“La spettroscopia di riflettanza risolta nel tempo (TRS ...iaa.entecra.it/WS2015/16...

“La spettroscopia di riflettanza risolta nel tempo (TRS): principi ed applicazioni”

Alessandro Torricelli

Winter School«Le tecniche spettroscopiche: strumenti innovativi applicati all’analisi dei settori

ambientale ed agro-alimentare –nuove sfide per il futuro»

28 January 2015, Milano

Politecnico di Milano (PoliMi)Teaching & Research University

PoliMi since 1863

1863 – 2013150 years

Nome relatoreA. Torricelli

150 years

EngineeringArchitectureDesign

Department of PhysicsPhotonics for Health, Food and Cultural Heritage

Head: Rinaldo CubedduStaff @PoliMi Antonio Pifferi

Paola TaroniAlessandro TorricelliGianluca ValentiniAndrea BassiDaniela ComelliCosimo D’AndreaDavide ContiniAlberto Dalla Mora

Alberto Dalla Mora

Staff @ IFN-CNR Lorenzo SpinelliAustin NevinAndrea Farina

Post-Docs Ilaria BargigiaGiovanna QuartoRebecca ReMaristella VanoliLucia Zucchelli + PhD Students

+ Undergraduate Students+ Facilities (mechanic and electronic workshop)

Photonics for Health, Food and Cultural Heritage Research activities: overview

Health In vivo Tissue Spectroscopy Optical Mammography Tissue Oximetry and Functional Imaging of the Brain Fluorescence Lifetime Imaging in biology and medicine

Food nondestructive assessment of

internal defects by pulsed NIR

Cultural Heritage Photoablation and Material Processing Fluorescence Spectroscopy and Imaging Multispectral Imaging and Colorimetry

internal defects by pulsed NIR nondestructive maturity

assessment at harvest

Photonics for Health, Food and Cultural Heritage Laboratories

Time-resolved systems mode-locking of dye, gas and solid state lasers time-correlated single-photon counting (TCSPC) time-gated imaging

Spectral-domain tunable laser sources broadband detectors

TEMPORAL⇒ fast!

SPECTRAL⇒ colored!

fNIRS Lab

NIRf Lab

• functional near infrared spectroscopy - fNIRS Lab• diffuse spectroscopy - DiffS Lab• optical mammography - Mammot Lab• molecular imaging - Molim Lab• near infrared spectroscopy for food - NIRf Lab• imaging spectroscopy for cultural heritage - ARTIS Lab• ultras for biomedicine - UB Lab

Spatial-domain scanning systems or camera multi-channel systems

Temporal-domain fast acquisition rate

SPATIAL⇒ wide!

DiffS Lab

Mammot Lab

Fluo Lab

UB Lab

Molim Lab

ISCH Lab

Can light penetrate biological tissues?

Georges de La Tour (1593 – 1652)

St Joseph, 1642, Louvre, Paris Thanks to Marco Ferrari (UnivAQ)

Light absorption in the NIRbiological tissue

The therapeutic and diagnostic window

Light absorption in the NIRfruit

Light propagation in diffusive media:absorption and scattering

clear diffusive

Scattering: related to tissue structure

Scattering coefficient:

Absorption: related to tissue components

Absorption coefficient:µa = 1/la (cm-1)

Linout

aeII µ−=

Scattering coefficient:µs = 1/ls (cm-1)

( )Linout

saeII µµ +−=

interplay between absorption and scattering

Time-resolved Reflectance Spectroscopy (TRS)Basics

Intensity

Laser pulse

10-100 ps duration

Remitted pulse

log10I

µ’s

Effect of scattering

Changes in peak position!

