WO2014184793A1

WO2014184793A1 - Method and system for use in inspection of samples by detection of optically excited emission from the sample

Info

Publication number: WO2014184793A1
Application number: PCT/IL2014/050418
Authority: WO
Inventors: Michael Elbaum; David KIRCHENBUECHLER
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2013-05-16
Filing date: 2014-05-11
Publication date: 2014-11-20

Abstract

An optical method and system is presented for use in inspection of a sample by detecting stimulated / optically excited emission from the sample, for example by fluorescence. The optical system comprises a lens arrangement and a spatial filter. The lens arrangement defines an illumination path for directing illuminating light of a first wavelength range onto an illumination region on a sample plane and a collection path for directing reflected illuminating light of the first wavelength range and emitted light of a second wavelength range propagating from the sample plane towards a detection plane. The lens arrangement comprises a plurality of lenses configured for focusing the reflected illumination light onto a conjugate aperture plane and imaging the illumination region by the collected emitted light onto a conjugate sample plane corresponding to the detection plane, thereby spatially separating optical paths of the collected reflected illumination and emitted light portion in the vicinity of a focal location of the reflected light. The spatial filter may define a light blocking region in the collection path in the conjugate aperture plane at a focal location of the reflected illumination light, thereby substantially preventing the illuminating light from reaching the detection plane.

Description

METHOD AND SYSTEM FOR USE IN INSPECTION OF SAMPLES BY DETECTION OF OPTICALLY EXCITED EMISSION FROM THE SAMPLE

TECHNOLOGICAL FIELD

The present invention is in the field of optical inspection/measurement/imaging of a sample based on detection of emission from the sample, e.g. microscopy such as fluorescent or Raman microscopy, and relates to a method and system for separating (distinguishing) between illuminating (exciting) and emitted (excited) light in such an optical system.

BACKGROUND

Optical systems of the type utilizing detection of light emitted/excited in a specimen in response to illuminating/exciting light (such as fluorescence microscopy or Raman microscopy) involve illumination of a specimen with one wavelength (color) of light, and acquisition of an image at a different wavelength (typically but not exclusively longer than that of the illumination). The most common mode of operation is by wide-field epi-illumination, in which the illuminating light enters an objective from the same side on which the image is formed.

The required separation between light emitted from a sample ("signal" to be detected) and illuminating light returned from the sample ("stray" to be removed) is typically performed by spectral separation using a matched set of colored filters. As shown more specifically in Fig. 1 illustrating a typical epi-fluorescence design of wide- field microscopes, the filters include an excitation filter, a dichroic mirror for reflecting the short wavelength of illuminating light and passing the longer wavelength emission to a detector (or vice versa), and an emission filter in the detection path that further selects the emission colors and, importantly, blocks leakage of the illuminating color. Light of the excitation wavelength is focused onto the specimen through the objective lens. The fluorescence emitted by the specimen is focused onto the detector by the same objective that is used for the excitation. Excitation light reflected from the specimen reaches the objective together with the emitted light. The emission filter between the objective and the detector can filter out the excitation light from fluorescent light.

GENERAL DESCRIPTION

There is a need in the art for a novel approach for inspecting samples by detection of stimulated / optically excited emission from a sample, enabling effective separation between illuminating and emitted radiations returned from a specimen/sample under inspection.

The problem with the existing, spectral separation based optical techniques is associated inter alia with the fact that blocking powers of spectral filters (e.g. dichroic mirrors, emission filters) are often weaker than required. Moreover, the need to block effectively all the illuminating light while maximally collecting the emission severely limits simultaneous acquisition of multiple color signals. Considering fluorescence microscopy techniques with spectral separation, the image quality is dominated by the quality of the filters, as the emission intensity is normally orders of magnitude weaker than the illumination intensity. Even a simple back-reflection of the illumination from a sample may be much stronger than the emitted signal of interest. Any improvement in spectral separation is translated immediately into improved signal to noise ratio, enabling detection of weaker and/or more transient signals.

The present invention provides a novel approach for use in optical inspection systems of the kind specified, i.e. systems where illuminating and emitted light are of different wavelengths and propagate along a common detection path.

It should be noted that the term "inspection" used herein refers also to any type of measurement/imaging or examination of a sample and should therefore be interpreted broadly.

According to the invention, separation between illuminating and emitted radiations returned from a specimen is achieved utilizing spatial separation, which is used preferably alternatively, or possibly additionally, to spectral separation between them. The invention is advantageously useful for inspecting specimens that are substantially reflective or transmitting with respect to illuminating light, rather than scattering. The invention may be used in an optical system operating in transmission or reflective mode, or the so-called trans-illumination or epi-illumination system. In such optical systems, an objective lens unit is mounted such that its front focal plane substantially coincides with a sample plane, and the objective lens unit collects illuminating light and emitted light and directs both, possibly attenuated by spectral filters, along a common light detection path to a detector. According to the invention, a spatial filter (beam stop or mask) is provided in the detection path and is placed substantially in a plane conjugate to a back focal plane of the objective lens unit.

