ITRM20100286A1 - SPECTRAL CONFOCAL MICROSCOPE IN REFLECTION WITH A WIDE BAND. - Google Patents

Description

Confocal spectral microscope in broadband reflectance

The present invention relates to a spectral confocal microscope in broadband reflectance.

More precisely, the present invention relates to a broad spectrum laser scanning confocal reflectance microscope. This tool can be used for both reflectance spectral analysis and morphometric imaging of optical sections of viable tissue samples (in vivo, ex vivo, in vitro), or materials science samples. Spectral data and images can be acquired simultaneously, in a very wide range of wavelengths, thanks to the use of a supercontinuum source (a particular type of laser, where the generation of radiation occurs in a photonic fiber, called also "white laser" or "white light laser").

Alongside the "traditional" confocal microscopy linked to fluorescence, today the laser scanning confocal microscopy solutions available on the market and used as a means for diagnostics belong to two families: reflectance microscopes and those with two-photon laser excitation. It should be noted that the development essentially concerned the application for dermatological diagnostics and therapy.

Confocal laser scanning reflectance microscopy (for example the "Vivascope" system from Mavig GmbH) is an imaging modality to visualize the most superficial layers of the skin in real time and in a non-invasive way but with high contrast and spatial resolution under the micrometer, therefore comparable to conventional histological analysis. The application of this tool to the visualization of the different layers of the skin has recently brought great advantages in dermatology, allowing to open a virtual window on the skin in physiological conditions, therefore without the need for biopsies or tissue treatment. The possibility of acquiring high-resolution images in a non-invasive way has wide applications in clinical and research areas, such as in the diagnosis of benign and malignant lesions, the identification of tumor margins, the evolution of the response to pharmacological, surgical or cosmetics and the pathophysiological study of inflammatory processes.

In confocal reflectance microscopes, the infrared and monochromatic radiation from the laser is focused on the skin sample. The radiation, passing between cellular structures with different refractive indices, is naturally reflected, is collected by the lens, sent to an acquisition system and recomposed into a two-dimensional image in gray tones by a computer software. By moving the focus of the microscope along the axis perpendicular to the skin surface, images can be obtained from planes at different depths in the skin. (Piergiacomo Calzavara-Pinton, Caterina Longo, Marina Venturini, Raffaella Sala and Giovanni Pellacani Reflectance Confocal Microscopy for In Vivo Skin Imaging, Photochemistry and Photobiology, 2008, 84: 1421-1430; Kishwer S. Nehal, Dan Gareau, and Milind Rajadhyaksha, Skin Imaging With Reflect ance Confocal Microscopy, Semin. Cutan. Med. Surg. 27: 37-43,2008).

On the other hand, the commercial multiphoton tomograph (eg "Dermainspect" from Jenlab GmbH) is based on the fluorescence caused by a two-photon excitation and second harmonic generation. The fields of use are early tumor diagnostics, analysis of the interaction between engineered and human tissues and in situ drug screening (Enrico Dimitrow, Iris Riemann, Alexander Ehlers, Martin Johannes Koehler, Johannes Norgauer, Peter Elsner, Karsten Koenig and Martin Kaatz, Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis, Experimental Dermatology, 18, 509-515, 2009).

In the case of two-photon excited fluorescence, two photons of equal wavelength in the infrared (spectral range 800-1200 nm wavelength) are absorbed simultaneously. Each photon provides half the energy that is normally required to excite the fluorophore into a higher energy electronic state. Hence the blue, green, yellow and red emissions of the fluorophores can be induced with photons in the near infrared, therefore with lower energy. With a three-photon process, photons in the near infrared can also induce ultraviolet fluorescence. The spectral range between 800 and 1200 nm corresponds to the optical window of cells and tissues, and is characterized by a high depth of penetration due to the low absorption and scattering coefficients.

A very particular luminescence, on the other hand, can be observed when the collagen is illuminated by ultra-short infrared laser pulses: in this case, the extracellular matrix proteins emit light at a wavelength that is half that of the incident laser light. This process is called second harmonic generation (SHG). It occurs only in some non-centrosymmetric molecular structures such as collagen, myosin or tubulin when they are hit by intense ultra-short laser pulses.