Slope does not change

µ’s , µa

ρtime (1-10 ns)

log10IEffect of absorption

Changes in slopePeak position does not change

Conservation of energy in a small volume dV in a given direction Ω

(1) (3) (4) (5)

Light source

Radiative Transport Equation (RTE)

+∫ )•′µ+µ−µ−∇•−=∂∂

4ssa dˆˆ(vvvˆv Ωnpnnn

sPhotons outPhotons in

Photons scattered to another direction

Photons scattered from another direction (s’) to direction of interest (s)

Light source

Photons absorbed

Radiative Transport Equation

Expansion methods

•PN approximation:

P0 = Diffusion Theory

P = Diffusing Wave

Stochastic methods

• Monte Carlo

Solutions of the RTE

P1 = Diffusing Wave

P3 = …

Discretisation methods

• Discrete ordinates

• 2-flux or Kubelka-Munk

• Adding-double method

• Finite Element Method

Hybrid methods

• Paasschens (1997)

• Martelli (2008)

• Kienle (2011)

Infinite Semi-infinite

Slab Parallelepiped

Photon Diffusionsolutions for other geometries

2 layer

N layers

Sphere Cylinder

F.Martelli et al. Photon migration through diffusive media:Theories and software (SPIE book, 2010)

inhomogeneity

Photon Diffusiontime-resolved reflectance

∑∞+

−∞=

−−

exp)4(2

,32/52/3

ρµρ

1.000laser pulseTRS datamodel

)Φ∂+)Φµ+)Φ∇− tSt

tttD ,(

1,(,( 0a

0 1000 2000 3000 4000 5000

time (ps)

µ’s , µa

Time Frequency

R(ρ,t)

R(ρ,ω)

Time, Frequency, Space and Angle domain measurein diffusive media

Time Frequency

AngleSpace

CW CWR(ρ)

Laboratory set-up for broadband TRS

Fully automated system

spectral range: 540 -1100 nm Pifferi et al., Review of Scientific Instrument 78, 053103 (2007)

Laboratory set-up for broadband TRS

Fully automated system

spectral range: 540 -1100 nm Pifferi et al., Review of Scientific Instrument 78, 053103 (2007)

Optical characterisation of biological tissuesfemale breast

HbO2 [43 uM]Hb [22 uM]Water [34%]Lipid [53%]

Σ= λελµ iicia

Empirical approximation to Mie theory

bcbnabxas −==≈

λλπµ r2'

Absorption spectrum and tissue constituents Scatteri ng spectrum and tissue structure

600 650 700 750 800 850 900 950 1000

wavelength (nm)

Σ= λελµ iicia

Beer’s law

600 650 700 750 800 850 900 950 1000

wavelength (nm)re

a ⇔ density of scatterers

b ⇔ equivalent size of scatterers

Breast Absorption spectra: Inter-subject variation

R3 PT-40 DC-29 EL-34 LT-23 FM-50 KE-31water 72% 48% 36% 31% 22% 12%lipids 5% 41% 35% 48% 66% 71%tHb/µM 15.3 25 11 20 16 15SO2 63% 75% 62% 73% 59% 81%

Fibrous Adipose

600 650 700 750 800 850 900 950 1000 1050

wavelength (nm)

BreastScattering spectra: Inter-subject variation

T PT-40 DC-29 EL-34 LT-23 FM-50 KE-31a 9.3 8.9 6.8 11.7 11.0 9.6b 2.0 1.4 2.0 0.7 0.7 0.7

µ’s = aλ-b

Density

Fibrous Adipose

600 650 700 750 800 850 900 950 1000 1050

wavelength (nm)

A.Pifferi et al., J. Biomed. Opt. 9:1143-1151, (2004)

Time-resolved optical mammograph

laser heads

905 nm

683 nm

785 nm

637 nm

driver

coupler

930 nm

975 nm

1060 nm

variable attenuators

• Continuous movement:

• Laser driver: “Sepia” (PicoQuant)

