output pattern, in this case a linear array of evenly spaced beamlets. These beamlets can be used to increase speed and/or increase sampling of imaging given that they allow for simultaneous exposure of multiple full power excitation points either in parallel or in series over portions of the field. (B) The SLM is dynamically controllable via computer and can create arbitrary patterns of output light to fit the spatial aspects of particular imaging fields (i.e., targeting particular neurites or cells). Furthermore, the SLM can have a new diffraction grating pattern thereby creating a new output pattern of beamlets every 16.7 ms (or even faster depending on type of the used SLM). SLMs allow scanless microscopy for both imaging as well as photostimulation experiments all at multiple spatial points simultaneously. Besides DOEs, which have an imprinted fixed pattern in them, we have used spatial light modulators (SLMs) (Nikolenko et al., 2008). SLMs are essentially DOEs made not of an etched medium, but instead a dynamic medium which can be controlled by a computer to create arbitrary diffraction patterns. This allows for a great deal of flexibility and customizability and we view it as particularly powerful for neuro-scientific experiments (Figures 1A vs 1B).\nWe utilize beam multiplexing approaches in three different ways: (1) Signal boost: increasing signal by oversampling, (2) Speed boost: increasing speed with parallel scanning, and (3) to create scanless dynamic SLM-based system; all of which will be described in detail in the following sections."}