Juan M. Bueno

Polarimetry using liquid-crystal variable retarders: theory and calibration

A Mueller-matrix polarimeter in transmission mode using two electronically controlled liquid-crystal variable retarders has been developed. Its design, theory and calibration are described. Although liquid crystals have been proposed earlier, this paper is focused on the process of the calculation of the Mueller matrix by a matrix-inversion method, oriented to static systems and in vitro samples. By driving the retarders with appropriate voltages, nine independent pairs of polarization states can be produced (incomplete polarimetry), while the other additional seven pairs are obtained by placing two quarter-wave plates (in the input and output optical paths respectively). This configuration allows extraction of 16 independent measurements of intensity. The Mueller matrix of the sample is calculated from them. The results of Mueller matrices for air, a linear polarizer and a quarter-wave plate are presented. Additional polarization parameters such as retardation, ellipticity or degree of polarization were also computed and some applications of the system are proposed.

Double-pass imaging polarimetry in the human eye

A Mueller-matrix imaging polarimeter was developed to measure spatially resolved polarization properties in the living human eye. The apparatus is a double-pass setup that incorporates two liquid-crystal variable retarders and a slow-scan CCD camera in the recording stage. Series of 16 images for the combinations of independent polarization states in the first and second passages were recorded for two experimental conditions: with the camera conjugated either with the retina or with the eyes pupil plane. Spatially resolved collections of Mueller matrices and the degree of polarization were calculated from those images for both retinal and pupil planes.

Spatially resolved polarization properties for in vitro corneas

Spatially resolved polarization properties of in vitro mammalian corneas have been studied by using a Mueller‐matrix imaging polarimeter in transmission mode. Sixteen images corresponding to independent combinations of polarization states in the illumination and analyzing pathways are recorded. Spatially resolved Mueller matrices of the samples are calculated from them. Results show that the birefringence of the cornea is almost linear. Although the magnitude of retardation depends on the sample, it is approximately constant at the center and increases towards the periphery. Dichroism and polarizing power are negligible. Maps of the degree of polarization indicate that the cornea basically does not depolarize the totally polarized incident light.

Measurements of the corneal birefringence with a liquid-crystal imaging polariscope

An imaging polariscope has been used to analyze the spatially resolved polarization properties of living human corneas. The apparatus is a modified double-pass setup, incorporating a liquid-crystal modulator in the analyzer pathway. Keeping the incident polarization state fixed (first passage), we recorded a series of three images of the pupils plane corresponding to independent polarization states of the analyzer unit. Azimuth and retardation at each point of the cornea were calculated from those images. Results show that the magnitude of retardation increases along the radius toward the periphery of the cornea. Left-right eye symmetry in retardation was also found. Maps of azimuth indicate that the direction of the corneal slow axis is nasally downward.

Measurement of parameters of polarization in the living human eye using imaging polarimetry

An imaging polarimeter using liquid-crystal variable retarders (Bueno, J. M., Artal, P. (1999). Double-pass imaging polarimetry in the human eye. Optics Letters, 24, 64-66) has been used to study the parameters of polarization in the living human eye. Retardation introduced by birefringent structures of the eye has been calculated by using a spatially resolved collection of Mueller matrices obtained from series of 16 double-pass retinal images. Results for images with a 2-mm pupil diameter show that although the retardation introduced by the eye in a double-pass varies among individuals, at the central cornea the slow axis is directed along the upper-temporal to lower-nasal line and the ellipticity is close to zero, which indicates the presence of linear birefringence. As pupil size increased, the measured retardation also increased, while ocular birefringence remained linear and azimuthal angle changed without a clear tendency.

Multiphoton microscopy of ex vivo corneas after collagen cross-linking.

