3 research outputs found
Matrix approach of Full-Field OCT for volumetric imaging of an opaque human cornea
Optical microscopy offers the possibility to image biological tissue with a diffraction limited resolution (~”m). However, the heterogeneity of biological tissues can strongly affect light propagation at large depths by distorting the initial wavefront. Large and short range fluctuations of the refractive index can induce aberration and multiple scattering, respectively. Inspired by a recent work [1], we have developed a matrix approach to Full-Field Optical Coherence Tomography (FF-OCT) to push back the fundamental limit of aberrations and multiple scattering. Here, we report on the application of this approach to the imaging of the human cornea and the quantitative measurement of the corneal transparency.
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Multi-Spectral Reflection Matrix for Ultra-Fast 3D Label-Free Microscopy
Label-free microscopy exploits light scattering to obtain a three-dimensional
image of biological tissues. However, light propagation is affected by
aberrations and multiple scattering, which drastically degrade the image
quality and limit the penetration depth. Multi-conjugate adaptive optics and
time-gated matrix approaches have been developed to compensate for aberrations
but the associated frame rate is extremely limited for 3D imaging. Here we
develop a multi-spectral matrix approach to solve these fundamental problems.
Based on an interferometric measurement of a polychromatic reflection matrix,
the focusing process can be optimized in post-processing at any voxel by
addressing independently each frequency component of the wave-field. A
proof-of-concept experiment demonstrates the three-dimensional image of an
opaque human cornea over a 0.1 mm^3-field-of-view at a 290 nm-resolution and a
1 Hz-frame rate. This work paves the way towards a fully-digital microscope
allowing real-time, in-vivo, quantitative and deep inspection of tissues.Comment: 27 pages, 4 figure
Approche matricielle de la microscopie optique : quantification et correction des aberrations et de la diffusion multiple
The basic principle of optical microscopy is to image the reflectivity of biological tissues from the scattered photons under a structured illumination. However, the propagation of incident and reflected light waves is often degraded by the heterogeneities of the medium itself. Theses heterogeneities can induce wavefront distortions (aberrations) and multiple scattering events that degrade the image resolution and contrast. Conventional microscopes have their penetration depth limited by the scattering mean free path (typically 10 to 200 ”m in biological tissues). However, this limit has been pushed back by confocal microscopy and OCT which spatially and/or temporally filter a large part of the multiply-scattered photons. A 3D image of biological tissues can thus be obtained over a few hundred ”m, but it remains strongly altered by aberrations and a predominant diffuse background. Initially developed in astronomy, adaptive optics has been transposed to microscopy to compensate for the aberrations induced by the medium heterogeneities. However, adaptive optics methods are complex to implement experimentally, limited to low-order aberrations and efficient only on the aberration invariance area. The isoplanatic patch size decreases with depth until the wave totally loses the memory of its initial direction after a transport mean free path (typically 1 mm in tissues). This is the fundamental limit of optical microscopy.In this thesis, we aim to go beyond adaptive optics by changing of paradigm. Inspired by past works in acoustics, this thesis develops a matrix imaging of biological media in optics. It is based on the measurement of a reflection matrix containing all the impulse responses between an emission and a reception plane, using for example a spatial light modulator at input and a camera at output. The principle of the matrix approach is then to apply a set of operations to this reflection matrix to extract relevant information on the medium according to the problem considered. A local quantification of aberrations and multiple scattering is thus possible, which opens a route towards a quantitative imaging of the medium. It also gives access to the transmission matrix that links the imaging system to all the voxels in the image. This matrix is valuable because it can be used in post-processing to locally compensate for all the aberrations encountered by the wave in the forward and return directions.During this thesis, two experimental setups have been developed. The first one combines matrix imaging and adaptive optics for a numerical and physical correction of aberrations. This setup, derived from Time-Domain OCT, allies the advantages of both methods. As a first ^proof-of-concept, very