This invention concerns a wide band reflectance spectral confocal microscope and its related spectral imaging method.

More precisely, this invention concerns a wideband reflectance confocal laser scanning microscope. This instrument can be used for the reflectance spectral image as well as for the morphometric imaging of optical sections of vital tissue samples (in vivo, ex vivo, in vitro), or for materials science samples. Spectral data and images can be acquired simultaneously, in an extremely wide interval of wavelengths, due to the utilisation of a supercontinuum type of source (a particular kind of laser where the radiation generation occurs in a photonic fibre, aka "white laser" or "white light laser").

Besides the "traditional" confocal microscopy linked to fluorescence, the current confocal laser scanning microscopy solutions available on the market and used as a diagnostic means belong to two families: reflectance microscopes and those with two- photon excitation lasers. It should be noted that developments have concerned mainly the application for dermatological diagnostics and therapy.

Reflectance confocal laser scanning microscopy (for example, the "Vivascope" system by Mavig GmbH) is an imaging mode for visualizing the more superficial cutis layers in real time and in a non-invasive manner, but with a high contrast and a spatial resolution under the micrometre, therefore comparable to conventional histological analysis. The application of this instrument for visualizing the various skin layers has produced recently great benefits to dermatology, enabling to open a virtual window on skin under physiological conditions, therefore without the need to perform a biopsy or tissue treatment. The possibility of acquiring high-resolution images, and in a non-invasive manner, has wide applications in clinical and research areas, as well as in the diagnosis of benign and malignant lesions, the detection of tumour margins, the evolution of the answer to pharmacological, surgical or cosmetic treatments, and the pathophysiological study of inflammatory processes.

In reflectance confocal microscopes, the monochromatic radiation of the laser is focused on the skin sample. The radiation, passing through the cellular structures with different refraction indexes, is naturally reflected, collected by the objective, sent to an acquisition system and recomposed by computer software into a grayscale two-dimensional image. Moving the microscope's focus along the axis perpendicular to the skin's surface allows to obtain images from different levels of skin depth (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 Reflectance Confocal Microscopy, Semin. Cutan. Med. Surg. 27:37-43, 2008).

On the other hand, a multiphotonic commercial tomograph

(e.g. "Dermainspect" by Jenlab GmbH) is based on two-photon excitation fluorescence and by second harmonic generation. The fields of utilisation are early detection of tumours, the analysis of the interaction between engineered tissues and human tissues, and pharmacological screening in situ (Enrico Dimitrow, Iris Riemann, Alexander Ehlers, Martin Johannes Koehler, Johannes Norgauer, Peter Eisner, 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 excitation fluorescence, two photons of the same infrared wavelength (wavelength spectral interval of 800-1200 nm) are absorbed simultaneously. Each photon supplies half the amount of energy normally required to excite the fluorophore in an electronic state with higher energy content. Therefore, the blue, green, yellow and red fluorophore emissions can be induced with photons in the near infrared, therefore at a lower energy. With a three-photon process, the photons in the near-infrared can also induce ultraviolet fluorescence. The spectral interval between 800 and 1200 nm corresponds to the optical window of cells and tissues, and is characterized by a high penetration depth because of the low absorption and scattering coefficients.

Conversely, a very specific luminescence can be observed when collagen is illuminated by ultrashort infrared laser pulses. In this case, the extracellular matrix proteins emit light at a wavelength that is half the wavelength of the incident laser light. This process is called Second Harmonic Generation (SHG). It happens only in some non-centrosymmetric molecular structures, such as collagen, myosin or tubulin when they are hit by ultrashort intense laser pulses.

Spectroscopy applied to a confocal design, instead, has so far been applied to the Raman and Cars confocal methods (Coherent anti-Stokes Raman spectroscopy) which, in the case of coupling to optical microscopes, is called 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 of (in the time and frequency domain) linked to the lifetimes of fluorochromes. Raman microspectroscopy, which in recent years has undergone great developments, combines the tradition of chemical analysis performed by means of vibrational spectroscopy with the possibility of carrying out testing on samples formed by single living cells immersed in their culture medium, with no addition of fixatives or external reagents. Differently from infrared spectroscopy, the spectral interval here utilised avoids the areas of high water absorption. The fundamental limit of Raman techniques is the intrinsic weakness of the signal, therefore imposing the choice of detectors that are adequately sensitive and of high intensity sources.

In both cases, however, a single wavelength is used to excite fluorochromes or specific molecules.

