Adaptive light-sheet fluorescence microscopy with a deformable mirror for video-rate volumetric imaging

Hong, Wenzhi; Wright, Terry; Sparks, Hugh; Dvinskikh, Liuba; MacLeod, Ken; Paterson, Carl; Dunsby, Chris

doi:10.1063/5.0125946

Cited by 8 publications

(3 citation statements)

References 20 publications

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“…However, the axial scan range of a DM is limited by the stroke length of the DM actuators. For example, for an objective with a numerical aperture (NA) of 0.8, the maximum axial scan range that DM-based techniques can generate is ∼100 μm 15,16 . Furthermore, using DM for focus control requires accurate alignment and complicated calibration of the DM to reduce the aberrations caused by imaging samples out of the nominal focal plane of the objective 9 .…”

Section: Mainmentioning

confidence: 99%

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Dibaji,

Nasaban Shotorban,

Grattan

et al. 2023

Preprint

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The ability to image at high speeds is necessary in biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging. However, owing to the incoherent and unpolarized nature of the fluorescence signal, manipulating this emission light through remote focusing is challenging. Therefore, remote focusing has been primarily limited to the illumination arm, using polarized laser light for facilitating coupling in and out of the remote focusing optics. Here we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light and lets them pass through the remote focusing module separately and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks, with an axial range of 70 μm and camera limited acquisition speed. Owing to its general nature, we believe this technique will find its application to many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging such as confocal, 2-photon, and light sheet variants.

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Section: Mainmentioning

confidence: 99%

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Dibaji,

Nasaban Shotorban,

Grattan

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…However, the axial scan range of a DM is limited by the stroke length of the DM actuators. For example, for an objective with a numerical aperture (NA) of 0.8, the maximum axial scan range that DM-based techniques can generate is ~100 µm 15 , 16 . Furthermore, using DM for focus control requires accurate alignment and complicated calibration of the DM to reduce the aberrations caused by imaging samples out of the nominal focal plane of the objective 9 .…”

Section: Introductionmentioning

confidence: 99%

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Dibaji,

Kazemi Nasaban Shotorban,

Grattan

et al. 2024

Nat Commun

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Rapid, high-resolution volumetric imaging without moving heavy objectives or disturbing delicate samples remains challenging. Pupil-matched remote focusing offers a promising solution for high NA systems, but the fluorescence signal’s incoherent and unpolarized nature complicates its application. Thus, remote focusing is mainly used in the illumination arm with polarized laser light to improve optical coupling. Here, we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light, lets them pass through the remote focusing module separately, and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks with an axial range of 70 μm and camera-limited acquisition speed. Owing to its general nature, we believe this technique will find its application in many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging, such as confocal, 2-photon, and light sheet variants.

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“…LS illumination 2,7,14,15 , where the sample is illuminated with a thin sheet of light at the image plane, has emerged as a simple and effective method of reducing fluorescence background, phototoxicity, and photobleaching of fluorophores. Several LS approaches involving the use of separate objectives for illumination and detection have been designed for single-molecule imaging in cells, however, they suffer from optical complexity, low numerical aperture (NA) objectives, steric hindrance, the inability to optically section adherent cells, incompatibility with microfluidic systems, or drift between the objectives 14,[16][17][18][19][20][21][22][23][24][25][26][27] . Previous single-objective LS designs have been demonstrated to reduce the complexity and limitations of multi-objective systems, but they have been limited by beam thickness 28 , the need for beam scanning 29,30 , and limited effective NA in the detection path and the requirement for post-processing due to an illumination beam that is not aligned to the detection axis 31 .…”

Section: Introductionmentioning

confidence: 99%

Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet

Saliba,

Gagliano,

Gustavsson

2023

Preprint

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Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample and for efficient and automated solution exchange. By combining these innovations with PSF engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets, we demonstrate whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.

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Adaptive light-sheet fluorescence microscopy with a deformable mirror for video-rate volumetric imaging

Cited by 8 publications

References 20 publications

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet

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