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# Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope

Andreas Deeg, Federico Trigo, Doriane Hazart, Brigitte Delhomme, Tchyia Zar, Thomas Naiser, Christian Seebacher, Adi Salomon, Clément Ricard, Rainer Uhl, et al.

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Andreas Deeg, Federico Trigo, Doriane Hazart, Brigitte Delhomme, Tchyia Zar, et al. .. Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope. 11th EOS (European Optical Society ) Annual Meeting (EOSAM 2023), Sep 2023, Dijon, France. pp.03002, 10.1051/epjconf/202328703002 . hal-04251722

HAL Id: hal-04251722

https://cnrs.hal.science/hal-04251722v1

Submitted on 20 Oct 2023

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# Fast, large-field fluorescence and second-harmonic generation imaging with a single-spinning disk two-photon microscope

Andreas Deeg,<fnref n="1" /> Federico Trigo,<fnref n="2" />,<fnref n="3" /> Doriane Hazart,<fnref n="2" /> Brigitte Delhomme,<fnref n="2" /> Tchyia Zar,<fnref n="4" /> Thomas Naiser,<fnref n="1" /> Christian Seebacher,<fnref n="1" /> Adi Salomon,<fnref n="2" />,<fnref n="4" /> Clément Ricard,<fnref n="2" /> Rainer Uhl,<fnref n="1" /> and Martin Oheim<fnref n="2" />,*

**Abstract.** Confocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitation and photo-bleaching risk can be somewhat relaxed in a spinning-disk geometry, due to shorter pixel dwell times and rapid re-scans during image capture. Alternatively, light-sheet microscopes rapidly image large volumes of transparent or chemically cleared samples. Finally, with infrared excitation and efficient scattered-light collection, 2-photon microscopy allows deep-tissue imaging, but it remains slow. Here, we describe a new optical scheme that borrows the best from three different worlds: the speed and direct-view from a spinning-disk confocal, deep tissue-penetration and intrinsic optical sectioning from 2-photon excitation, and a large field of view and a low invasiveness of a selective-plane illumination microscope – all with a single objective lens. We validate the performance of our 2-photon spinning-disk microscope in various applications that have in common to simultaneously require a large depth penetration, high speed and larger fields of view. Beyond biological fluorescence, we demonstrate an application in material science, imaging coherent non-linear scattering from a 3-D nano-porous network.

## 1 Introduction

Confocal spinning disk microscopes excel in fast, direct-view, 3-D live-cell imaging but multi-photon variants of this multi-spot scanner have remained marginal. This is because: (i), the co-linear arrangement of microlenses and confocal pinholes on a tandem spinning disk is difficultly optimised for both infrared (IR) excitation and visible fluorescence so that spatial resolution must be traded against signal; (ii), for deep-tissue imaging, interference of IR excitation light emerging from multiple pinholes results in high-intensity nodes above and below the focal plane (Talbot effect), which invalidates a fundamental assumption of 2-P imaging, that all generated fluorescence comes from a single focal plane; finally, (iii), in the classical Yokogawa design [1], the primary dichroic is located in the non-infinity space, between the two disks, inducing chromatic variations across the field-view during scanning and prescribing the use of objectives having small pupil diameters, high magnification and low working distance. A variant in which the pinhole disk is omitted and only the microlens disk kept results in a loss of image contrast with depth, due to the increased collection of scattered fluorescence. Having these limitations in mind, we conceptualised, prototyped and characterised a new multi-photon multi-spot microscope geometry, in which the microlenses, pinholes and dielectric coating are all combined on a

<caption>**Fig. 1.** Optical layout of the OASIS Multi-spot Multi-photon Microscope. Ti:Sapph- titanium-sapphire fs-pulsed IR laser, DS- DeepSee pulse compressor, BE- beam expander, M-mirror, L- lens, dic- dichroic, SD-single disk, TL- tube lens, OL- objective lens, CDE- corrective distance element, sCMOS) scientific complementary metal oxyde sensor.</caption>

single, micro-machined disk [2]. The expanded and flattened beam of a fs-pulsed IR laser beam is transmitted by the disk, focused by the microlenses and imaged onto the sample without pinhole filtering. 2-P excited multi-

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<footnote n="1">¹TILL I.D. GmbH, Am Klopferspitz 19a, D-82152 Martinsried, Germany, http://www.till-id.com;</footnote>

<footnote n="2">²Université Paris Cité, Saint-Pères Paris Institute for the Neurosciences, CNRS, 45 rue des Saints Pères, F-75006 Paris, France, https://sppin.fr;</footnote>