The patenting document US2007/114362 describes a confocal microscope for spectral reflectance microscopy, in particular for microarray. This microscope would have enabled to obtain high resolution images and fast scanning speed, when the two features are generally conflicting. The microscope utilises lenses for the optics of the beam projection and focusing. The light source utilised may include more than one radiation source, for example lasers that are each one capable of generating radiation at different wavelengths. The emission signals coming from distinct examined samples may be collected simultaneously or in sequence. The excitation radiation generated by each source of radiation may pass through filters and radiation combiners before being directed toward an expander. Different emissions, however, due to the microscope's structure, cannot be referred unambiguously to an exact point in space, i.e. response signals do focus all on the same point.

The purpose of this invention is to provide a reflectance confocal microscope that resolves the problems and overcomes the inconveniences of previous techniques.

A further specific purpose of this invention is an imaging method utilising a scanning confocal microscope.

A further specific purpose of this invention is the utilisation of the confocal microscope for the acquisition of spectral images of a sample.

It is subject-matter of the present invention is a wideband spectral reflectance confocal microscope, comprising the following: a laser source; a beam expander inserted immediately after the laser source; a beam splitter in a material that is adequately transparent through the whole spectral interval emitted by the laser source; a focusing system for the laser beam on the sample with a working distance greater than 5 mm and a numerical aperture greater than 0.25; a system of collection and analysis of the laser beam reflected by the sample and transmitted by the beam-splitter, whereby such analysis system includes a chromatic dispersion subsystem capable of forming the spectrum to be analysed. The microscope is characterized by the following:

said laser source is a source emitting simultaneously at several wavelengths in the visible and infrared regions;

said focalization system is free of chromatic aberration;

said beam-splitter is positioned at 45° in respect of the laser beam direction;

said collection system is free of chromatic aberration;

so that at each analysed point of the sample corresponds unambiguously a frequency response spectrum collected by the collection system.

According to the invention, this beam-splitter preferably comprises a lamina in a material with an n _B s refraction index and a thickness, in order to obtain:

a minimal deviation of the beam escaping from the beam-splitter with respect to the optical axis of the system, as a rigid translation with respect to the optical axis, or as an angular deviation with respect to this same optical axis:

a minimal chromatic aberration introduced by the beam-splitter:

so that at each analysed point of the sample shall correspond unambiguously a frequency response spectrum over essentially the whole interval emitted by the laser source.

In this context, "minimal deviation" and "minimal aberration" mean respective values so as to achieve the scope whereby at each analysed point of the sample shall correspond unambiguously a frequency response spectrum essentially over the whole interval emitted by the laser source. A typical technician in this field, through routine experiments, will be able to determine therefore the values of the refraction index and of the thickness required to ensure this effect, in each application case.

Preferably, according to the invention, said laser source shall be of continuous spectrum.

Preferably, according to the invention, said laser source is a multiple source emitting several wavelengths through spectral multiplexing.

Spectral multiplexing here means the combination of diverse sources which beams then propagate simultaneously along the same optical path.

Preferably, according to the invention, said lamina material is Calcium Fluoride, Zinc Sulphide, or optical glass of adequate optical transmittance.

Preferably, according to the invention, said lamina material is BK7 and the thickness c½ is less or equal to 160 μηι.

Preferably, according to the invention, said focusing system is achromatic.

Preferably, according to the invention, said focusing system is a first reflective objective free of chromatic aberration.

Preferably, according to the invention, the focusing objective is an objective with Cassegrain-Schwarzschild design.

Preferably, according to the invention, the spectrum band of said focusing system is comprised between 200 nm and 20 μητι.

Preferably, according to the invention, the focusing system presents a working distance greater than 8 mm, and an aperture greater than 0.40.

Preferably, according to the invention, the microscope comprises means capable of centring the focus of the focusing system at the entrance of a monomodal fibre, mounted at the extreme opposite on a miniaturized mechanical system that comprises a system for the suction and securing to the sample, and a piezoelectric control for scanning the fibre in respect of the sample; in particular, such means may be coupled with endoscopic systems.

Preferably, according to the invention, the aperture of said pinhole is variable and controllable in a continuous and repeatable manner by means of piezoelectric or mechanical means of control, or it is fixed at an adequate value.

Preferably, according to the invention, said second objective for focusing the beam escaping from the sample on the pinhole, is of a reflective type free of chromatic aberration.

Preferably, according to the invention, the pinhole comprises or is constituted by a pair of laminae with a main body and a dovetail suitably worked and arranged with the dove-tails facing and in symmetrical contact on both sides of a plane surface, the laminae of this pair being capable of being moved continuously along the plane in respect of their common midpoint, thus defining a rhomboidal hole of a variable diameter between 0 and 200 μΐτι.

