ADAPTIVE LY CORRECTED GRIN OBJECTIVE FOR MICROSCOPY

Technical Field The present invention is concerned with techniques for optimization in microscopy, in particular in-vivo microscopy and microendoscopy.

Background Art Definition of GRIN fiber objective:

a relay of graded index (GRIN) fibers used as a high numerical aperture conventional lens but with a very small lateral dimension making it effective for in-vivo insertions. It is combined with a conventional illuminating lens emulating a collimated microscope objective.

Properties of GRIN fiber objective: such objective can simulate the optical action (light focusing) of a conventional lens by means of the graded distribution of refractive index in the fiber optical medium. This is opposed to the conventional lens case where a graded change of shape in the optical medium, with fixed refractive index, produces the focusing property. These properties allow the design of relatively high numerical aperture (N.A.) objectives (typical N.A. =0.5) with small lateral dimension (< 1 mm) which can be used in combination with a conventional transmitted light or confocal microscope.

Definition of aberrations of a GRIN fiber objective:

Fabrication defects, material unhomogeneity and other construction problems (e.g. illuminating optics mismatches and relative misalignments between illumination beam, illuminating lens and GRIN fiber) introduce departures of the performance of the GRIN fiber objective from the predictions of optical theory and/or ray tracing algorithms, which result in blurring/distortions of the image produced by the GRIN objective.

Definition of Adaptive Optics:

an adaptive optics system can modify, in commanded coherent way, the wavefront shape of an incoming collimated beam of light. Basic part of an adaptive optical system is the wavefront deformation device; there are several technologies available to this purpose, subdivided between transmissive and reflective devices.

Definition of Wavefront Sensor:

a wavefront sensor is a metrological device that can detect wavefront differences from a pre-assigned reference wavefront (typically flat) in a set of points of an incoming beam of light. Differences can be expressed in terms of mathematical functions such as Zernike polynomials. Definition of wavefront control: wavefront control is the control loop which permits the correction of optical distortions present in an optical device by the measurement of distortions and the corrective action taken using an adaptive optics device.

Point Spread Function optimization: in the field of medicine and biological microscopy (with the only exception of ophthalmology) it is impossible to operate a wavefront sensor on the light produced by an observed specimen. This is a direct consequence of the unavailability of a point-like reference source and consequent lack of a pupil where phase can be analyzed.

An alternative way, found in microscopy and further developed through the invention herein presented, is to make a pre-calibration of the measurement set-up in a configuration not involving specimens but a reference medium. With this set-up it is possible to act in modal way on the deformable device observing the behavior of a merit function based on the measure of the final point spread function. Obtained optimization tables are then iteratively applied during the real observation.

Summary of Invention

By means of the combination of an adaptive optics system, typically a deformable mirror, and a specific error minimization algorithm, here described, it is possible to adaptively correct distortion inherent in the images formed by a generic GRIN fiber objective for microscopic observations, greatly improving image quality.

Technical Problem

GRIN fiber objectives are hybrid devices made by the terminal fiber itself combined with a fiber illuminating optics based on an aspheric element (FIG. 3). They suffer from several optical distortions as mentioned above.

Solution to Problem In previous GRIN fiber configurations, moveable mechanisms have been applied to the illuminating lens in order to overcome the problem; the solution here presented is to insert in the input collimated laser beam, before the intervention of the scanning unit, an adaptive optics system (FIG. 2) capable of correcting distortions induced by the GRIN objective, and even wavefront distortions present in the laser beam itself.

The GRIN objective system here presented (FIG. 3) has no moveable parts thanks to the presence of the aberration compensator based on the adaptive optics system mentioned above (e.g. deformable mirror). The relays of GRIN fibers plus the illuminating lens are connected by simple mechanics allowing set-and-forget regulation of the distance between fiber relay and lens by means of a micrometric screw. The GRIN objective is connected to the conventional microscope-objective attachment by means of a suitably threaded adapter.

The adaptive optics (deformabie mirror, DM) shown in FIG. 2 belongs to the category of thin membranes electrostatically activated mirrors, several models of which are commercially available together with drive electronics. A deformabie mirror system with a limited number of actuators, 37 in the present embodiment, can then be used to correct the system.

A pair of beam compressor/expanders, also commercially available, has been used in order to match the diameter of the input collimated laser beam (3 mm) to the diameter of the deformabie mirror (10 mm). Beam expanders, which introduce unavoidable aberrations in the beam wavefront, might be disposed of to provide even better results, if adaptive optics are constructed using the new generation of MEMS based electrostatic devices, with reduced dimensions more closely matched to the diameter of the laser beam. Optical distortions introduced by the combined microscope plus GRIN objective system are of low order (defocusing, astigmatism, coma and spherics) and chiefly due to assembly misalignments. Compared to other fields of application (i.e. observational astronomy or ophthalmology) adaptive optics implementations in the fields of microscopy and microendoscopy present with the known problem of estimating wavefront distortions on the light emitted by the examined specimen. This is chiefly due to the unfeasibility to have on the system, during the actual observation on the specimen, a true pupil produced by a point-like source to be projected inside a waveform analyzer. The classic solution of a wavefront-analyzer in loop with a deformable mirror is thus not effective.