• Repetition rate: 20 MHz

• Pulse duration: 180-400 ps

• Average incident power: 1-1.5 mW

fiber bundle

TCSPC board

NIR PMT

VIS PMT

collimator

cut-off filter variable attenuators

optics

XY translation stage

personal computer

TCSPC board

• Continuous movement:stepping motors & Counter/Timer PC board

• Maximum scan area: 180 mm x 240 mm

• Step interval: 1 mm

• Measurement time: 25 ms per point

• Photomultipliers (Hamamatsu, KK):

VIS: R5900U-01-L16, 150 ps TTS, λ < 850 nm

NIR: H7422P-60, 450 ps TTS, λ < 1100 nm

• TCSPC board (SPC-134, Becker & Hickl):

4 independent channels

16 MHz max count rate

Optical MammographyOPTIMAMM Project FP5 (2000-2003)

Patient #47, oblique view

age: 36 ythickness = 5.7 cmLesion size = 3.0 cmLesion type = tumor

S02 tHb

R LR L

52%-89% 17 - 91µM62%-95% 16 - 66µM

Type View Cases Detection rate FailuresCorrected

detection rate

Cancer 2 41 73% 80%1 9 89% 4 96%

0 6 11%

Cyst 2 59 72% 8 83%1 5 78% 3 90%

0 18 22%

Fibroadenoma 2 17 33% 2 39%1 5 43% 5 50%

0 29 57%Taroni et al., TRTC 4:527-537 (2005).

Clinical study (225 lesion)

Multi-channel time-domain system for functional nea r infrared spectroscopy (fNIRS)

µCHIP

2x2 fused splitter

2x4 fusedsplitter

820 nm

690 nm

Laserdriver

variable ND

1x9 fiber switch

PicoQuantPDL800

PiezojenaF-SM19

OZOpticsVISNIR5050

µCHIP

2x2 fused splitter

2x4 fusedsplitter

820 nm

690 nm

Laserdriver

variable ND

1x9 fiber switch

PicoQuantPDL800

PiezojenaF-SM19

OZOpticsVISNIR5050

µCHIPS16

4 anodesPMT-1

4 anodesPMT-2

4 anodesPMT-3

4 anodesPMT-4

4 chrouter-1

4 chrouter-2

4 chrouter-3

4 chrouter-4

8 champ-1

8 champ-2

TCSPC-1

TCSPC-2

TCSPC-3

TCSPC-4

HamamatsuR5900-20-M4

Becker & Hickl, SPC-134, HRT-41, HAFC-26

Microchip TechnologydsPIC30F6014

Contini et al. Opt Express14:5418-5432 (2006)

µCHIPS16

4 anodesPMT-1

4 anodesPMT-2

4 anodesPMT-3

4 anodesPMT-4

4 chrouter-1

4 chrouter-2

4 chrouter-3

4 chrouter-4

8 champ-1

8 champ-2

TCSPC-1

TCSPC-2

TCSPC-3

TCSPC-4

HamamatsuR5900-20-M4

Becker & Hickl, SPC-134, HRT-41, HAFC-26

Microchip TechnologydsPIC30F6014

Contini et al. Opt Express14:5418-5432 (2006)

Can light penetrate biological tissues?

Georges de La Tour (1593 – 1652)

St Joseph, 1642, Louvre, Paris Thanks to Marco Ferrari (UnivAQ)

Noninvasive imaging of brain function and disease b y pulsed near infrared light (nEUROPt FP7 2008-2012)

healthy ULD patientsA, D:O2Hb and HHb time-courses in themost reactive channel and thecorresponding GLM activationmaps.

In collaboration with: I.Gilioli, S.Franceschetti, F. Panzica, E.Visani @ IRCCS Besta Milan, Italy

B, E:BOLD signal extracted from theactive cluster and fMRI maps.

An optical neuro-monitor of cerebral oxygen metabol ismand blood flow for neonatology (BabyLux ICT CIP 620 996)

• The BabyLux project aims to provide a

precise, accurate, and robust

integrated system to continuously

monitoring cerebral blood flow,

oxygen metabolism, and oxygenation

in critically ill newborn babies.