Preferably, according to the invention, the sample is moved in respect of the focusing system's focus by a piezoelectrically controlled lateral and axial scanning system.

Preferably, according to the invention, the sample is fixed, while the focus of the focusing system is moved by a piezoelectrically or electromagnetically controlled lateral and axial scanning system.

Preferably, according to the invention, the pinhole is coupled optically to a collection system which, through an optical fibre or a free path, conveys the signal to a chromatic dispersion system and to a successive linear spectrometric acquisition and analysis section composed by one or more arrays or matricial sensors. A further specific subject-matter of the invention is a spectral imaging method of a sample by means of a wideband spectral reflectance confocal microscope, characterized by the fact that the microscope comprises a laser source, a beam expander inserted right after the continuous spectrum laser source in the visible and infrared wavelengths, a beam-splitter in a material that is adequately transparent through the whole spectral interval emitted by the laser source, a focusing system of the laser beam on the sample with a working distance greater than 5 mm and a numerical aperture greater than 0.25 and free of chromatic aberration, a collection system free of chromatic aberration and an analysis system that includes a chromatic dispersion subsystem capable of producing the spectrum to analyse, the laser beam reflected by the sample and transmitted by the beam-splitter positioned at 45° in respect of the laser beam direction, and by the fact of performing the following phases:

send a continuous spectrum laser beam on the sample through the said focusing system;

receive the radiation reflected by said sample through said collection system;

acquire the spectral data relating to the radiation reflected by said sample, through said acquisition system, assigning to each point of the sample a frequency response spectrum. A further specific subject-matter of the invention is the utilisation of the wideband spectral reflectance confocal microscope subject-matter of the invention, for the acquisition of spectral images of a sample, where each analysed point of the sample is assigned unambiguously a frequency response spectrum.

The invention is now illustrated to exemplify, but is not intended to be limited by this, with particular reference to the drawings in the attached pictures, where:

Picture 1 shows the construction outline of the confocal microscope, according to the invention: Picture 2 shows the pinhole outline, according to the invention, in three different views: (a) section; (b) in perspective; (c) from above.

This invention concerns a new quadri-dimensional optical microscopy in which the single pixel is represented by a whole reflectance spectrum (the wavelength coordinate, or other physically equivalent ones, represent the fourth coordinate) drawn from an X-Y-Z position, located even in depth of a given sample, drawn on a microscopic scale down to a dimension of 1 μ ^η τι ³ through a wideband spectral reflectance confocal microscope.

It is essential that that an entire spectrum might be associated unambiguously to the same physical point. To this purpose, the invention requires and is constrained to a microscope free of chromatic aberration and, more generally, to an achromatic microscope.

This allows achieving the invariance of the focal point, irrespective of the wavelength used.

The instrument, according to the invention, is a laser scanning confocal microscope. It is fed by a wideband and continuous band LS laser source, in the visible and infrared wavelengths

The beam escaping from the photonic fibre generating it gets expanded through a BE achromatic beam expander (required to preserve the possibility of sending all the wavelengths generated by the source, free of chromatic aberration), which is essential for exploiting the construction features of the reflective objective.

To follow, through an M flat mirroring system, the light beam is sent to a BS beam-splitter of a material that is adequately transparent through the whole spectral interval emitted by the source, and is positioned at 45° in respect of the beam direction.

The function 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 originating from the source: the first time in reflection, and the second time in transmission. Various constructive elements have been taken into consideration, as listed below.

To begin with, in principle, the reflection/transmission sequence could be inverted. In the beam path, however, it is worth reserving precedence to reflection, as it is necessary to ensure a minimal optical distortion with respect to the objective facing the sample. This choice has also an effect on reducing the incident intensity in a manner that is determined by the reflection coefficient of the lamina, in order to avoid a thermal overcharge of the sample.

The innovative solution of this invention, linked to the utilisation of a wideband beam source such as the photonic fibre here used, is also in the way the radiation intensity deposited on the sample is reduced through a beam splitter, according to the invention.

This intensity, in fact, cannot be regulated by acting simply on the power of the laser source used. Actually, the laser controller would allow a simple control of the emission power but, at the same time, an excessive reduction can modify severely the emission spectrum, eliminating a great part of the visible spectrum. In fact, the spectrum extension of supercontinuum lasers is linked in a complex manner to non-linear phenomena and, therefore, below a certain power threshold the spectrum would result being severely compromised.

