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# Biphotonic Microscopy for in vitro, in vivo tissue phenotyping and cell tracking

Romain Fleurisson, Laurence Dubreil

► To cite this version:

Romain Fleurisson, Laurence Dubreil. Biphotonic Microscopy for in vitro, in vivo tissue phenotyping and cell tracking. Gen2bio2017, Mar 2017, Nantes, France. hal-01547559

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https://hal.science/hal-01547559

Submitted on 5 Jun 2020

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Biphotonic Microscopy for in
vitro, in vivo tissue
phenotyping and cell tracking

Romain Fleurisson et Laurence Dubreil
# APEX UMR 703 INRA Oniris: two main original features

## ❖ Synergy between two fields of expertise

- Veterinary pathology

- Fluorescence bio-imaging

<figure><img src="image_4.png" /></figure>

## ❖ A large spectrum of investigated animal species including animal models and target species

<figure><img src="image_5.png" /></figure>

## ❖ Multiscale fluorescence bio-imaging

<figure><img src="image_6.png" /></figure>
# APEX : Multiscale fluorescence Bio-imaging

Quantitative pathology workstation

<figure><img src="image_7.png" /><figcaption>Brain</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Heart</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Neurons network</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Collagen network</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Multilabeling</figcaption></figure>

<figure><img src="image_12.png" /></figure>

Macroscope

500 nm

Intravital -
Biphotonic
Microscopy

250 nm

Mantra
Wide field
multispectre

1 µm

<figure><img src="image_13.png" /></figure>

Multiscale
fluorescence
Bio-imaging

<figure><img src="image_14.png" /></figure>

PALM/STOR
M
microscopy

20/40 nm

<figure><img src="image_15.png" /></figure>

TIRF
microscopy

80/100 nm

<figure><img src="image_16.png" /></figure>

Confocal
spectral
microscopy

200 nm

Confocal
Microscopy

200 nm

<figure><img src="image_17.png" /></figure>

Neurons (nanoscale
architecture)

<figure><img src="image_18.png" /><figcaption>Neuron (focal adhesion)</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Neuron (spectral unmixing)</figcaption></figure>

<figure><img src="image_20.png" /></figure>
# Biphotonic Microscopy

<figure><img src="image_21.png" /></figure>

* **High sensitive GaAsp Non Descanned Detectors**

* Auto laser alignment

* **Tunable laser range 680-1300 nm, pulse width<120fs**
  0,6W (1300nm) and 1,3W (900 nm).

* Dual wavelength option IR: Insight Deepsee, tuning with 1040 nm (fixed)

* Multimodal imaging, Multiphotonic fluorescence, SHG, THG

<figure><img src="image_22.png" /></figure>

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* 1931, Discovery Two Photon absorption. M. Goeppert-Mayer (1906-1972)

1Photon excitation

<figure><img src="image_26.png" /></figure>

Excitation 488 nm
E=2,5eV

2Photon excitation

<figure><img src="image_27.png" /></figure>

Excitation 976 nm
E=1,25eV

Fluorescence
ermision

2Photon excitation Condition

* **Temporal confinement**

* **Spatial confinement**
# Biphotonic Microscopy

## 2Photon excitation Condition

* Temporal confinement

* Spatial confinement

* Two photon excitation needs pulsed lasers

* A pulsed laser allows to get High Power in Small time

* Each of the two photons provides half the energy necessary to the electronic transition of the excited molecule

<figure><img src="image_28.png" /></figure>
# Biphotonic Microscopy advantages

## 2Photon excitation Condition

* Temporal confinement

* Spatial confinement

* Focal plane of the objective

<figure><img src="image_29.png" /></figure>
# Biphotonic Microscopy : Many contrast modes

* Combination of 2P emission fluorescence and second harmonic generation (SHG)

* 2PEF = localisation of fluorophores

* SHG = dense structure **without centro symmetry**
  Collagen Type I

  $\lambda_{em} = \lambda_{IR} \text{ excitation}/2$

* THG = **sub micrometric interface**
  Lipid bodies

  $\lambda_{em} = \lambda_{IR} \text{ excitation}/3$

<figure><img src="image_30.png" /></figure>
# Biphotonic Microscopy advantages

Major Inputs of 2Photon excitation:

- Deeper **tissue penetration**
  **IR excitation wavelength**
  Long working distance objective

- Reduction of the **photoxicity**
  Only focal excitation

- Imaging of **living tissue**
  Low photoxicity

- Imaging of **harmonic signals**
  Free labeling
# Deep Investigation of Tissue

## Combination of clearing methods and biphotonic microscopy :

* ScaleA2, 4 M urea, 10% glycerol and 0.1% Triton X-100, (Hama et al., 2011)

* Clarity, acrylamide and bisacrylamide, clearing tissue, Chung et al., 2013)

* uDISCO, BENZYLE ALCOOL (BA), BENZYLE BENZOATE (BB) ET DBE, P. CHENCHEN ET AL., 2016)

* LUCID-A, 2,2'Thiodiethanol (Y. Aoyagi et al., 2015)

<figure><img src="image_31.png" /></figure>

(1 to 2 mm)
Tissular Phenotyping by using 2P Microscopy

for pathology investigation

and

therapeutic efficacy assesment
# Pompe Disease : sarcomeric anomalies, 2P microscopy

* Glycogen storage in lysosomes
deficiency in acid-α-1,4-glucosidase (GAA)

* 1/40 000 birth

* infants form : cardiomyopathy+ myopathy+ neuronal
effects (DeRuisseau et al, 2009)

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Feeney et al. Acta Neuropathologica Communications 2014. 22
http://www actaneurocommis org/content/2/1/2

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ACTA NEUROPATHOLOGICA
COMMUNICATIONS

RESEARCH

Open Access

The value of muscle biopsies in Pompe disease:
identifying lipofuscin inclusions in juvenile- and
adult-onset patients

Erin J Feeney¹, Stephanie Austin<fnref n="2" />, Yin-Hsiu Chien<fnref n="3" />, Hanna Mandel<fnref n="4" />, Benedikt Schoser<fnref n="2" />, Sean Prater<fnref n="2" />,
Wuh-Liang Hwu<fnref n="3" />, Evelyn Ralston⁵, Priya S Kishnani⁶* and Nina Raben¹*

Interruption of the myosin bands
= black holes of autophagic area

<figure><img src="image_34.png" /></figure>

Detection and imaging of non-contractile inclusions and sarcomeric anomalies
in skeletal muscle by second harmonic generation combined with two-photon
excited fluorescence

E. Ralston A*, B. Swaim b, M. Czapiga c, W.-L. Hwu d, Y.-H. Chien d, M.G. Pittis e, B. Bembi e, O. Schwartz c,
P. Plotz f, N. Raben f

<figure><img src="image_35.png" /><figcaption>Sarcomeric anomalies</figcaption></figure>

Fig. 2. A 50-year-old woman with a history of family consanguinity. She presented with a left lower limb, upper extremity, and trunk spasticity and had a left hemiparesis. She had a left hemiparesis. She had a left hemiparesis.

<footnote>*Light Imaging Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Building 50, Room 1535, Bethesda, MD 20892-8023, USA</footnote>

<footnote n="2">²Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA</footnote>

<footnote n="3">³Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA</footnote>

<footnote n="4">⁴Department of Pediatrics and Medical Genetics, National Taiwan University Hospital, Taiwan</footnote>

<footnote>*Pediatric Hospital Burlo Garfunkel, Trieste, Italy</footnote>
# Duchenne Myopathy: fibrosis investigation by 2P microscopy

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Microscopy and imaging science: practical approaches to applied research and education (A. Méndez-Vilas, Ed.)

## Multiphotonic and Harmonic generation microscopy: an attractive label free imaging and non-destructive observation of collagenic and adipose tissues in pathological muscle context

Laurence Dubreil <fnref n="1" />,<fnref n="2" />, Mireille Ledevin <fnref n="1" />,<fnref n="2" />, Claire Lovo <fnref n="1" />,<fnref n="2" />, Thibaut Larcher <fnref n="1" />,<fnref n="2" />, Romain Fleurisson <fnref n="1" />,<fnref n="2" />

Lydie Guigand <fnref n="1" />,<fnref n="2" /> and Karl Rouger <fnref n="1" />,<fnref n="2" />