Spectroscopy applied to a confocal design, on the other hand, has so far been applied to confocal Raman and CARS methods (Coherent anti-Stokes Raman spectroscopy), called in the case of coupling to optical microscopes Raman microspectroscopy (R. J. Swain and M. M. Stevens, Raman microspectroscopy for non-invasive biochemical analysis of single cells, Biochemical Society Transactions (2007) Volume 35, part 3) or applied to fluorescence spectroscopy (in the time or frequency domain) linked to the life times of fluorochromes. Raman microspectroscopy, which has undergone great development in recent times, combines the tradition of chemical analysis using vibrational spectroscopy with the possibility of carrying out the investigation on samples represented by single live cells immersed in their culture medium without adding fixatives or reagents. external. Unlike infrared spectroscopy, the spectral range used avoids areas of great water absorption. The fundamental limitation of Raman techniques is the intrinsic weakness of the signal which therefore requires the choice of suitably sensitive detectors and high intensity sources.

In both cases, however, only one wavelength is used to excite specific fluorochromes or molecules.

The object of the present invention is to provide a confocal reflectance microscope which solves the problems and overcomes the drawbacks of the prior techniques.

The object of the present invention is a confocal spectral microscope in broadband reflectance, comprising a laser source, a beam splitter, a system for focusing the laser beam on the sample, a system for detecting the laser beam reflected from the sample and transmitted by the divider. beam, characterized by the fact that:

said laser source is a continuous spectrum source in the visible and infrared wavelengths;

- a beam expander is inserted immediately after the laser source;

- said beam splitter is made of suitably transparent material throughout the spectral range emitted by the source and is placed at 45 ° with respect to the direction of the beam;

- said focusing system is achromatic and has a spectral band between 200nm and 20μπι, a long working distance greater than 5mm and a numerical aperture greater than 0.25.

Preferably according to the invention, the material of the beam splitter is calcium fluoride.

Preferably according to the invention, the material of the beam splitter is Zinc Sulfide.

Preferably according to the invention, said focusing system is a first reflective objective.

Preferably according to the invention, said focusing objective has a Cassegrain-Schwarzschild design.

Preferably according to the invention, the spectral band of said focusing system is comprised between 400nm and 3μπι.

Preferably according to the invention, the working distance is greater than 8mm, and the opening is greater than 0.40.

Preferably according to the invention, the sample is moved, with respect to the focus of the focusing system, by a lateral and axial scanning system with piezoelectric control.

Preferably according to the invention, the microscope comprises means suitable for centering the focus of the focusing system at the inlet of a single-mode fiber, which is mounted at the other end on a miniaturized mechanical system which comprises a system for suction and anchoring to the sample and a piezoelectric control for scanning the fiber with respect to the sample, in particular said means being able to be coupled to endoscopic systems.

Preferably according to the invention, the focusing system is adapted to collect the light reflected by the sample, which passes through the beam splitter and is conveyed through an optical system capable of maintaining the achromaticity of the beam and therefore achromatically focused through a second lens on a pinhole whose opening is variable and controllable in a continuous and repeatable manner by means of piezoelectric control means.

Preferably according to the invention, said second objective for focusing the beam leaving the sample on the pinhole is of the reflective type and has a Cassegrain-Schwarzschild design.

Preferably according to the invention, said pinhole is constituted by a pair of plates with a main body and a dovetail suitably machined arranged with the dovetails facing and in contact symmetrically on one side and on the other of a plane, the plates of said pair of laminae being able to be moved continuously along the plane with respect to their common center, thus delimiting a rhomboid hole with a diameter ranging from 0 to 200 μπι.

Preferably according to the invention, the pinhole is optically coupled to a collection system which, through an optical fiber or a free path, conveys the signal to a chromatic dispersion system and to a subsequent linear spectrometric acquisition and analysis section formed by one or more multiple array or matrix sensors.

The invention will now be described for illustrative but not limitative purposes, with particular reference to the drawings of the attached figures, in which:

- figure 1 shows the constructive scheme of the confocal microscope according to the invention.

- figure 2 shows the pinhole scheme according to the invention, in three different views (a) section, (c) from above, and (b) in perspective

The instrument according to the invention is a confocal laser scanning microscope. It is powered by an LS laser source with a broad and continuous spectrum in the visible and infrared wavelengths.