For all the above reasons, the material selected for the BS component is Calcium Fluoride (CaF ₂ ). This choice is dictated by a complex of factors. The main aim is to have a material with good optical qualities (absence of absorbance) for the extremely extended spectral interval of the photonic laser utilised (450 nm-2.5 μΐτι), and a good structural and chemical stability. Other choices do not appear possible, unless giving up some fundamental properties. However, limiting the operational spectral range strictly between 350 nm and 2.3 μητι, even a thin glass lamina of BK7 type is acceptable. Table 1 shows the disadvantages or the main characteristics of other possible materials. The only material that can be used as an alternative is Zinc Sulphide (ZnS), also known under its commercial name CLEARTRAN™ (by CVD Inc.), which has similar characteristics, but excludes the possibility of working with sources that can extend into the ultraviolet region for fluorescence experiments. This material could represent an interesting alternative. However, the high refraction index (2.27 at 1 μητι) produces a high tendency to chromatic aberration and a lower transmittance (about 70%), hence a higher reflectance which, although increasing in theory the available signal, would impose a reduction of the power input on samples that were particularly sensitive to localized heating. As already mentioned, such variation might imply a profound modification of the emission spectrum of the photonic fibre laser. The more common glasses were excluded as opaque in the deep infrared.

The proposed solution, according to this invention, is therefore the utilisation of Calcium Fluoride as a BS beam divider material in order to obtain a spectral window accessible between 0.15 μΐη and 6 μΐη, with a transmittance higher than 90% of such interval, an optimal grade of insolubleness in water, with a consequential stability in time, the absence of defects that might be centres for the diffusion or absorbance by intense laser beams, and the absence of birefringence which, with coherent sources in the given configuration, would produce detrimental modulations in the optical response.

Material Transparency Region Notes

(μηι)

Barium Fluoride 0.15÷12.5 Slightly hygroscopic, sensitive to temperature

Caesium Bromide 0.22÷40 Hygroscopic and soft Caesium Iodide 0.25÷55 Hygroscopic and soft

Calcium Fluoride 0.13÷10 Virtually not hygroscopic

Germanium 1.8÷23 Opaque in the non-visible

Lithium Fluoride 0.12÷8.5 Hygroscopic, plastic

Magnesium Fluoride 0.11÷7.5 Birefringent

Potassium Bromide 0.23÷25 Hygroscopic

Potassium Chloride 0.23÷25 Hygroscopic

Rubidium Chloride / 0.21÷20 / 0.22÷35 / Hygroscopic

Bromide / Iodide 0.3÷50

Sapphire 0.15÷5.5 Birefringent

Silicon 1.2÷10 Opaque in the visible

Silver Bromide 0.43÷35 Partly opaque in the visible

Silver Chloride 0.4÷30 Partly opaque in the visible, subject to various effects if exposed to visible radiation

Sodium Chloride 0.2÷20 Hygroscopic

Sodium Fluoride 0.15÷14 Hygroscopic

Thallium compounds 0.6÷40 (typical values) Partly opaque in the visible, toxic

Zinc Selenide 0.5÷22 Partly opaque in the visible

Zinc Sulphide 0.37÷14 Partly opaque in the visible

Table 1

The refraction index of the crystal is such that a part of the beam, as described above, gets reflected toward the OB1 achromatic reflective objective and then focus by it on the S sample.

Another important feature of the microscope, according to the invention, is represented by the OB1 objective that focuses the beam on the sample. For the purposes of this confocal microscope, the objective must be characterised by a spectral wide band, a long working distance and a high numerical aperture allowing focusing the beam on a very restricted area on the focal plan, on the surface, or inside the sample being analysed.

The following has been utilised on the realized prototype: an objective with a spectral band between 200 nm and 20 μιη, a working distance of 10.4 mm, and a numerical aperture of 0.52. Preferably, this band is included between 450 nm and 2.5 μηη, the working distance is greater than 5mm, and the aperture is greater than 0.25.

The solution, according to the invention, utilises a reflective objective. This kind of objective, which can be characterized by a Cassegrain-Schwarzschild design, responds to the above mentioned requisites, and it does not find a general application in confocal microscopes as it requires a particular care in the beam geometry. Furthermore, in general, there is no need for such a wide spectral interval as in the exposed case. Although other solutions cannot be excluded, refractive objectives, which are normally used, would suffer a focus shift in function of the wavelength.

An XYZs piezoelectric control axial and lateral scanning system moves the sample in respect of the focus of the objective.

