Large-field high-resolution two-photon digital scanned light-sheet microscopy

doi:10.1038/cr.2014.124

Comment

. 2015 Feb;25(2):254-7.

doi: 10.1038/cr.2014.124. Epub 2014 Sep 26.

Large-field high-resolution two-photon digital scanned light-sheet microscopy

Weijian Zong¹, Jia Zhao², Xuanyang Chen², Yuan Lin², Huixia Ren², Yunfeng Zhang³, Ming Fan⁴, Zhuan Zhou², Heping Cheng², Yujie Sun⁵, Liangyi Chen²

Affiliations

¹ 1] The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China [2] China Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China.
² The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
³ School of Electronics Engineering and Computer Science, Peking University, Beijing 100871, China.
⁴ China Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China.
⁵ Biodynamic Optical Imaging Center, Peking University, Beijing 100871, China.

PMID: 25257466
PMCID: PMC4650563
DOI: 10.1038/cr.2014.124

Comment

Large-field high-resolution two-photon digital scanned light-sheet microscopy

Weijian Zong et al. Cell Res. 2015 Feb.

. 2015 Feb;25(2):254-7.

doi: 10.1038/cr.2014.124. Epub 2014 Sep 26.

Authors

Weijian Zong¹, Jia Zhao², Xuanyang Chen², Yuan Lin², Huixia Ren², Yunfeng Zhang³, Ming Fan⁴, Zhuan Zhou², Heping Cheng², Yujie Sun⁵, Liangyi Chen²

Affiliations

¹ 1] The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China [2] China Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China.
² The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
³ School of Electronics Engineering and Computer Science, Peking University, Beijing 100871, China.
⁴ China Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China.
⁵ Biodynamic Optical Imaging Center, Peking University, Beijing 100871, China.

PMID: 25257466
PMCID: PMC4650563
DOI: 10.1038/cr.2014.124

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Figures

**Figure 1**
**(A)** A schematic illustration of 2P3A-DSLM. We used a commercial Ti-sapphire femtosecond laser (Chameleon Version II, Coherent) as the illumination light source, and expanded the beam by a pair of concave mirrors. The focal length of the TAG lens was modulated sinusoidally at frequency of 100-500 kHz (diopter, ±30), which rapidly scanned the laser focus along the optics axis (x-direction) in front of the illumination objective (IO, 40× NA 0.8, Nikon). The GSM scanned the laser beam vertically to the illumination objective (y-direction). The TAG and the GSM were positioned at two conjugate planes of the back focal plane (BFP) of the illumination objective by L1 (50 mm), L2 (100 mm), L3 (100 mm) and L4 (200 mm). This configuration minimized the distortion and divergence of illumination intensity during scanning. The detection objective (DO, 40× NA 0.8, Nikon), emission filter, tube lens (200 mm) and sCMOS camera (flash 4.0, 2 048 × 2 048, Hamamatsu) were carefully aligned to record fluorescence images. A USB-controlled 3D stage (Thorlabs, MT3-Z8) combined with a fast Piezo Z-stepper (Edmund, Nanopositioning Piezo) was used to translate the sample for fast 3D imaging. The structure of a TAG len is given in the inset^,. EF, emission filter; TL, tunable lens. (B-D) Tailorable light sheet size from less than 10 × 10 μm² to 170 × 170 μm² driven by intensity modulation of the TAG. **(E)** The DOF was linearly related with the increase of TAG modulation intensity. **(F)** The thickness of the light sheet at the near-end and the middle remained nearly constant below 1 μm, while the thickness at the far-end increased to 1.5 μm when DOF reached 170 μm. **(G)** The axial resolution is below 1 μm under all circumstances except at the far-end of the 170 μm DOF (TAG modulation, 35%). (H-I) Dynamic imaging of mitochondria in live mt-cpYFP transgenic *C. elegans* pharynx on adult day 3 (also refer to Supplementary information, Movie S1) revealed fast split in the mitoflash fluorescence trace. (J-L) 2P3A-DSLM 3D imaging of DAPI-labeled (2 μg/ml) nuclei in the heart of a live zebrafish. **(J)** A selected section from the Z-stack movie (Supplementary information, Movie S3); **(K)** Selected area in J shows cells in division phase and inter-phase. Dividing chromosomes within single nucleus were clearly resolved; **(L)** The fluorescence profile of the red line imposed on the chromosome shown in K. Images in H, J and K were deconvolved using ImageJ RL algorithm. Scale bar: 100 μm (B-D), 20 μm **(H)**, 50 μm **(J)** and 5 μm **(K)**.

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Comment on

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References

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1. 2Keller PJ, Schmidt AD, Wittbrodt J, et al. Science 2008; 322:1065–1069. - PubMed
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[1] 1Pampaloni F, Reynaud EG, Stelzer EH. Nat Rev Mol Cell Biol 2007; 8:839–845. - PubMed

[2] 1Pampaloni F, Reynaud EG, Stelzer EH. Nat Rev Mol Cell Biol 2007; 8:839–845. - PubMed

[3] 2Keller PJ, Schmidt AD, Wittbrodt J, et al. Science 2008; 322:1065–1069. - PubMed

[4] 2Keller PJ, Schmidt AD, Wittbrodt J, et al. Science 2008; 322:1065–1069. - PubMed

[5] 3Truong TV, Supatto W, Koos DS, et al. Nat Methods 2011; 8:757–760. - PubMed

[6] 3Truong TV, Supatto W, Koos DS, et al. Nat Methods 2011; 8:757–760. - PubMed

[7] 4Gao L, Shao L, Higgins CD, et al. Cell 2012; 151:1370–1385. - PMC - PubMed

[8] 4Gao L, Shao L, Higgins CD, et al. Cell 2012; 151:1370–1385. - PMC - PubMed

[9] 5McLeod E, Arnold CB. Appl Opt 2008; 47:3609–3618. - PubMed

[10] 5McLeod E, Arnold CB. Appl Opt 2008; 47:3609–3618. - PubMed

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Large-field high-resolution two-photon digital scanned light-sheet microscopy

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Large-field high-resolution two-photon digital scanned light-sheet microscopy

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