High-speed 2D and 3D fluorescence microscopy of cardiac myocytes

Kumar, Sunil; Wilding, Dean; Sikkel, Markus B.; Lyon, Alexander R.; MacLeod, Ken; Dunsby, Chris

doi:10.1364/oe.19.013839

Cited by 72 publications

(64 citation statements)

References 22 publications

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“…However, the use of 2 objectives is only practical with relatively small samples that can be aligned in the focal plane of both objectives. Dunsby et al [30][31][32] have developed an oblique plane microscope that uses 1 objective that can illuminate tissue at oblique angles (eg, 60°). Although the oblique plane microscope architecture sacrifices numeric aperture, it may be able to resolve sarcomeric structures.…”

Section: Discussionmentioning

confidence: 99%

Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Botcherby¹,

Corbett

Burton

et al. 2013

Circulation Research

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Objective: We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample. Methods and Results:We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS. Conclusions:

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Section: Discussionmentioning

confidence: 99%

Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Botcherby¹,

Corbett

Burton

et al. 2013

Circulation Research

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“…By launching the light sheet from a fiber that was mechanically coupled to the detection lens, image stacks of brain tissue over a range of 250 µm were recorded in 2 s already a few years ago [9]. Using a motorized detection lens, imaging over a range of 38 µm at a speed of 21 volumes per second has been demonstrated [10]. The system was based on oblique plane illumination, where an inclined light sheet to illuminate the sample is launched by the detection lens itself [11].…”

Section: Comparison Of Different Approaches For Volume Imaging In Spimmentioning

confidence: 99%

Rapid 3D light-sheet microscopy with a tunable lens

et al. 2013

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The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such samples as it records high-contrast images of entire planes within large samples at once. However, in order to obtain images of different planes, it has been necessary to move the sample relative to the fixed focal plane of the detection objective lens. This mechanical movement limits speed, precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have thereby achieved flexible volume imaging at much higher speeds than previously reported. Our high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume scans per second. Movements, shape changes and signals across the entire volume can be followed which has been impossible with existing reconstruction techniques. Abstract: The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such samples as it records high-contrast images of entire planes within large samples at once. However, in order to obtain images of different planes, it has been necessary to move the sample relative to the fixed focal plane of the detection objective lens. This mechanical movement limits speed, precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have thereby achieved flexible volume imaging at much higher speeds than previously reported. Our high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume scans per second. Movements, shape changes and signals across the entire volume can be followed which has been impossible with existing reconstruction techniques.

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“…These combined effects offer a technique that allows the user to minimize both photobleaching and phototoxicity [10,[12][13][14], while allowing for long-term imaging studies (up to several days). This is an essential characteristic required for all sorts of developmental biology studies [15][16][17][18], such as organogenesis [19], cell migration [20,21], cardiac development [22][23][24][25], vascular development [26,27], neuro-development [28,29], and generally any kind of in vivo studies. Drosophila melanogaster [2,[30][31][32] and zebrafish [19,26,30,[33][34][35][36] have traditionally been the most widely used samples in this field, but it fits well many others, such as worm embryos [28,37] and other small organisms or plants [38][39][40].…”

Section: 1a Lsfm Is Low Photodamage and Low Phototoxicmentioning

confidence: 99%

“…Finally, higher resolutions (NA > 1.1) can be obtained by using a conventional microscope with a single objective, which creates the LS and collects the fluorescence generated. Examples of these are the OPM technique [23,77,78] and the soSPIM [79][80][81][82] techniques that have been used for observing whole cells and cell aggregates in combination with superresolution (SR) localization microscopy.…”

Section: 1e Lsfm Allows Imaging Of Single Cells At High Resolutionmentioning

confidence: 99%

Light-sheet microscopy: a tutorial

et al. 2018

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This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.

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High-speed 2D and 3D fluorescence microscopy of cardiac myocytes

Cited by 72 publications

References 22 publications

Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Rapid 3D light-sheet microscopy with a tunable lens

Light-sheet microscopy: a tutorial

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