The beam coming out of the photonic fiber that generates it is expanded through a beam expander or achromatic "beam expander" BE (necessary to maintain the possibility of sending all the wavelengths generated by the source without chromatic aberration), which is essential to exploit the construction characteristics of the reflective lens.

Then, through a system of plane mirrors M, the light beam is sent to a beam splitter or "beam splitter" BS of suitably transparent material in the entire spectral range emitted by the source and placed at 45 with respect to the direction of the beam. .

The role of the beam splitter is particularly critical for the microscope according to the invention. In fact, this component must be crossed twice by the laser beam coming from the source, a first time in reflection and a second time in transmission. Several constructive elements have been taken into consideration, as listed below.

First, the reflection / transmission sequence could in principle be reversed into transmission / reflection. However, reflection should be given precedence in the beam path as minimum optical distortion must be ensured towards the target facing the sample. This choice also has the effect of reducing the incident intensity in a manner determined by the reflection coefficient of the foil, to avoid thermal overload of the sample.

The innovative solution of the present invention, linked to the use of a broad spectrum source such as the photonic fiber laser used here, also lies in the way of reducing the intensity of radiation deposited on the sample, through a beam splitter according to the invention.

In fact, this intensity cannot be simply adjusted by acting on the power of the laser source used. The laser controller would allow, in effect, an easy control of the emission power, but, at the same time, an excessive reduction can profoundly modify the emission spectrum by eliminating a large part of the visible spectrum. In fact, the extension of the "supercont inuum" laser spectrum is linked in a complex way to non-linear phenomena and, therefore, under a certain power threshold the spectrum would be profoundly altered.

For all these reasons, the material chosen for the BS component is Calcium Fluoride (CaF2). This choice is dictated by a series of factors. Mainly, we want to have a material with good optical properties (absence of absorption) for the extremely extended spectral range of the photonic laser used (450nm-2.5μπι) and a good structural and chemical stability. No other choices seem possible, apart from giving up some fundamental properties. Table 1 shows the disadvantages or characteristics for other possible materials. The only material that can be used as an alternative is Zinc Sulfide (ZnS), known by the trade name CLEARTRAN ™ (from CVD Ine.), Which has similar characteristics, but excludes the possibility of working with sources that can extend into the ultraviolet region. for fluorescence experiments. Such material could represent an interesting alternative. However, the high refractive index (2.27 a Ιμπι) produces a lower transmission (about 70%), and therefore a greater reflection which, if it would theoretically increase the available signal, would force a decrease in the input power for samples. very sensitive to local heating. As already mentioned, this variation can lead to a profound modification in the emission spectrum of the photon fiber laser. The most common glasses were excluded as they are opaque in the infrared.

The solution proposed according to the present invention is therefore the use of Calcium Floride as material for the BS beam splitter to have an accessible spectral window between 0.15μπι and 6μπι, with a transmission greater than 90% in this range, an excellent degree of insolubility in water, with consequent stability over time, the absence of defects that could be centers of diffusion or absorption by intense laser beams, and the absence of phenomena of birefringence which, with coherent sources in the given configuration, would produce deleterious modulations in the optical response.

Table 1

The refractive index of the crystal causes a part of the beam, as described, to be reflected towards the achromatic reflective objective OBI and therefore focused by it on the sample S.

Another important point of the microscope according to the invention is constituted by the OBI objective which focuses the beam on the sample. For the purposes of this confocal microscope, the objective must be characterized by a wide spectral band, long working distance and high numerical aperture to allow the beam to be focused on a very narrow area on the focal plane, on the surface or inside the sample. analyses.

In the prototype we used an objective with a spectral band between 200nm and 20μπι, a working distance of 10.4mm, and a numerical aperture of 0.52. Preferably, the band is between 400nm and 3μπι, the working distance is greater than 5mm, and the aperture is greater than 0.25.

The solution according to the invention uses a reflective lens. This type of objective, which can be characterized by Cassegrain-Schwarzschild design, meets the above requirements, and does not find general application in confocal microscopes as it requires particular care in the geometry of the beam. Furthermore, there is generally no need for such a large spectral range as the case described above. Although other solutions cannot be ruled out, commonly used refractive lenses would suffer from a shift of focus as a function of wavelength.

A piezoelectrically controlled XYZs lateral and axial scanning system moves the sample relative to the focus of the objective.