<footnote n="3">³Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida Italia 3318. CP 11600, Montevideo, Uruguay, https://www.gub.uy/ministerio-educacion-cultura/iibce;</footnote>

<footnote n="4">⁴Institute of Nanotechnology and Advanced Materials (BINA), Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900 Israel, https://nano.biu.ac.il;</footnote>

<footnote>* Corresponding author: martin.oheim@u-paris.fr</footnote>
spot fluorescence is collected by the same objective,
acquires an optical path length difference compared to
the excitation light on the way back to the detector, and
it is focused to pass through the tiny pinholes etched in
the dielectric coating on the rear side of the disk,
**Fig. 1**. This optical layout permits the use low-mag, high-
numerical aperture objectives [3], for example a Nikon
×25/1.1 water dipping lens, offering <400-nm xy- and
<2-µm z resolution over a >200-µm field-of-view [2].
We previously successfully used this microscope for fast
3-D imaging of brain organoids [2] as well as label-free
second-harmony generation (SHG) and autofluorescence
imaging across the entire thickness of the mouse intes-
tinal wall, [4]. Our multi-photon, multi-spot microscope
permits a 3- to 4-fold deeper imaging than a 1-photon
spinning disk confocal, it is 10 to 100 times faster than a
point scanner, and it has a two- to 3-fold better axial
sectioning than a typical light sheet microscope.

## 2 Results

### 2.1 Imaging the dendritic tree of a Purkinje cell

Purkinje cells (PCs) are the principal output neurones of
the cerebellar cortex. The have a large, flat and highly
branched dendritic tree and a single long axon that forms
an inhibitory projection to the deeper cerebellar nuclei. It
has been difficult to record PC calcium (Ca²⁺) activity
and the summation of dendritic inputs throughout the
entire arborisation of these large cells. In 200-µm thick
sagittal cerebellar slices we filled a single PC with
Oregon-Green-BAPTA488 fluorescent Ca²⁺indicator,
using the whole-cell patch-clamp technique, and imaged
its entire dendritic tree in less than 0.5 s.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Imaging the dendritic tree of an OGB-1 filled Purkinje neuron. *Left*, maximum-intensity projection of a mini-stack (3 slices) and zoom, *right*. Exposure was 480 ms, whole frame.</figcaption></figure>

### 2.3 Towards medical imaging of intrinsic signals

The enteric system is a quasi-autonomous nervous net-
work of interconnected plexuses organized in a 3-D
mesh, lining the gastro-intestinal tract. Our microscope
allowed us the detailed label-free visualisation of enteric
neurones and glia at much greater depths [4] and with

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Comparative confocal (**left**) and non-linear autofluo-
rescence/SHG (**right**) imaging of unlabelled mouse intestinal
wall, depth 27µm. See ref. [4] for details.</figcaption></figure>

better contrast than on a confocal microscope, illustra-
ting the interest for diagnostic imaging.

### 2.3 SHG hotspots in a 3-D metallic network

3-D nanoporous metallic networks [5] generate rando-
mized hot spots at different frequencies and they are
attractive substrates for photocatalysis, sensing or struc-
tured illumination microscopy. The OASIS microscope
allowed us fast, large-field inspection of such 3-D struc-
tures by imaging the SHG signal generated in the nano
holes and silver nano beads present in this volume.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Electron micrograph of a silver nanoporous network, *left*, and SHG hotspot generation, *right*, in a deep layer, imaged on the OASIS microscope, taken with a ×20 air lens. Image diagonal 400 µm, text = 480 ms. $\lambda_{ex}=850$ nm, detection in the blue-green band.</figcaption></figure>

Supported by CNRS (to MO), the EU (Eureka! Eurostars E!
10266 to MO & RU), a CNRS International Emerging Action
(to FT & MO), a Chateaubriand Fellowship, FranceBioImaging
and the Israel Science Foundation (ISF 1231/19) to AS.

## References

1. T. Shimozawa, K. Yamagata, T. Kondo, Y. Mimori-Kiyosue, PNAS **110**, 9 (2013).

2. I Rakotoson, B. Delhomme, P. Djian, A. Deeg, M. Brunstein, C. Seebacher, R. Uhl, C. Ricard, M. Oheim, Front. Neuroanat. **13** (2019).

3. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, J. Neurosci. Meth. **111**, 1 (2001).

4. D. Hazart, B. Delhomme, M. Oheim, C. Ricard, Front. Neuronat. **16** (2023).

5. R. Ron, T. Zar, A. Salomon, Adv. Opt. Mater. **11**, 5 (2022).