• The instrument must be easy to

operate by busy clinical staff and

must be ready for integration into a

generic neonatal intensive care

environment.

• The proposed solution will integrate

two advanced photonic techniques:

time resolved near-infrared

spectroscopy (TRS) and diffuse

correlation spectroscopy (DCS).

Started on 1st January 20149 European partnersCoordinated by PoliMi

Photonics for Health Photonics for Food

Photonics for Food @ PoliMiResearch activities

DIFFRUIT, EU FP4, 1996-1999 TRS APPLE, MAFF (UK), 2000 AGROTEC, MIUR (I), 2000-2002 CUSBO, LASERLAB, EU FP5+FP6+FP7, 2004-2014 INSIDEFOOD, EU FP7 2009-2013 TROPICO, Regione Lombardia (I), 2010-2012 3D Mosaic, EU ICT-AGRI, 2011-2013 USER-PA EU ICT-AGRI 2013-2016

Projects

Publications (2001-2013) >30 papers published on peer reviewed international journals >30 papers on international books and proceedings

Collaborations

>30 papers on international books and proceedings >30 talks on international conferences

CRA-IAA, Milan (I), Paola Eccher-Zerbini , Anna Rizzolo, Maristella Vanoli, Maurizio Grassi Laimburg (I), Angelo Zanella Agricultural Research Organization, Bet Dagan (Israel), Susan Lurie, Victor Alchanitis Wageningen Universiteit (NL), Pol Tijskens, Olaf Van Kooten, Rob Schouten Planteforsk, Lofthus (N), Eivind Vangdal Potsdam (D), Manuela Zude-Sasse Leuven (B), Bart Nicolai, Bert Verlinden, Wouter Sayes, Pieter Verboven, Maarten Hertog UPM, ETSI Agronomos Madrid (E), Margarita Ruiz-Altisent, Constantino Valero Horiculture Research International, East Malling, (UK) - David Johnson, Colin Dover

Photonics for Food @ PoliMiMain applications

Non destructive optical characterisation of internal optical propertiesand correlation with quality parameters• Basic studies in apples, kiwifruits, nectarines, to matoes, …• Changes in optical properties during growth in Elst ar apples and Tophit plums• Texture in Jonagored apples, Braeburn apples and Pin k Lady apples during storage

Non destructive detection of internal disorders and defects• Browning in Granny Smith apples, Braeburn apples an d Conference pears• Watercore in Fuji apples

• Watercore in Fuji apples• Mealiness in Braeburn apples and Jonagored apples• Chilling injuries in Jubileum plums and Morsiani nect arines

Non destructive assessment of fruit maturity at harvestand correlation with quality parameters• Basic studies in apples, kiwifruits, nectarines, pe aches, mangoes, …• Sensory attributes, aroma composition, ethylene pro duction Ambra nectarines• Softening prediction (based on biological age) in S pring Bright nectarines

and in Tommy Atkins mangoes

Optical characterization of foodsabsorption and scattering spectra

kiwifruit

tterin

kiwifruit

650 700 750 800 850 900 950 1000

wavelength (nm)

650 700 750 800 850 900 950 1000

wavelength (nm)

tterin

Cubeddu et al., Applied Optics 40:538-543 (2001)

tterin

Optical characterization of foodseffect of skin: Apple (cv. Golden Delicious)

peeledintact

600 625 650 675 700

wavelength (nm)

tterin

peeledintact

600 625 650 675 700

wavelength (nm)

Optical characterization of foodseffect of skin: Mango (cv. Palmer)

Green skin

500 550 600 650 700 750 800 850 900

Red skin

500 550 600 650 700 750 800 850 900

wavelength (nm)500 550 600 650 700 750 800 850 900

wavelength (nm)

500 550 600 650 700 750 800 850 900

wavelength (nm)

500 550 600 650 700 750 800 850 900

wavelength (nm)

0.0350 400 450 500 550 600 650 700 750

wavelength (nm)

350 400 450 500 550 600 650 700 750

wavelength (nm)

Optical properties during growthAbsorption spectra

Plum Apple

GrapefruitChlorophyll breakdown in plum and apple, almost no changes in grapefruit.

