Confidential

Healthcare Business Unit

Current Status and Future Perspectivesof Microscopic Imaging

So Nishikawa

Section ManagerProduct Planning Section, Healthcare Business Unit, Nikon Corporation2019/12/18

1Confidential

About us

2Confidential

About me working 20 years with optical microscope

2003

Ph.D., Biophysics, Osaka University

2004-2006

Postdoctoral Fellow, CREST JST and Osaka Univ.

2007-2009

Assistant Professor, Osaka University

& Senior Lecturer, Osaka University

A couple of research budget supported by JST

2009-2015

Design Department, Nikon Corporation

2016

Marketing Department, Nikon Corporation

Omni-directional TIRF microscopy

Uniform laser illumination by piezo tip-tilt mirror

for quantitative single molecule measurement

Total internal reflection Dark field microscopy

27k frame/sec acquisition with nm precision

for ultra fast single gold nano particle tracking

Full model change of Nikon Inverted Microscope series

recognized with the iF Gold Award,

a globally prestigious design recognition

3Confidential

Why Microscope ? the more your goal clearly focused, the more help your discovery

2D information of Myosin steps

1D position vs time with improved spatial resolution

step

Nishikawa S, Arimoto I, Ikezaki K, et al. Switch between large hand-over-hand and small inchworm-like steps in myosin VI. Cell. 2010;142(6):879–888.

1st

2nd

4Confidential

About Nikon support scientific research activity with power of visualization

https://www.nikon.com/about/corporate/introduction/

Scientific Research = Discovery + Understanding + Explanation + Communication

5Confidential

About microscope

6Confidential

Microscope for Understanding on the comprehensive network between human organs

Deeper

Faster

Larger

Higher Contrast and Resolution

What type of microscopy should we prepare NEXT

7Confidential

Live Imaging ?

8Confidential

If live imaging not required for deeper imaging

Chung, K., Deisseroth, K. CLARITY for mapping the nervous system. Nat Methods 10, 508–513 (2013) Voigt, F.F., Kirschenbaum, D., Platonova, E. et al. The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue. Nat Methods 16, 1105–1108 (2019)

Clearing

9Confidential

If live imaging not required for higher resolution

Wassie, A.T., Zhao, Y. & Boyden, E.S. Expansion microscopy: principles and uses in biological research. Nat Methods 16, 33–41 (2019)

Expansion Microscopy

10Confidential

If live imaging required, Toolbox guideline

Yang, W., Yuste, R. In vivo imaging of neural activity. Nat Methods 14, 349–359 (2017) doi:10.1038/nmeth.4230

Scattering ?

Photo sensitive ?

Sectioning ?

Epi FL

CF Scanning

Large FOV ?

Light Field

Light Sheet

Free animal ?

Image required ?

2P Random

2P Scanning

Fast XY, Z

Fast volumetric

Multiplexing

Super Deep

Adaptive Optics

3 photon

Weight acceptable ?

Miniature Mic.

Fiber scope

NC. elegans, Zebrafish, Drosophila

YBrain, intestines etc.

N

Y

or “CF Spinning”

“Temporal focusing” is another candidate. but VERY High-peak-power laser pulses required.

N

Y

Galvano, Resonance scanner, AOD for XY,Piezo, ETL, SLM, Remote focusing for Z

Ultrasound, TAG lens

N

Y

N

Y

N

Y

N

Y

11Confidential

Transparent Sample ?

12Confidential

Confocal Scanning for Transparent samples, Optical sectioning with High resolution

Epi Fluorescence CF Scanning

13Confidential

Confocal Scanning for Faster, Larger, Higher

14Confidential

Confocal Scanning for Faster, Larger, Higher

The imaging area of the A1 HD25/A1R HD25 is nearly twice the conventional FOV of 18 mm, enabling the user to obtain significantly more data by capturing more of the specimen in each shot.

The large FOV reduces both the required number of images for stitching large images and image acquisition times, enabling efficient, high-throughput imaging of even large-scale samples.

25mm

15Confidential

Confocal Scanning for Faster, Larger, Higher

While resonant scanning at very short exposure times usually requires averaging to reduce Poisson shot noise contributions, users now instead can employ Denoise.AI to remove the shot noise contribution. The results are multi-fold:

1. Fewer imaging loops results in a longer imaging .2. Sampling frequency increases with less averaging for faster biological events.3. Acquisitions with low signal are appreciably improved.

16Confidential

Confocal Scanning for Faster, Larger, Higher

1024 x 1024 pixels enables acquisition of high-resolution, high-quality images at lower magnifications, enabling compatibility with a wide range of samples.

x1 Zoom x6 Zoom

17Confidential

Confocal Scanning for Faster, Larger, Higher

Higher resolution images can be generated with a single click. The unique image processing technology increases image resolution beyond that of a conventional confocal image (resolution can be improved 1.5 times (XY), 1.7 times (Z)).

Apical surfaces of auditory epithelia of mouse cochleae were stained by Atto-565-phalloidin at postnatal day 2.

Image courtesy of: Dr. Hideru Togashi, Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine.

18Confidential

Confocal Scanning for Faster, Larger, Higher

Spectral images at a high wavelength resolution of at least 2.5 nm. 32 channels of fluorescence spectra (up to a 320 nm wavelength range) can be acquired with a single scan, enabling fast imaging at up to 24 fps (512 x 32 pixels).