PURPOSE To investigate changes in the morphology of the corneal stroma after collagen cross-linking (CXL) treatment in bovine and porcine eyes using a nonlinear microscope providing both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) corneal images. METHODS Freshly enucleated eyes were imaged using a tomographic nonlinear imaging method that was highly suitable to track temporal changes in corneal structures. CXL (riboflavin instillation plus UV irradiation) was applied on the enucleated eyes using similar protocols as in the clinic. A set of eyes without treatment were measured to be used as control. RESULTS In control corneas, SHG images showed a regular distribution of lamellae across the stroma that appeared stable for at least 6 hours postmortem. CXL changed the collagen distribution pattern showing some abnormal structures. TPEF revealed a large reduction in corneal thickness in CXL corneas immediately after treatment. The changes in the distribution of collagen bundles appeared also in corneas treated with riboflavin only, but not followed by UV irradiation. SHG tomography also revealed a partial recovery of the corneal thickness with time. CONCLUSIONS Nonlinear microscopy (in both tomographic and regular XY imaging configurations) was used to study spatial and temporal changes in the cornea during and after CXL on intact ocular globes. SHG imaging showed changes in the morphology of anterior corneal stroma after CXL. Regular collagen patterns turned into random distributed structures with thicker bundles at some localized areas. This might be a consequence of the corneal thickness decrease as a result of riboflavin-dextran instillation.

Analysis of corneal stroma organization with wavefront optimized nonlinear microscopy.

Purpose: To investigate the organization of stromal collagen in healthy ex vivo corneas of different species from second harmonic generation (SHG) microscopy images. Methods: A custom backscattered nonlinear microscope has been used to study the corneal structure of different species: porcine, bovine, rabbit, rat, chicken, and humans. The instrument uses a femtosecond laser for illumination, a scanning unit, and a photon-counting detection device. It also includes a wavefront aberration control module. SHG signals produced by collagen within the cornea were acquired. A motorized stage allowed optical sectioning across the entire corneal thickness. Samples were neither fixed nor stained, and they were fully scanned. Results: SHG images revealed the microscopic organization of the lamellae of collagen fibers. Despite absorption, for all corneal depths, images could be analyzed. The anterior stroma was similar in all samples, showing interwoven short bands of collagen randomly distributed. The lamellae at the central and posterior stroma were densely packed and often presented longer bundles lying predominantly parallel to the corneal surface with characteristic spatial distributions for each species. In particular, collagen bundles in bovine and porcine corneas were interweaved. In the chick cornea, the stromal arrangement had an orientation changing regularly with depth. In human corneas, lamellae were longer and had similar orientation than their neighbors. Conclusions: Using a unique wavefront aberration-controlled backscattered nonlinear microscope, changes in corneal morphology as a function of depth were characterized for different species (including humans). This allowed a direct comparison among species, which might help to establish the basis of collagen distribution in animal models or to understand diseased corneas.

Adaptive optics multiphoton microscopy to study ex vivo ocular tissues

We develop an adaptive optics (AO) multiphoton microscope by incorporating a deformable mirror and a Hartmann-Shack wavefront sensor. The AO module operating in closed-loop is used to correct for the aberrations of the illumination laser beam. This increases the efficiency of the nonlinear processes in reducing tissue photodamage, improves contrast, and enhances lateral resolution in images of nonstained ocular tissues. In particular, the use of AO in the multiphoton microscope provides a better visualization of ocular structures, which are relevant in ophthalmology. This instrument might be useful to explore the possible connections between changes in ocular structures and the associated pathologies.

Confocal scanning laser ophthalmoscopy improvement by use of Mueller-matrix polarimetry

A new technique for improving the signal-to-noise ratio and the contrast in images recorded with a confocal scanning laser system is presented. The method is based on the incorporation of a polarimeter into the setup. After the spatially resolved Mueller matrix of a sample was calculated, images for incident light with different states of polarization were reconstructed, and both the best and the worst images were computed. In both the microscope and the opthalmoscope modes, the best images are better than the originals. In contrast, the worst images are poorer. This technique may be useful in different fields such as confocal microscopy and retinal imaging.

Wavefront optimized nonlinear microscopy of ex vivo human retinas

A multiphoton microscope incorporating a Hartmann-Shack (HS) wavefront sensor to control the ultrafast laser beams wavefront aberrations has been developed. This instrument allowed us to investigate the impact of the laser beam aberrations on two-photon autofluorescence imaging of human retinal tissues. We demonstrated that nonlinear microscopy images are improved when laser beam aberrations are minimized by realigning the laser system cavity while wavefront controlling. Nonlinear signals from several human retinal anatomical features have been detected for the first time, without the need of fixation or staining procedures. Beyond the improved image quality, this approach reduces the required excitation power levels, minimizing the side effects of phototoxicity within the imaged sample. In particular, this may be important to study the physiology and function of the healthy and diseased retina.