WO2008071240A1

WO2008071240A1 - Optical image duplicator

Info

Publication number: WO2008071240A1
Application number: PCT/EP2007/006185
Authority: WO
Inventors: Fabio Mammano; Michele Giarin
Original assignee: OPTICAL ELECTRONIC SOLUTIONS Srl
Current assignee: OPTICAL ELECTRONIC SOLUTIONS Srl
Priority date: 2006-12-15
Filing date: 2007-07-12
Publication date: 2008-06-19
Anticipated expiration: 2009-06-15
Also published as: ITPD20060452A1

Abstract

An image duplicator (1) is described, for duplicating an incoming image relating to a spacial object examined into two images in the same image plane which have different spectral characteristics, comprising dispersion means (5) for subdividing a light signal (F) relating to the incoming image into two separate beams (T, R), each beam (T; R) having different optical characteristics from the other (T;R); image forming means (12) for obtaining an output signal including two separate images formed on the same image plane (P2), said separate images having different spectral characteristics but representing the same spatial object; wherein the dispersion means (5) comprise a single reflecting mirror (11) for directing one of the separate beams (T; R) towards the image forming means (12).

Description

Optical image duplicator DESCRIPTION Technical field

The present invention relates to an optical image duplicator, i.e a device suitable for producing multiple images of one object that correspond to different spectral components of the object itself. Technological background

In the technical field of reference of image spectroscopy, it is a known requirement to obtain from one object various images that are different from one another on the basis of the wavelength and/or the polarisation. The multiple images should preferably be obtained simultaneously and be focused on the same detector.

The duplication of images is particularly important in the FRET

(Fluorescence Resonance Energy Transfer) spectroscopic techniques in which the interactions of two proteins in vivo are studied by means of the use of two different fluorescent molecules, of acquisition of high resolution images of double colorant preparations, etc.

Various techniques are used to obtain two spatial images of portions of the spectrum emanating from an object. Typical systems are based on dispersive elements which, positioned along the optical path, subdivide the components at different wavelengths. A typical dispersive element is a prism or a grating, in which dispersion takes piace respectively by means of refraction or diffraction of the light.

In United States Patents US 5926283 and US 5982497 in the name of Optical Insights, an optical duplicator is described which includes a dichroic filter for dividing the incoming light beam into two independent beams having wavelengths respectively above and below the cut-off frequency of the dichroic filter. The device described further comprises two adjustable lenses for directing the two beams on two separate optical paths to form two spatial images, identical but spectrally different, of the object on the detector. Summary of the invention

A principal aim of the present invention is to provide an optical image duplicator which is simpler in construction, is easier to set up and consequently makes it possible to obtain greater efficacy of the instrument itself. At the same time, a subject of the invention is a duplicator the results obtainable from which, in terms of quality and resolution of the image, are at least equal to, if not better than, the devices of the prior art.

The problem underlying the present invention is that of providing an image duplicator structurally and functionally designed to remedy the limitations described above with reference to the prior art cited.

This problem is solved by the present invention by means of an image duplicator having the characteristics mentioned in the following claims. Brief description of the drawings

The characteristics and advantages of the invention will become clearer from the detailed description of a preferred exemplary embodiment thereof, illustrated by way of non-limiting example with reference to the appended drawings, in which:

Figure 1 is a diagrammatic perspective view of an image duplicator according to the present invention, - Figure 2 is an exploded view of the image duplicator of Figure 1, Figure 3 is a diagrammatic view of the optical path of two marginal light rays within the duplicator of Figure 1,

Figure 4 represents two images of the same type of cells obtained one by means of the duplicator of the present invention and the other with a duplicator according to the prior art.

Preferred embodiment of the invention

With initial reference to Figures 1 to 3, an optical image duplicator produced according to the present invention is indicated generally by 1.

The optical duplicator 1 comprises an aperture 2 for the entry of a light signal, preferably corresponding to an image, issuing for example from a microscope connected to the duplicator 1 itself. The incoming image signal, represented by the arrow F in Figure 3, relates to a sample spatial object (not shown) examined by means of the microscope. Preferably, in proximity to the aperture 2, the duplicator 1 comprises first connection means 3 for the attachment of the microscope.

The microscope in question used in combination with the duplicator 1 may be of any type, and generally comprises an objective lens (not shown) for receiving light from the object examined so as to create an image which, as stated before, is then sent to the aperture 2 of the duplicator 1. Preferably in FRET spectroscopic experiments, the light used for the illumination of the object examined is a monochromatic light, emitted for example by a Polichrome V® monochromator produced by TILL PHOTONICS GmbH.