The light reflected by the S sample is collected by the OB1 objective (condenser), goes through the BS beam-splitter and is conveyed through an M optical system capable of maintaining the absence of chromatic aberration in the beam (combination of plane and parabolic mirrors, all with a surface that has been optimized for the whole spectrum in use) and then focused (always in an achromatic manner, free of chromatic aberration) by means of the OB2 objective on a PH pinhole which aperture is variable and controllable through piezoelectric technology.

The PH pinhole is the characterizing and critical element for all confocal systems. Its existence ensures the possibility of effecting an optical sectioning of the sample, as it ignores from the detection the signal that does not originate from the focus volume of the objective.

The system, or OB2 objective, must not introduce chromatic aberration in order to have the focus centred on the pinhole, independently from the wavelength. The pinhole, in fact, defines the "working" area of a confocal microscope for the axial part (along Z), but also laterally (XY); this facilitates, therefore, the concept of a constant focus point for the different wavelengths.

Many systems provide the possibility of choosing between different fixed dimension pinholes. This invention has anticipated the need to work with very different levels of signal intensity and very different axial resolutions.

The solution, according to this invention, is represented in Figure 2 and consists of the project of an original design composed of a pair of laminae 110,120, with a man body 111 ,121 and a dovetail 112,122, adequately worked, which can be moved in a symmetric manner in respect of a 200 plane and in a continuous manner in respect of the central point 130, delineating in this way a 140 rhomboidal forum of a variable diameter between 0 and 200 μΐτι. The movement, as already mentioned, is managed and controlled by piezoelectric assembled and calibrated on this specific system, hence enabling to vary the pinhole aperture in a continuous and repeatable manner and to adapt the detection to different experimental needs.

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

Alternatively to scanning with the system of piezoelectrically control motorized axes, in the case of samples that cannot be placed on the motorized axes it is possible to centre the focus of the objective or of other achromatic optical devices at the entrance of a monomodal fibre which, mounted at the exit on an miniaturized mechanical system for endoscopy that includes a suction and securing system to the tissue, can effect a piezoelectrically controlled three-dimensional scanning on the sample. This instrument, coupled to endoscopic systems in such a way, enables to reach the internal surfaces of the human body (applications on the whole digestive tract, but also of in gynaecology, urology, or major vessels).

This system, in fact, allows the positioning of the source and the detector even at a certain distance from the investigation sample, without the need for a rigid mechanical connection between the parts (see US Patent N. 6341036, "Confocal miniaturized system incorporated into a compact probe"). In this case, the light reflected by the sample is then recollected by the fibre and, on exit from it, collected by the OB1 objective or by another achromatic optical device and, from this point forward, it follows the same path as described above.

The innovative proposed solution consists in the concurrent utilisation of all the wavelengths acquired. In contrast with other projects, the whole design of the machine is directed to this aim. In addition, it is essential to use a focussing system and possible filters free of achromatic aberration. For this reason, based on the current state of the art for lenses, they categorically must not be used.

Custom software manages the control of the scanning, acquisition and data recording.

The instrument, according to the invention, is innovative as it makes it possible to unite reflectance spectroscopy in the visible and near infrared on samples of arbitrary dimensions, with submicrometric resolution by means of confocal imaging techniques.

Confocal imaging, which traditionally has been differentiated between fluorescence microscopy, utilised mainly for research, and reflectance microscopy, utilised in clinical practice, enables to visualize respectively fluorescent molecules and tissue architecture.

The instrument, according to the invention, provides chemical/physical information with an extremely high spectral resolution and high spatial resolution, carried by the spectroscopic response, to the structural information carried by the imaging that is typical of the confocal approach, due to the simultaneous elaboration of a spectrum of frequencies rather than a single frequency.

The possibility of associating unambiguously an entire spectrum to each scanning point (corresponding to physical dimensions in the micron range or smaller) enables to overcome one of the previous 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, for example, 2000 different wavelengths equals to the possibility of having 2000 different contrast modes, such as in fluorescence or in histology different probes are utilised to characterize different targets. Spectral and imaging approaches, synergically related, therefore lead to the possibility of obtaining 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 several areas connected with endoscopy. The possibility of varying the laser emission power on extremely defined tissue regions, even in an automated way, expands potential applications from diagnostics to highly selective therapies for several pathologies, and to applications that are typical of lasers for "cosmetic" use.

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

All of the above describes the preferred forms of realization and suggests variations to this invention, but it is clear that a person skilled in the art can make modifications or amendments without departing from the scope of protection, as defined by the appended claims.