<footnote n="1">¹ INRA UMR703 PAnTher, F-44307 Nantes, France</footnote>

<footnote n="2">² LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, Nantes, F-44307, France</footnote>
# Arterial wall structure, 2P microscopy

## Micrometer Scale *Ex Vivo* Multiphoton Imaging of Unstained Arterial Wall Structure

T. Boulesteix,<fnref n="1" /> A.-M. Pena,<fnref n="1" /> N. Pagès,<fnref n="2" /> G. Godeau,<fnref n="3" /> M.-P. Sauviat,<fnref n="1" /> E. Beaurepaire,<fnref n="1" /> and M.-C. Schanne-Klein<fnref n="1" />*

Received 1 July 2005; Revised 26 August 2005; Accepted 31 August 2005

View on Wiley Online Library →

Collagen

Artery wall

Elastin

Multiphoton
microscopy

2PEF

SHG

150 µm

Transmitted-light
microscopy

150 µm

150 µm

f

Adventitia

Media

Intima

Lumen of the aorta

<figure><img src="image_38.png" /></figure>

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Calibration x 0.78 µm, y 0.78 µm, z 1.00 µm

Width: 399.35 µm Height: 399.35 µm Depth: 10.00 µm

SHG collagen

Elastic fibers
autofluorescence

<figure><img src="image_40.png" /></figure>

b

<figure><img src="image_41.png" /></figure>

e

<figure><img src="image_42.png" /></figure>

g

<figure><img src="image_43.png" /></figure>

Effect of residual doses of pesticide on the vessel wall

<figure><img src="image_44.png" /></figure>

Biogenouest

RIOGUDON2

<footnote n="1">¹Laboratory for Optics and Biosciences, CNRS/INSERM, Ecole Polytechnique, 91128 Palaiseau, France</footnote>

<footnote n="2">²Louis Pasteur University, 74 Route du Rhin, 67 401 Illkirch, France</footnote>

<footnote n="3">³Laboratory of Unmineralized Tissues Physiopathology, Paris V University, 1 rue Maurice Arnoux, 91 120 Montrouge, France</footnote>
# Long term intravital imaging of autophagosome accumulation in Pompe skeletal muscle during pathologic development and for therapeutic assessment

<figure><img src="image_45.png" /></figure>

Julien Pichon/Carine Ciron, PhD project U1089/UMR703
Vascular permeability, 2P microscopy

<figure><img src="image_46.png" /></figure>

Received
5 April 2013

Accepted
1 May 2013

Published
4 June 2013

SCIENTIFIC REPORTS

Intravital analysis of vascular
permeability in mice using two-photon
microscopy

Gyohei Egawa, Satoshi Nakamizo, Yohei Natsuaki, Hiromi Doi, Yoshiki Miyachi & Kenji Kabashima

Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

<figure><img src="image_47.png" /><figcaption>Figure 3 | Vascular hyper-permeability induced by injection of histamine.</figcaption></figure>

Discussion

In this study, we reported an intravital evaluation system for vascular permeability with two-photon microscopy and characterized the basal size-limitation of skin microvessels in mice. The major advantages of this method are as follows; (1) the dynamic kinetics of vascular permeability is determined in real-time, (2) the permeability could be evaluated without introducing inflammations, (3) the types of blood vessels, such as arterioles, venules, and capillaries are readily distinguished. Thus this method should provide a novel convenient way to study vascular biology *in vivo*. Though we examined only the characteristics of skin microvessels, this method may be applicable to examine the vessel permeability in other internal tissues such as in the brain.
Cell tracking by using nanoparticles

and biphotonic microscopy,

application for Cell Therapy
Electromagnetic/fFluorescence Nanoparticles

<table><thead><tr><th>NPs</th><th>Imaging modalities</th><th>Resolution</th><th>Imaging depth</th></tr></thead><tbody><tr><td></td><td>Magnetic resonance imaging</td><td>10-100µm</td><td>Whole body</td></tr><tr><td></td><td>Fluorescence imaging</td><td>0,25 µm</td><td>300µm</td></tr></tbody></table>

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /><figcaption>Elena ISHOW CEISAM Univ. Nantes</figcaption></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>
# Nanoparticules : BiFeO3 non centrosymmetric nanocrystals, range 100-120 nm SHG and THG signals.

