Converting lateral scanning into axial focusing to speed up 3D microscopy

Chakraborty, Tonmoy; Chang, Bo-Jui; Daetwyler, Stephan; Sapoznik, Etai; Chen, B; Km, Dean; Fiolka, Reto

doi:10.1101/2020.03.06.981472

Cited by 5 publications

(4 citation statements)

References 19 publications

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“…The major caveat of our system is that the empirically determined resonant frequency sets the speed of image acquisition. This limit can be resolved by employing a TAG lens [36][37][38] or remote focusing [39]. In support of this notion, the resonance frequency of a commercially available TAG lens is at 70 kHz (Mitutoyo), which can enhance the acquisition speed of our eLSCM to hundreds of frames per second.…”

Section: Discussionmentioning

confidence: 86%

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

Mac

Qureshi

et al. 2021

Preprint

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In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and suffers a reduced acquisition rate, which is critical to document neuronal dynamics properly. In this study, we implemented an electro-tunable lens (ETL) in the line-scan confocal microscopy, enabling the volumetric acquisition at the rate of 20 frames per second with the maximum volume of interest of 315 × 315 × 80 μm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 μm and 90.4 ± 2.1 μm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth elongation by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of cleared mouse brain ex vivo samples and in vivo brains. The current study foregrounds the successful application of resonant ETL for constructing a basis for a high-performance 3D line-scan confocal microscopy system, which will enhance our understanding of various dynamic biological processes.

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Section: Discussionmentioning

confidence: 86%

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

Mac

Qureshi

et al. 2021

Preprint

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“…subcellular resolution for intra-cellular structures or neuron axonal or dendritic structures) and possibility to combine our current efforts with higher near-isotropic resolution. In such efforts, both dual view deconvolved approaches and single view axially scanned approaches will deserve consideration for large sample brain and prostate imaging [30,31].…”

Section: Novel Mosaic Scans Of Large Cleared Human Tissue Samples Wit...mentioning

confidence: 99%

Efficient 3D light-sheet imaging of very large-scale optically cleared human brain and prostate tissue samples

Schueth

Hildebrand

Samarska

et al. 2022

Preprint

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The ability to view human tissue samples in 3D, with both cellular resolution and a large field of view (FOVs), can considerably improve fundamental and clinical investigations. Here, we demonstrate the feasibility of light-sheet imaging of ~50 x 35 x 3 mm (i.e., ~5 cm3) sized formalin fixed human brain and up to ~40 x 35 x 5 mm (i.e. ~7 cm3) sized formalin fixed paraffin embedded (FFPE) prostate (prostatectomy) cancer samples, which have been processed with the FFPE-MASH clearing and labelling protocol. To this end, we present a light-sheet microscopy prototype, the cleared-tissue dual view Selective Plane Illumination Microscope (ct-dSPIM), capable of fast, high-resolution acquisitions, as well as cubic centimetre-scale (many cm in lateral size and up to 5 mm thick) imaging of cleared tissue in 3D. To focus on fast 3D overview scans of entire tissue samples or cubic centimetre-scale selected regions of Interest (ROIs), we explored the use of stage scanned acquisitions with slice spacing on the order of the axial resolution (i.e. light-sheet thickness) and corresponding in-plane down sampling to achieve isotropic resolution, which we term Mosaic scans. These Mosaic scans allow for fast 3D overview scans of entire tissue samples (Mosaic 16 with 16.4 microns isotropic resolution) at approx. 8-16 hrs, ~1 cm3/h, or higher resolution overviews of large, selected ROIs (Mosaic with 4.1 microns isotropic resolution) 3-4 hrs, ~1 cm3/h, speed. We were able to select and visualise ROIs around the border of human brain area V1 and V2, as well as 3D prostate morphology in human prostatectomy cancer biopsies. We show that ct-dSPIM imaging, including Mosaic scans is an excellent technique to assess entire MASH prepared large-scale human tissue samples and enable multiscale investigations and quantification (e.g. cell count) in 3D.

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“…It has pushed the limits of observability in Cell Biology 3 , Developmental Biology [4][5][6][7] , and Neuroscience 8,9 . The main reasons for this success are: its low photo-invasiveness 2,3,6 , its high spatio-temporal resolution 3,10,11 , and its ability to capture images of large and thick specimens such as embryos and organoids 4,[12][13][14] . Lightsheet microscopy has been particularly impactful in Developmental Biology allowing the first in-toto volumetric reconstructions of embryonic development of model organisms such as Drosophila, Zebrafish, and Mouse 1,2,15 .…”

Section: Introductionmentioning

confidence: 99%

High-Resolution, Large Imaging Volume, and Multi-View Single Objective Light-Sheet Microscopy

Yang

Lange

Millett-Sikking

et al. 2020

Preprint

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Light-sheet microscopy has become the preferred method for long-term imaging of large living samples because of its low photo-invasiveness and good optical sectioning capabilities. Unfortunately, refraction and scattering often pose obstacles to light-sheet propagation and limit imaging depth. This is typically addressed by imaging multiple complementary views to obtain high and uniform image quality throughout the sample. However, multi-view imaging often requires complex multi-objective configurations that complicate sample mounting, or sample rotation that decreases imaging speed. Recent developments in single-objective light-sheet microscopy have shown that it is possible to achieve high spatio-temporal resolution with a single objective for both illumination and detection. Here we describe a single-objective light-sheet microscope that achieves: (i) high-resolution and large field-of-view imaging via a custom remote focusing objective; (ii) simpler design and ergonomics by remote placement of coverslips; (iii) fast volumetric imaging by means of light-sheet stabilised stage scanning – a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iv) multi-view imaging by means of dual orthogonal light-sheet illumination. Finally, we demonstrate the speed, field of view and resolution of our novel instrument by imaging zebrafish tail development.

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Converting lateral scanning into axial focusing to speed up 3D microscopy

Cited by 5 publications

References 19 publications

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

Efficient 3D light-sheet imaging of very large-scale optically cleared human brain and prostate tissue samples

High-Resolution, Large Imaging Volume, and Multi-View Single Objective Light-Sheet Microscopy

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