The solution adopted on this invention embodiment is to pre-calibrate the FIG. 1 setup in two consecutive steps:

1- the wavefront correction system (FIG. 2) is calibrated by retrieval of the actuator voltage patterns corresponding to the natural modes of deformation;

2- the GRIN objective is focused on a fluorescent sample and the returned fluorescence is light focalized on a digital camera, as shown in FIG. 4. By suitable choice of the sample, calibration is possible for both single- and multi-photon applications.

Step 1 requires the construction of a 'solicitation matrix', which is derived by activating each deformable mirror actuator at minimum and maximum value and measuring the resulting wavefront distortion on a modal wavefront analyzer placed after the mirror. The next action involves the analytical retrieval of the actuator voltage patterns corresponding to DM membrane natural modes and even Zernike modes, i.e. :

C. = M x ^k ,y Φ x,y

where c _k is the electrode voltage pattern (k = 1, 37) corresponding to a required deformable membrane shape phase b _{x y} and the 'influence matrix' * contains the mirror natural deformation modes U as implicit in the decomposition

M _x ^k = V x S ^'l x U ^T . Step 2 is based on a recursive minimization algorithm based on the standard simplex (Nelder-Mead) method that iteratively imposes modal perturbations to the mirror, starting from the lowest order, minimizing a merit function such as FWHM + FWHM]

ε = —

Max after computation of full width at half maximum (FWHM) and maximum (Max) of the light spot focalized on the calibration arm of Fig.4. The light spot is an image of the point spread function of the system provided that the GRIN objective:

- is focused on a thin film of molecules for single-photon applications; - is immersed in a solution of uniformly distributed fluorescent molecules excited by the input laser beam for multi-photon applications; The invention is, by proper nature, modifiable and adaptable to several microscopy and microendoscopy observational techniques and is not limited to the peculiar embodiment hereinafter described.

Additional features and advantages of the invention will be apparent from the following detailed description in conjunction with the accompanying drawings, illustrating by way of example the features of the invention.

FIG. 1 reports on the general invention concept. A commercial laser scanning microscope body provided with a collimated beam of laser light for illumination, an x-y scanning unit, and a punctual fluorescence light receptor (photo-tube) is modified for in-vivo applications by mount of:

- an adaptive-optics system interposed on the collimated illumination beam;

- a custom GRIN based objective ;

- a planar detector on the fluorescence returning light path.

The three modifications made are reported in FIGURES 2, 3, 4 respectively. Advantageous Effects of Invention

The important advantages of the present invention are the peaking of the gathered light in single and multi-photon confocal microscopy applications, the sharpening of the illumination PSF and, generally, the minimization of wavefront distortions produced by the GRIN objective optics.

The key-point for the optimization algorithm used on the present invention is that deformable mirror stimuli are not applied on a single electrode base but are instead activating a limited set of patterns contained inside the

K,

representing the orthogonal set of natural flexure modes for the mirror membrane.

The main advantage is to make possible the convergence of the merit function minimization in a limited number of trials. A second advantage is that a complete table of minimizations can be created moving the service laser beam in different positions of the field of interest acting on the microscope scanning unit.

During normal operation the calibration arm is removed and the obtained set of tables are directly applied to the deformable mirror during the specimen scan.

Brief Description of Drawings The invention may be more completely understood by consideration of the accompanying drawings here described in brief :

FIG. 1 shows an example of arrangement for in-vivo microscopy and microendoscopy based on a GRIN fiber objective, an adaptive-optics system and removable post-calibration optics;

FIG. 2 shows the adaptive optics system based, on this invention embodiment, on a commercial electrostatic mirror plus a pair of beam expander/reducers;

FIG. 3 shows the GRIN fiber objective based on a mechanical mount supporting the fiber and an aspheric lens;

FIG. 4 shows the post-calibration device based on a folding mirror placed on the sample return beam plus an optical system producing a conjugate image of the sample and a matrix detector (e.g. a CMOS or CCD camera). Industrial Applicability

Areas of application of the adapti vely-corrected GRIN objective are microscopy and micro-endoscopy operated by means of: transmitted light, single and multi-photon confocal laser scanning apparatuses, OCT (Optical Coherence Tomography).

Citation List

Patent Literature

The following are patents related to the present invention:

U.S. Pat. No. 2009/0054791 Ralf Wolleschensky, Robert Grub (2004) :

Applications of adaptive optics in microscopy

U.S. Pat. No. 2009/0054791 Benjamin Flusberg, Mark Jacob Schnitze (2009) : Microendoscopy with corrective optics