The aperture 2 is preferably arranged perpendicularly to the optical axis of the duplicator and has a preferably rectangular slot configuration, however its configuration is not essential for the application of the teachings of the present invention. The aperture 2 preferably has a field stop function, and serves to limit the field of view of the subject of the image.

The positioning of the aperture 2 coincides with a first image plane Pl (see

Figure 3) in which the image of the object in question issuing from the microscope is formed, in order to delineate clearly the outlines of the region of interest in this plane.

The duplicator 1 further comprises an outlet 6, in proximity to which preferably second connection means 7 are further positioned for the connection of the duplicator 1 to a sensor 15 (visible in Fig. 3). The output signal from the duplicator 1 comprises two separate images (half-images) of the same object derived from the incoming image, but having different spectral characteristics.

The width of the aperture 2 is selected according to the dimensions of the sensor

15 on which the two half-images are projected and may be varied, for example by controlling it manually or electronically. The sensor 15 connected to the duplicator 1 is suitable for storing and analysing the two images derived. For example, a sensor 15 used in the present invention may be a CCD or MOS sensor.

Downstream of the aperture 2, relative to the direction of the light signal

F, a lens 4 is positioned, for example in a cylindrical lens holder 4a, for collimating each light ray of the signal coming from the aperture 2, so as to render them parallel. The collimated rays emerging are then directed towards dispersion means 5 capable of separating the incident signal into several components, as outlined hereinafter. In particular, the incident image F is separated into its different spectral components. The dispersion means 5 comprise a dichroic filter 10 which transmits a portion of the light incident on it. In detail, a dichroic filter is such that it subdivides the incident light signal into two beams separate from each other: a first beam contains all the wavelengths present in the incident beam that are above the cut-off wavelength of the dichroic filter, while the second beam contains all the wavelengths below the cut-off wavelength. The original image is therefore subdivided into two separate images, containing different spectral components, which are propagated along two different optical paths. Generally, the beam with wavelengths longer than the cut-off length is the transmitted beam, while the beam with wavelengths below the cut-off length is the reflected beam (represented by the beams T and R of Figure 3).

In the prior art, in a fluorescence microscope, a dichroic filter is generally used for separating the fluorescent emission light from the excitation light. A dichroic filter transmits around 90% of the incident light having a wavelength above the threshold value and reflects around 90% of the incident light having a wavelength below the threshold value.

Alternatively, provision is made to position a prism or a grating in place of the dichroic filter 10 in order to obtain the separation into two beams having different spectral characteristics.

The dichroic filter 10 is preferably positioned at an angle of 45° with respect to the optical axis of the duplicator 1, and in particular at an angle of 45° with respect to the direction of propagation of the incident beam on the dichroic 10, collimated by the collimating lens 4.

The dispersion means 5 further include means for redirecting the transmitted beam, in particular a reflecting mirror 11. In the definition of reflecting mirror given here there is also included a different optical element such as a prism.

The beam T transmitted by the dichroic 10 is propagated in a substantially unvaried direction with respect to the direction of propagation of the collimated beam, while the reflected beam R, which is initially propagated perpendicularly to the collimated beam, is redirected by the reflecting mirror 11 by a further 90° so as to end up parallel to the transmitted beam T. The positioning of the reflecting mirror 11 is adjustable, or it is mounted on suitable adjusting means 11a for suitably angling the mirror 11 with respect to the optical axis of the duplicator 1 so as to adjust and correctly define one of the two images issuing from the duplicator so that it forms on the sensor 15.

Both the transmitted beam T and the reflected beam R, after its redirection by means of the reflecting mirror 11, are then optionally incident on two separate optical filters 13 and 14, arranged in a filter holding member 13a.

Each optical filter 13, 14 is preferably an interference filter, more precisely an emission filter, such as to better define the content, in terms of wavelengths present, of the reflected beam R and transmitted beam T. These filters are therefore used in the case where a greater definition of the wavelengths present in the two beams, reflected R and transmitted T, is desired (for example two defined wavelength intervals) with respect to the simple sub-division "above/ below a certain threshold" effected by the dichroic 10. Each filter 13, 14 is therefore selected such as to allow only one wavelength interval of interest to pass.

The two beams, reflected R and transmitted T, optionally filtered, are incident on the image forming means 12, capable of forming two images that are spatially identical but spectrally different, and which constitute the output signal of the duplicator 1. In Figure 3 can be seen the lens-holder 12a of the adjusting means 12. Both the images are formed in the same second image plane P2, which is preferably coincident with the plane defined by the recording sensor 15, for the acquisition of the images. The image forming means 12 may be constituted by a single optical element or by several elements spatially separated from one another, such as a plurality of lenses, for example an achromatic doublet.