Specimen courtesy of: Dr. Tadashi Karashima, Department of Dermatology, Kurume University School of Medicine

Unmixed5 color fluorescence labeled HeLa cells

19Confidential

Light-sheet for Transparent samples, Optical sectioning with Gentle and Large Field of View

Epi Fluorescence and CF scanning Light-sheet

Confocal sectioning method scan the laser focal spot across the sample and using a pinhole to reject out-of-focus fluorescence.

Although out-of-focus photons are not used, they can contribute to phototoxicity.

Light-sheet microscopy uses a thin sheet of light projected into the sample from the side, exciting only a two-dimensional (2D) section of the sample.

The emitted fluorescence signal is then imaged in an orthogonal direction to the excitation plane.

20Confidential

Light-sheet for Transparent samples, Optical sectioning with Gentle and Large Field of View

From subcellular to embryo Simple add-on version

Wan Y, McDole K, Keller PJ. Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes. Annu Rev Cell Dev Biol. 2019;35:655–681.

https://www.mizarimaging.com/

21Confidential

Scattering Sample ?

22Confidential

2 photon Scanning for Scattering samples, Optical sectioning with High resolution

2P scanning

Minker KR, Biedrzycki ML, Kolagunda A, et al. Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens. Microsc Res Tech. 2018;81(2):141–152

23Confidential

2 photon Scanning for Scattering samples, Optical sectioning with High resolution

Deep brain imaging in in vivo mouse

Captured with GaAsP NDD for 1300 nm and CFI75 Apochromat 25XC W 1300 objective (NA 1.10, WD 2.0 mm)Excitation wavelength: 1040 nm

① Pyramidal cells in layer V

② White matter

③ Alveus

④ Hippocampal pyramidal cells

⑤ Hippocampus 3D zoom image

Photos courtesy of: Drs. Ryosuke Kawakami, Terumasa Hibi and Tomomi Nemoto, Research Institute for Electronic Science, Hokkaido University

24Confidential

2 photon Scanning for Scattering samples, Optical sectioning with High resolution

CFI90 20XC Glyc

Includes a correction collar that accommodates refractive indices ranging from 1.44 to 1.50 and is compatible with a variety of immersion media and tissue-clearing agents.

Working distance: 8.2 mmNumerical aperture: 1.00Chromatic aberration correction: from 588nm to 1300nmNano Crystal Coat applied.

CFI Plan Apochromat 10XC Glyc

Correction for refractive indices from 1.33 to 1.51 enables deep 3D-imaging of tissues cleared with a variety of optical clearing agents.

Working distance: 5.50 mmNumerical aperture: 0.50Chromatic aberration correction: from UV through to near IRNano Crystal Coat applied.

CFI75 Apochromat 25XC W 1300

Perfect for deep multiphoton imaging, achieving both a high NA and long working distance, as well as correcting spherical aberrations caused by sample thickness.

Working distance: 2.0 mmNumerical aperture: 1.10Chromatic aberration correction: from visible to 1300nmNano Crystal Coat applied.

25Confidential

2 photon Scanning for Faster imaging

Botcherby EJ, Smith CW, Kohl MM, et al. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc Natl Acad Sci U S A. 2012;109(8):2919–2924.

Various scanning methods are available to minimize the transition time from pixel to pixel.

XY:Galvano scanners (<10 fps)Resonant scanners (>30 fps)

Z:Piezo objectives (400 μm in <20 ms), ETLs (<10 ms), SLMs (>500 μm in <3 ms)TAG lens (Resonance frequency >450 kHz)

Other option:Remote focusing, the mirror at the auxiliary unit is light weight and can be rapidly moved, the focal spot can be scanned in z at high speed across the sample.

26Confidential

2 photon Random Access for Faster measurement

Sofroniew, N. J., Flickinger, D., King, J., & Svoboda, K. (2016). A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. eLife, 5, e14472.

A fast resonant scan is moved over the specimen in a flexible manner using a galvanometer scanner, allowing rapid sampling of activity in widely dispersed brain regions.

Macro imagingLow magnification image from a mouse expressing GCaMP6f. Sampling rate, 4.3 Hz.

Random access imagingTraces for 16 neurons extracted from the four separate regions. Sampling rate, 9.6 Hz.

27Confidential

Imaging between Organs ?

28Confidential

Dual axis 2P Microscope with 16 mechanical degrees-of-freedom

Wagner MJ, Kim TH, Kadmon J, et al. Shared Cortex-Cerebellum Dynamics in the Execution and Learning of a Motor Task. Cell. 2019;177(3):669–682.e24.

Might be transferred to Simultaneous neuronal recording

between human organs?

20 x obj. for the cortex 2 photon imaging40 x obj. for the cerebellum 2 photon imaging

Each microscope arm had six mechanical DOFs.

3 translational DOFs to position the objective tip in space2 rotational DOFs to adjust the orientation of the optical axis 1 fine, piezo-controlled movement along the objective axis

29Confidential

Miniature 2P Microscope for freely moving animal

Zong, W., Wu, R., Li, M. et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat Methods 14, 713–719 (2017)

Miniaturized 2 photon microscopethat resolves single-spine activity in freely behaving animals.

Weighing 2.15 g0.64 μm laterally and 3.35 μm axially40 Hz at 256 × 256 pixelsFOV of 130 × 130 μm2

Might be transferred to Simultaneous neuronal recording

between human organs?

30Confidential

Perspective for Understanding on the comprehensive network between human organs

Deeper

Faster

Larger

Higher Contrast and Resolution

Should be realized with Next Innovation of Multi-photon Microscopy