Optical properties during growthScattering spectra

Grapefruit

Large changes during growth in the scattering properties for plum and grapefruit, minor changes for apple.

Optical propertiesEffect of layered structure in grapefruit

IRF (a.u.)

DTOF_1.5cm

DTOF_2.5cm

albedo

0 1000 2000 3000 4000 5000 6000 7000 8000

time (ps)

Shorter distance: early photons travel in the albedo, late photons in the pulpLonger distance: photon-path is mainly in the pulp

Nondestructive detection of internal defectsBrown heart in Conference pears

R3 (BH)

R3 (sound)

R3 (BH - sound)

µa (cm-1)@ 720 nm

710 730 750 770 790 810 830 850

wavelength (nm)

µa (cm-1)@ 720 nm

Eccher Zerbini et al., Postharvest Biology and Technology 25:87-99 (2002)

Measurement campaignsNondestructive assessment of maturity at harvest

0.090.10.140.180.20.270.30.390.42overripe

ready-firmtransportable

dangerously hardnever ripe

ready-ripe

0.090.10.140.180.20.270.30.390.42overripe

ready-firmtransportable

dangerously hardnever ripe

ready-ripe

( ) minminmax

*minmax1

Ff ttFFk+

∆+⋅−⋅

=∆ β

α 1log*a

maxa,Ft

5 6 13

spots with decay

overly soft spots

0 2 4 6 8 10 12

days at 20°C after harvest

0 2 4 6 8 10 12

days at 20°C after harvest

overripe ready-ripe

ready-firm

trans-portable

danger.hard

neverripe

class of usability

softer

seems soft

very firm

Eccher Zerbini et al., Postharvest Biology and Technology 39:223-232 (2006) Tijskens et al., Int. J. Postharvest Technology and Innovation, 1 (2), 178-188 (2006)

Tijskens et al., Postharvest Biology and Technology 45:204-213 (2007)Rizzolo et al., Biosystems Engineering (2009 in press)

TRS measurements:Absorption spectra: µ a

Day 0 Day 5

Maturity at harvest and shelf lifein Tommy Atkins mangoes

chlorophyll

carotenoids

Nome relatoreA. Torricelli IC NIRS 2013 – A1 O 094

540 nm

650 nm

TRS measurements:Absorption spectra: µ a

Maturity at harvest and shelf lifein Tommy Atkins mangoes

chlorophyll carotenoids

Chlorophyll-a and carotenoids estimation from TRSOpen problem: extinction coefficient for carotenoids in vivo

TRS set-up @PoliMi (Photonics for Food)

Laboratory based set-up (time sharing with Photonic s for Health):1) Broad band (450-1700 nm) TRS

ps supercontinuum laser, fast detectors, and TCSPC

2) fs laser + streak camera TRSoperative since September 2013

Transportable set -up

Transportable set -up3) Dual-wavelength (670 nm, 780 nm) TRS

ps diode laser heads, metal channel dynode PMT, and TCSPC

4) Multi-wavelength (14 wavelengths in the 500-900 nm range) TRSps supercontinuum laser, hybrid PMT, and TCSPC

1) Broad band (450-1700 nm) TRS

ps white light laser

NKT Photonics SuperK Extreme

Bassi et al., Opt Expr (2007)

Becker-HicklSPC130

Hybrid PMTNIR PMT1NIR PMT2

2) fs laser + Streak camera

Hamamatsu Photonics

+++ temporal resolution+ sensitivity

http://sales.hamamatsu.com/en/products/system-division/ultra-fast/streak-systems.php