The optical system suitable for using the invention preferably utilizes a small light source, such as a laser. In some embodiments of the invention the optical system is configured such that illuminating light incident on a sample is collimated light. Considering an optical system of any known type of the kind specified, utilizing a relay lens associated with an imaging lens of a detector, a device of the invention in its simplest configuration includes a spatial filter defining a small light blocking region placed in or near the Fourier plane of the relay lens. Here, the illuminating light returns to a point, as it was focused to the objective back aperture/focal plane. The light blocking region in the spatial filter has an appropriate dimension (for illumination by extended sources, the blocking region is appropriately larger) to cover the spot of light, and is aligned so as to overlap with it. In the case of laser illumination, this is a negligible fraction of the system aperture for the image, meaning that the loss of emission light is very small. The inventors have found that the use of such a device is extremely effective using laser illumination, to the extent that the emission filter may sometimes be removed entirely without deterioration in image signal to noise ratio.

Modern optical microscope design is based on two sets of conjugate planes: the conjugate sample planes, and the objective back-focal or "aperture" planes. In order to illuminate the sample field uniformly, the illuminating light is brought to focus at the back-focal plane. The objective forms an image of the sample at the primary image plane, either directly or in combination with a telan lens in so-called "infinity-focus" systems. The eyepiece or camera sensor (detector) is placed at this image plane. Geometric constraints, or the need to introduce additional magnification, often require an optical relay to re -project the image to a more distant plane. The lens that relays the image also relays the conjugate aperture plane. Technically, it is found at a distance of one focal length Fr from the relay lens. It is often called the Fourier plane because the distribution of light there presents a Fourier transform of the image.

Thus, generally, in such optical system utilizing the invention, these two sets of conjugate planes (conjugate sample planes and aperture planes) are arranged such that excitation/illuminating light is focused where excited/emitted light is parallel (collimate) or vice versa, and a spatial filter defining a small light blocking region is placed at or near the conjugate aperture plane, re -projected after the primary image plane, so as to block the excitation light with minimal effect on the emission.

The optical system utilizing the invention may provide a significant cost reduction, and moreover vastly enhanced flexibility in collection or spectroscopy on the emission colors. For example, the invention makes it possible to concurrently illuminate several fluorescent dyes with several excitation colors, where the excitation of one overlaps the emission of another. This is not possible with the spectral separation alone, and creates a demand for very complex "multi-band" filter/mirror combinations. In a system utilizing the invention, it may yet be possible to replace a wavelength-selective filter (dichroic mirror) by a simple partial mirror, if care is taken to minimize scattering of the illumination light.

Thus, according to one broad aspect of the invention, there is provided an optical system for use in inspection of a sample by detecting optically excited emission from the sample, the optical system comprising:

a lens arrangement defining an illumination path for directing illuminating light of a first wavelength range onto an illumination region on a sample plane and a collection path for directing reflected illuminating light of said first wavelength range and emitted light of a second wavelength range propagating from the sample plane towards a detection plane, said lens arrangement comprising a plurality of lenses configured for focusing the reflected illumination light onto a conjugate aperture plane and imaging the illumination region by the collected emitted light onto a conjugate sample plane corresponding to the detection plane, thereby spatially separating optical paths of the collected reflected illumination and emitted radiations in the vicinity of a focal location of the reflected light; and

a spatial filter defining a light blocking region accommodated in the collection path in the conjugate aperture plane at a focal location of said reflected illumination light, thereby substantially preventing the illumination light from reaching the detection plane.

In a simple example, the spatial filter may be implemented as a mask in the form of a thin transparent (e.g. glass) plate with a small light blocking (opaque) region.

According to another broad aspect of the invention, there is provided a method for inspection of a sample by detecting optically excited emission from the sample, the method comprising: illuminating a region on the sample with light in a first wavelength range to cause emission from the sample in a second wavelength range; collecting light reflected from the sample and light emitted by the sample propagating substantially along the same optical path and directing the collected light towards a detection plane and spatially separating between said reflected light and said emitted light in the vicinity of a focal location of the reflected light, thereby substantially spectrally separating between the reflected illuminating light and the emitted light at the detection plane.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

Fig. 1 illustrates the design of a conventional wide-field epi-fluorescence microscope;

Fig. 2 illustrates schematically an example of the configuration of and light propagation scheme in an optical system utilizing the invention; and

Fig. 3 illustrates schematically another example of the configuration of and light propagation scheme in an optical system utilizing the invention. DETAILED DESCRIPTION OF EMBODIMENTS

Fig. 1 shows the design scheme of a modern state of the art epi-fluorescence microscope, which requires multiple spectral filters for reducing illumination spectrum in the detected light.

Referring to Fig. 2, there is schematically illustrated an example of an optical system 10 utilizing the invention. The optical system 10 is configured generally similar to that of an epi-illumination microscope. It should be understood that the principles of the invention are limited neither to microscopes nor to epi-illumination arrangements of optical systems. The principles of the invention may be used in any optical system where excited (emitted) light, of a wavelength different from exciting (illuminating) light, is to be effectively detected/observed.