## BiFeO3 non centrosymmetric nanocrystals

<figure><img src="image_52.png" /></figure>

THG (Chi3) is proportional to (Chi2) SHG for BFO NPs, Chi2>10⁻¹²m/V

<figure><img src="image_53.png" /><figcaption>Luigi BONACINA
GAP Biophotonics,
University of Geneva,
Switzerland</figcaption></figure>

L Bonacina et al. APB 2007

Y. Nakayama et al. Nature 2007

Comprehensive and review papers:

D. Staedler ACS Nano (2012)

L. Bonacina, Molecular Pharmaceutics (2013)

A. Rogov et al. IOP J. of Optics (2015)

<figure><img src="image_54.png" /></figure>

Heterobifunctional PEG amino and azide end groups for further functionalization

<figure><img src="image_55.png" /><figcaption>Sandrine Gerber</figcaption></figure>

<figure><img src="image_56.png" /></figure>
# BFO-PEG : HNPS highly brightness and multiwavelength excitation

Dual excitation
1040/1300

Emission

HNPS, highly brightness = Tracking of HNPS in depth

SHG 1040 nm

$\lambda_{exc}/2$

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

SHG 1300 nm

$\lambda_{exc}/2$

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

THG 1300 nm

$\lambda_{exc}/3$

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

Thickness : 1,4 mm
# hMustem Cells, therapeutic agent for Duchenne Myopathy

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

## Cell therapy of muscle diseases

**Karl Rouger**

INRA UMR 703, Oniris, Nantes, France

<figure><img src="image_66.png" /></figure>
# BFO-PEG cell labeling

## HNPs cell labelling efficacy

<figure><img src="image_67.png" /></figure>

* % of HNPs labelled cells counted for the two concentrations.
  Day 1 corresponds to 24 hrs after labelling procedure.

## HNPs cytotoxicity

<figure><img src="image_68.png" /></figure>

## HNPs Subcellular localization

<figure><img src="image_69.png" /></figure>
# BFO-PEG labelled MuStem cell tracking in muscle, proof of concept

## HNPs labeled cell tracking

Intramuscular
Injection

<figure><img src="image_70.png" /></figure>

BFO-PEG
labelled
hMuStem
cells

<figure><img src="image_71.png" /></figure>

Intramuscular
Injection

<figure><img src="image_72.png" /></figure>

24 hours

<figure><img src="image_73.png" /></figure>

Muscle
cryosections

<figure><img src="image_74.png" /></figure>

Immunolabeling
to improve the
tracking of cells

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

Anti-human
Lamin A/C antibody
Red : human nuclei
Blue : Nanoparticles (SHG)

<figure><img src="image_77.png" /></figure>

lexc 950 nm

<figure><img src="image_78.png" /></figure>

lexc 1040/1300 nm
# APEX : New offer for cell tracking

Harmonic Nanoparticules
for cell labeling

+

Biphotonic microscopy
to investigate cell imaging
In living tissue

<figure><img src="image_79.png" /></figure>

Order on www.h-np.ch

<figure><img src="image_80.png" /></figure>

APEX UMR 703 INRA Oniris

Biodistribution of cells in organ with high resolution

## APPLICATIONS

* Cell therapy
* Immunotherapy
Aknowledgments :

UMR703 INRA ONIRIS
Marie-Anne Colle
Karl Rouger

Karim Bey
Carine Ciron
Johan Deniaud
Lydie Guigand
Lydie Lagalice
Thibaut Larcher
Mireille Ledevin
Isabelle Leroux
Claire Lovo
Julien Pichon
Virginie Maurier
Cindy Schleder

<figure><img src="image_81.png" /></figure>

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<figure><img src="image_84.png" /></figure>

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<figure><img src="image_86.png" /></figure>

University of Geneva,
Switzerland
Luigi Bonacina
GAP Biophotonics,

<figure><img src="image_87.png" /></figure>

EPFL Lausanne
Sandrine Gerber

<figure><img src="image_88.png" /></figure>

TIBIO company
Switzerland
Davide Städler

CEISAM
UMR CNRS 6230

Elena Ishow