3) Dual-wavelength transportable system for TRS

laser heads

750 nm

670 nmdriver

fiber optic swtch

PMTampTCSPCsync

filters and optics

Cubeddu et al., Appl Spectroscopy 55:1368-1374 (2001)Torricelli et al. Sens. & Instrumen. Food Qual. 2:82–89 (2008)

Laser source:

Supercontinuum fiber laser

− spectral range: 450-1600 nm

− power: 6 W

− frequency: 40 MHz

Wavelength selection:

Filter wheel

− spectral range: 540-900 nm

Detector:

Hybrid PMT

− no afterpulse

− time response: 250 ps

4) Multi-wavelength (500-900 nm range) transportable TRS

Supercontinuumlaser

grin fiber∅ =100μm

Large areadetector

Objective 10x Step-index fiber∅ =1mmSupercontinuum

laserSupercontinuum

lasergrin fiber∅ =100μm

Large areadetector

Objective 10x Step-index fiber∅ =1mm

TCSPCSYNC

Time resolution:

Time-Correlated Single-Photon Counting:

− high dynamic range

− suitable for faint signal

− time resolution: up to 1 ps

Filter wheelSample

Filter wheelFilter wheelSampleSample

Filter wheel

8 absorption nominal values:

0 : 0.07 : 0.49 cm-1 (label 1 -> 8)

4 reduced scattering nominal values:

5 : 5 : 20 cm-1 (label A -> D)

Robust calibrations….Solid phantoms: Epoxy based

Well established solutionabsorption

New series: 2.5, 7.5, 12.5 17.5 cm-1 nominal reduced scattering

Development of a transportable TRS set-upfor measurements in the orchards

2x1 combiner

100um GIλ1PD

laser head

variable attenuator fiber bundle

1 mm POF GI

cfdSPAD

lens, IF filter, shutter

SPADlens,

IF filter, shutter

TCSPC board

cfdTCSPC board

Scheme of the TRS setup

light source: pulsed diode laser (LDH series PicoQuant GmbH, Germany)detection: SPAD detectors (Excelitas Inc., Canada)acquisition: TCSPC board (Becker&Hickl GmbH, Germany)power supply: portable (gasoline), inverter technology (i20, Honda, Japan)

80 MHz sync

shutter

Development of a transportable TRS set-upfor measurements in the orchards

Photo of the TRS set-up

Field trials 2014Preliminary results

Absorption coefficient

Chlorophyll absorption decreases during fruit growth(agreement with Seifert et al. Physiol Plantarum 2014)

Scattering coefficient

Scattering decreases during fruit growth(agreement with Seifert et al. Physiol Plantarum 2014)

Evolution of TRS set-upTowards a smart photonic chip for TRS

Transportable>2013

Handheld>2016

Canopy>2018

Reducing size and cost ...

Evolution of TRS set-upThe future is near …

fNIRS – brain response to finger tapping

1 wavelength1 channelExternal power supply

Conclusion and future perspectives

• Time-resolved reflectance spectroscopy (TRS) naturally yieldsdiscrimination between light absorption (related to tissue constituents)and light scattering (related to tissue structure)

• Physical and mathematical models for TRS are available and allowquantitative data analysis for non destructive fruit quality assessment

• We have demonstrated in the last years several applications of TRS inthe food sector, mainly at research level

• We are at the forefront of a new era where recent advances inphotonic technologies might allow TRS to bridge the gap betweenresearch and market (the development is mostly driven by thebiomedical sector)

Acknowledgements

• CNR-IFN, Milan (Italy) Lorenzo Spinelli

• Politecnico di Milano - Dipartimento di Fisica, Milan (Italy) Antonio Pifferi Davide Contini Alberto Dalla Mora

• CRA-IAA (ex IVTPA), Milan (Italy)

• CRA-IAA (ex IVTPA), Milan (Italy) Anna Rizzolo Maristella Vanoli Maurizio Grassi (Paola Eccher Zerbini)

“La spettroscopia di riflettanza risolta nel tempo (TRS ...iaa.entecra.it/WS2015/16...

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