The images which form on the sensor 15 are constitutively coplanar and are aligned by acting on the mirror 11 via the adjustment means 11a. The output signal of the duplicator 2 therefore comprises two images, which both form in the same plane P2, arranged such that each image covers substantially half of the total useful area of the sensor 15. This latter therefore registers and processes the images, by means of standard analytical techniques in an electronic processor. The image of the object examined therefore coming from the microscope enters the image duplicator 1 where it is subdivided into two separate images which are formed on the same detector. The two images differ in their spectral composition.

Figure 4 shows the output signals of two different image duplicators: the image duplicator of the present invention (photograph on the left) and an image duplirator of the prior art commercially available under the name Dual View ™ of Optical Insights, LLC (photograph on the right). For each duplicator, the images shown represent cells of the same type in three different fields.

In detail, the object analyzed by the Olympus BX51 fluorescence microscope with 8OX enlargement (final enlargement at the sensor with field of 125 μm and 10 mm sensor) connected to the duplicator is provided with CHO cells (Chinese Hamster Ovarian Cells) rendered fluorescent by CFP protein (Cyan Fluorescent Protein) and YFP protein (Yellow Fluorescent Protein), excited at λ=430±5 nm. The two proteins cited are fluorophors which offer a reasonable spectral separation and a luminosity that are compatible with FRET measurements, without requiring ultraviolet excitation which is harmful to the cells. The CFP donor is excited by using the monochromator via the duplicator 1 and the duplicator Dual View of the prior art, and the CFP and YFP emission are registered simultaneously on a single high sensitivity CCD as in Figure 4. HQ480/40m and HQ535/30m and dichroic 515dcxr filters were used.

As can be seen from Figure 4, the quality and resolution of the images obtained with the duplicator of the present invention are of superior quality to those obtained through the Dual View duplicator, although the duplicator which is the subject of the present application is endowed with a greater simplicity of production owing to a smaller number of constituent components.

The invention therefore solves the problem posed, obtaining numerous advantageous described above compared to the prior art cited.

Claims

C L A I M S

1. An image duplicator (1) for duplicating an incoming image relating to a spatial object examined into two images in the same image plane that have different spectral characteristics, comprising :

5 - dispersion means (5) for subdividing a light signal (F) relating to said incoming image into two separate beams (T,R), each beam (T; R) having optical characteristics different from the other (T; R), image forming means (12), for obtaining an output signal including two separate images formed on the same image plane (P2), said separateo images having different spectral characteristics but representing said same spatial object examined, characterized in that said dispersion means (5) comprise a single reflecting mirror (11) for directing one of said separate beams (T; R) towards said image forming means (12). s 2. An image duplicator (1) according to claim 1, wherein said dispersion means (5) comprise a dichroic filter (10) upstream of said reflecting mirror (11), relative to the direction of said incoming light signal (F), said dichroic filter (10) subdividing said incoming signal into said separate beams (T, R), a reflected beam (R) and a transmitted beam (T). o

3. An image duplicator (1) according to claim 1 or claim 2, wherein said image plane (P2) is arranged at a recording sensor (15).

4. An image duplicator (1) according to ciaim 2 or ciaim 3, wherein said dichroic filter (10) is positioned at an angle substantially of 45° with respect to the axis of propagation of said incoming signal (F).

5 5. An image duplicator (1) according to one or more of the preceding claims comprising, upstream of said dichroic filter (10), a collimating lens (4), for collimating said incoming signal.

6. An image duplicator (1) according to one or more of claims 2 to 5, comprising, downstream of said dichroic filter (10), at least one optical filter

5 (14; 13) for filtering said reflected signal (R) and said transmitted beam

(T).

7. An image duplicator (1) according to claim 6, comprising two optical filters (13, 14) for filtering said reflected beam (R) and said transmitted beam (T).

8. An image duplicator (1) according to claim 5 or claim 6, wherein said opticalo filter (14; 13) is an interference filter.

9. An image duplicator (1) according to one or more of the preceding claims, comprising an aperture (2) for the entry of said incoming signal.

10. An image duplicator (1) according to claim 9, wherein said aperture is a slot. s

11. An image duplicator (1) according to one or more of the preceding claims, comprising first connection means (3) for the connection to a microscope.

12. An image duplicator (1) according to one or more of the preceding claims, including adjusting means (lla) for adjusting said mirror (11) so as to angle said mirror (11) suitably with respect to said incoming signal. o

13. An image duplicator (1) according to one or more of the preceding claims, wherein said image forming means (12) comprise an achromatic doublet.

14. An image duplicator (21) according to one or more of the preceding claims, including a recording sensor (15), coinciding with said image plane (P2).

15. An image duplicator (1) according to claim 14, wherein said recording5 sensor (15) is a CCD sensor.