The light reflected by the sample S is collected by the objective (condenser) OBI, passes through the beam splitter BS and is conveyed through an optical system M capable of maintaining the achromaticity of the beam (combination of paraboloid and flat mirrors all with optimized surface for the entire spectral range in use) and then focused (always in achromatic way) through the 0B2 objective on a PH pinhole whose aperture is variable and controllable by piezoelectric technology.

The pinhole PH is a characterizing and crucial element for every confocal system. Its presence ensures the possibility of optical sectioning of the sample, because it excludes from the detection the signal not coming from the focus volume of the objective.

Many systems provide a choice of several fixed size pinholes. In the case of the present invention, the need to work with very different levels of signal intensity and need for axial resolution has been envisaged.

The solution according to the present invention is represented in figure 2 and consists in the design of an original design consisting of a pair of sheets 110,120 with a main body 111,121 and a dovetail 112,122 suitably machined which can be moved symmetrically with respect to a plane 200 and continuous with respect to the central point 130 thus delimiting a rhomboidal hole 140 of variable diameter between 0 and 200 μπι. The movement, as already mentioned, is managed and controlled by a piezoelectric assembled and calibrated on this specific system which allows therefore to vary in a continuous and repeatable way the opening of the pinhole to adapt the detection to the different experimental needs.

The pinhole is optically coupled to a collection system which, through an optical fiber or a free path, conveys the signal to a chromatic dispersion system and to a subsequent acquisition formed by one or more AS array sensors (Array-Spectrometer, or Linear Spectrometer; the sensors can be diodes, CMOS, CCD, or any other equivalent device) which allows the simultaneous acquisition of all wavelengths reflected from the same point (the focus point).

As an alternative to scanning with the piezoelectrically controlled motorized axes system, for samples that cannot be deposited on the motorized axes, it is possible to center the focus of the objective or other achromatic optical devices at the input of a single-mode fiber which, mounted at the output on a miniaturized mechanical system for endoscopy that includes a tissue suction and anchoring system, perform a three-dimensional piezoelectric control scan on the sample. This instrument thus coupled to endoscopic systems allows to reach the internal surfaces of the human organism (applications on the entire digestive tract, but also of the gynecological, urological or large vessels type).

In fact, this system allows the source and the detector to be positioned even at a certain distance from the sample under investigation, without the need for a rigid mechanical connection between the parts (see in this regard the US patent No.

6,341,036, "Confocal miniaturized System incorporated into a compact probe"). In this case, the light reflected by the sample is then again collected by the fiber and exiting this collection by the OBI objective or other achromatic optical device and from here it follows the same path indicated above.

The innovative solution proposed lies in the simultaneous use of all the acquired wavelengths. Unlike other designs, the entire design of the machine is aimed at this purpose.

A specially developed software controls the scanning, acquisition and storage of data.

The instrument according to the invention is innovative because it makes it possible to combine visible and near infrared reflectance spectroscopy on samples of arbitrary size with submicrometric resolution with confocal imaging techniques.

Confocal imaging, which has traditionally been divided between fluorescence microscopy, used primarily for research, and reflectance microscopy, used in clinical practice, allows visualization of fluorescent molecules and tissue architecture, respectively.

The instrument according to the invention combines physico-chemical information with very high spectral resolution and high spatial resolution brought by the spectroscopic response to the structural information carried by imaging typical of the confocal approach, thanks to the simultaneous processing of a frequency spectrum instead of a single frequency.

The possibility of associating an entire spectrum to each scanning point (corresponding to physical dimensions of the order of a micron or lower) overcomes one of the past limitations of single wavelength reflectance microscopy: the lack of specificity for organelles and ultrastructures (Kishwer S. Nehal, Dan Gareau, and Milind Rajadhyaksha, Skin Imaging With Reflectance Confocal Microscopy, Semin. Cutan. Med. Surg. 27: 37-43,2008).

The possibility of observing the same physical surface under eg. 2000 different wavelengths is equivalent to the possibility of having 2000 different contrast modes, as in fluorescence or histology different probes are used to characterize different targets. The spectral and imaging approaches, combined in a synergistic way, lead to the possibility of having multimodal images from optical sections with the possibility of obtaining a structure-specific contrast.