priomising results have been obtained on a mouse brain slice which is particularly diffusive. The second setup is a polychromatic matrix imaging device derived from Frequency-Domain OCT. A multi-spectral reflection matrix can be recorded at a fast rate (1-100 Hz). Thanks to the matrix approach we are able to realize a three-dimensional confocal image of the sample with an ideal resolution and an optimal contrast in each voxel of the image. The proof of concept consists in the 3D imaging of a particularly opaque human cornea. Finally, the access to a large number of spectral degrees of freedom paves the way towards a spatio-temporal control of light in post-processing and thus a fine compensation of multiple scattering phenomena in the medium that goes beyond the correction of snake photon trajectories.Le principe de la microscopie optique est dâimager la rĂ©flectivitĂ© des tissus biologiques Ă partir des photons diffusĂ©s par ces derniers sous une illumination structurĂ©e. Cependant, la propagation des ondes lumineuses incidente et rĂ©flĂ©chie est souvent dĂ©gradĂ©e par les hĂ©tĂ©rogĂ©nĂ©itĂ©s du milieu. Elles peuvent induire des distorsions du front d'onde (aberrations) et des Ă©vĂšnements de diffusion multiple qui dĂ©gradent la rĂ©solution et le contraste de l'image. Les microscopes conventionnels voient leur profondeur de pĂ©nĂ©tration limitĂ©e par le libre parcours moyen de diffusion (typiquement de 10 Ă 200 ”m dans les tissus biologiques). Cette limite a toutefois Ă©tĂ© repoussĂ©e par la microscopie confocale ou lâOCT qui permettent de filtrer spatialement et/ou temporellement une grande partie des photons multiplement diffusĂ©s. Une image 3D des tissus biologiques peut ainsi ĂȘtre rĂ©alisĂ©e sur quelques centaines de ”m mais elle reste toutefois fortement altĂ©rĂ©e par les aberrations et un fond diffus prĂ©dominant. Initialement dĂ©veloppĂ©e en astronomie, lâoptique adaptative a Ă©tĂ© transposĂ©e Ă la microscopie pour corriger les aberrations induites par le milieu. Cependant, les mĂ©thodes dâoptique adaptative sont complexes Ă mettre en Ćuvre expĂ©rimentalement, limitĂ©es Ă des faibles ordres dâaberrations et efficientes seulement sur la zone d'invariance de l'aberration. Cette aire d'isoplanĂ©tisme voit sa taille diminuer en profondeur jusqu'Ă ce que l'onde perde totalement la mĂ©moire de sa direction initiale au bout d'un libre parcours moyen de transport (typiquement 1 mm dans les tissus). Cela constitue la limite fondamentale de la microscopie optique.Dans le cadre de cette thĂšse, nous souhaitons dĂ©passer lâoptique adaptative en changeant de paradigme. InspirĂ©e par des travaux passĂ©s en acoustique, cette thĂšse dĂ©veloppe une imagerie matricielle des milieux biologiques en optique. Celle-ci est basĂ©e sur la mesure dâune matrice de rĂ©flexion contenant lâensemble des rĂ©ponses impulsionnelles entre un plan dâĂ©mission et de rĂ©ception, Ă lâaide par exemple dâun modulateur spatial de lumiĂšre en entrĂ©e et une camĂ©ra en sortie. Le principe de l'approche matricielle consiste ensuite Ă appliquer un ensemble d'opĂ©rations Ă cette matrice de rĂ©flexion pour en extraire une information pertinente sur le milieu en fonction du problĂšme considĂ©rĂ©. Une quantification locale des aberrations et de la diffusion multiple est ainsi possible, ce qui ouvre la voie Ă une imagerie quantitative du milieu. Elle permet en outre d'accĂ©der Ă la matrice de transmission qui relie le systĂšme dâimagerie Ă l'ensemble des voxels de lâimage. Cette matrice est prĂ©cieuse car elle permet en post-traitement de compenser localement tous les phĂ©nomĂšnes aberrations subies par lâonde Ă lâaller et au retour.Au cours de cette thĂšse, deux dispositifs expĂ©rimentaux ont Ă©tĂ© dĂ©veloppĂ©s. Le premier permet de combiner lâapproche matricielle et lâoptique adaptative pour une correction numĂ©rique et physique des aberrations. Ce montage, dĂ©rivĂ© de lâOCT temporel, permet de combiner les avantages de deux mĂ©thodes. De premiers rĂ©sultats particuliĂšrement encourageants ont Ă©tĂ© obtenus sur une tranche de cerveau de souris particuliĂšrement diffusante. Le second montage est un dispositif dâimagerie matricielle polychromatique dĂ©rivĂ© de lâOCT frĂ©quentiel. Une matrice de rĂ©flexion multi-spectrale peut ainsi ĂȘtre enregistrĂ©e Ă une cadence rapide (1-100 Hz). L'approche matricielle permet ensuite de rĂ©aliser une image confocale tridimensionnelle de l'Ă©chantillon avec une rĂ©solution idĂ©ale et un contraste optimal en chaque voxel de l'image. La preuve de concept consiste en lâimagerie 3D dâune cornĂ©e humaine particuliĂšrement opaque. Finalement, lâaccĂšs Ă un grand nombre de degrĂ©s de libertĂ© spectraux permet dâentrevoir un contrĂŽle spatio-temporel de lâonde lumineuse en post-traitement et ainsi une compensation des phĂ©nomĂšnes de diffusion multiple plus complexe que les seuls photons serpentiles