The foreseeable applications of this technology involve fields such as dermatology, cosmetology, ophthalmology, pharmacology (both as pharmacodynamics and pharmacokinetics), tissue bioengineering, but also many issues related to endoscopy. The possibility of being able to vary the emission power of the laser on extremely defined regions of tissue, even automatically, extends the potential applications from diagnostics to highly selective therapy of various pathologies and to the typical applications of lasers for "cosmetic" use.

Another feature of the instrument according to the invention is the possibility of combining conventional laser scanning confocal microscopy or Raman spectroscopy with the described techniques, to increase the diagnostic potential and the related applications.

In the foregoing, the preferred embodiments have been described and variants of the present invention have been suggested, but it is to be understood that those skilled in the art will be able to make modifications and changes without thereby departing from the relative scope of protection, as defined by the claims attached.

Claims (13)

CLAIMS 1) Confocal spectral microscope in broadband reflectance, comprising a laser source (LS), a beam splitter (BS), a system (OBI) for focusing the laser beam on the sample (S), a detection system (M, 0B2, PH, AS) of the laser beam reflected from the sample (S) and transmitted by the beam splitter (BS), characterized in that: said laser source (LS) is a continuous spectrum source in the visible and infrared wavelengths; immediately after the laser source (LS) a beam expander (BE) is inserted; - said beam splitter (BS) is made of suitably transparent material throughout the spectral range emitted by the source and is placed at 45 ° with respect to the direction of the beam; - said focusing system (OBI) is achromatic and has a spectral band between 200nm and 20μπι, a working distance greater than 5mm and a numerical aperture greater than 0.25. 2) Microscope according to claim 1, characterized in that the material of the beam splitter (BS) is Calcium Fluoride (CaF2). 3) Microscope according to claim 1, characterized in that the material of the beam splitter (BS) is Zinc Sulfide (ZnS). 4) Microscope according to any one of claims 1 to 3, characterized in that said focusing system (OBI) is a first reflective objective. 5) Microscope according to claim 4, characterized in that said focusing objective (OBI) has a Cassegrain-Schwarzschild design. 6) Microscope according to any one of claims 1 to 5, characterized in that the spectral band of said focusing system (OBI) is comprised between 400nm and 3μπι. 7) Microscope according to any one of claims 1 to 6, characterized in that the focusing system (OBI) has a working distance greater than 8mm, and an aperture greater than 0.40. 8) Microscope according to any one of claims 1 to 7, characterized in that it comprises a lateral and axial scanning system with piezoelectric control (XYZs) suitable for moving the sample (S) with respect to the focus of the focusing system (OBI). 9) Microscope according to any one of claims 1 to 7, characterized in that it comprises means suitable for centering the focus of the focusing system (OBI) at the inlet of a single-mode fiber, which is mounted at the other end on a system mechanical miniaturized which comprises a system for sucking and anchoring to the sample (S) and a piezoelectric control for scanning the fiber with respect to the sample (S), in particular said means being able to be coupled to endoscopy systems! . 10) Microscope according to any one of claims 1 to 9, characterized in that the focusing system (OBI) is adapted to collect the light reflected by the sample (S), which passes through the beam splitter (BS) and is conveyed through an optical system (M) capable of maintaining the achromaticity of the beam and therefore is achromatically focused through a second objective (OB2) on a pinhole (PH) whose aperture is variable and controllable in a continuous and repeatable manner by means of control piezoelectric. 11) Microscope according to claim 10, characterized in that said second objective (OB2) for focusing the beam leaving the sample on the pinhole is of the reflective type and has a Cassegrain-Schwarzschild design. 12) Microscope according to claim 10, characterized by the fact that said pinhole (PH) comprises or consists of a pair of plates (110,120) with a main body (111,121) and a dovetail (112,122) suitably worked arranged with the tails dovetails (112,122) facing and in contact symmetrically on both sides of a plane (200), the blades of said pair of blades being able to be moved continuously along the plane (200) with respect to their common center ( 130), thus delimiting a rhomboid hole (140) of variable diameter between 0 and 200 μπι. 13) Microscope according to claim 10 or 12, characterized in that the pinhole (PH) is optically coupled to a collection system which, through an optical fiber or a free path, conveys the signal to a chromatic dispersion system and to a next section of acquisition and linear spectrometric analysis (AS) formed by one or more array or matrix sensors.