The optical system 10 is associated with a light source unit 12 for directing illuminating (exciting / pumping) light Lm from the light source unit 12 towards a sample plane SP where a sample under inspection is located (typically on a stage or table, or any other support surface). The optical system 10 includes an objective lens unit 14, and a lens system for directing light towards a detection plane. The detection plane may be constituted by a light sensitive surface of an imager (camera), or an eyepiece for visual observation, as the case may be. The light directing lens system includes an image-forming collection lens unit 18 (telan lens, one or more lenses) and possibly also a relay lens unit 20. Telan lens (or tube lens), generally at least one, forms an image at the camera plane in an "infinity corrected" optical system. Also provided in the optical system 10 is a wavelength-selective filter, e.g. a dichroic mirror 16, in the optical path of illuminating light propagating towards the sample plane. Such a dichroic mirror may be used for selecting a desired wavelength for illumination (exciting) so as to cause emission from the given sample. In some cases, the dichroic mirror might not be used at all, or may be replaced by a partially reflecting mirror in applications where a broadband light source is used without interference from the part of the spectrum that overlaps the emission wavelengths.

In the specific but not limiting example of Fig. 2, the epi-illumination configuration is used, in which case the objective lens is located close to the sample plane such that both the reflection of the illuminating light Lm and the emitted light L_em on their way from the sample first pass through the objective 14 and then interact with the dichroic mirror 16. It should be understood that in case of trans-illumination configuration, the dichroic mirror 16 might not be used.

Thus, optical system 10 defines an illumination channel for directing illuminating light Lm from the light source unit 12 to the sample plane SP, and a light collection channel for collecting light propagating from the sample and appropriately directing at least part thereof towards the detection plane. The light propagating from the sample along the detection channel includes light formed by reflected illuminating light Lm of one (first) wavelength range and light formed by emitted light L_em of a different (second) wavelength range.

The illumination channel includes the objective lens unit 14, and possibly includes a focusing lens unit 15 associated with the light source unit, and further possibly includes the dichroic mirror 16. Generally speaking, the system 10 is preferably configured so as to provide wide-field illumination of the sample (e.g. by collimating the light Lm illuminating the sample). The light collection channel is formed by lens units 18 and 20, and in the epi-illumination set up also by objective 14 and dichroic mirror 16.

It should be noted that in the example of Fig. 2, the normal incidence epi- illumination set up is illustrated, and accordingly the illumination and collection channels have common optical elements. It should, however, be understood that the principles of the invention can be used in an oblique incidence system configuration as well.

The optical elements of the system are configured and arranged so as to define conjugate aperture planes CAPi and CAP₂, and conjugate sample planes CSPi and CSP₂. The objective lens unit 14 is located such that its front focal plane substantially coincides with the sample plane SP. Conjugate aperture plane CAPi is the back focal plane of the objective 14. According to the invention, a spatial filter 22 designed as a beam stop with respect to sample's reflection of illuminating light, is positioned in the conjugate aperture plane at a focal location of illuminating light reflected from an illumination region IS. In this example of epi-illumination set up, the beam stop 22 is located in the aperture plane CAP₂. The spatial filter 22 defines a light blocking region (beam stop) having a dimension corresponding to that of the focal projection of the reflected illumination light in the respective plane. Thus, light Lm from the light source 12 defining an illumination aperture is focused by lens 15 onto the back focal plane CAPi of the objective 14, and the so created collimated light L_m is directed by objective 14 onto the illumination spot IS on the sample, thereby causing emission L_em from the sample together with creating reflection of this light Lm from the sample. The objective 14 collects these light portions and directs collimated beam of emitted light L_em and focused illumination reflection Lm (e.g. through dichroic mirror 16 in this example) towards the telan lens 18 which operates together with the relay lens 20 to create an image of the illumination region by the light emitted from the sample in the conjugate sample plane / detection plane CSP₂ and focus the illumination reflection on the conjugate aperture plane CAP₂ where the beam stop is located. By this, the illumination light Lm is substantially prevented from reaching the detection plane, thus significantly increasing signal to noise ratio of detection.

Reference is made to Fig. 3 illustrating schematically another example of the system configuration. To facilitate understanding, the same reference numbers are used to identify components that are common in all the examples. The system 10 of Fig. 3 is generally similar to that of Fig. 2, but exemplifies a folded collection path, as the case may be to serve a specific application. To this end, an additional reflector 24 is provided, e.g. between the lenses 18 and 20. Also, the light source unit 12 may include a light emitter and a beam expander to provide a desired cross-sectional dimension of illuminating light beam Lm, and an illumination aperture 26 at the output of the light source unit. The light propagation scheme is shown in Fig. 3 in a self explanatory manner.

It should be noted that the present invention can be easily implemented in an optical set up (e.g. microscope) of a given configuration. As exemplified in Fig. 3, the invention may provide a device installable in the optical system at a certain location with respect to the objective lens 14 and telan lens 18, and being formed by a relay lens 20 of a predetermined focal length and a beam stop (mask) 22 located at a certain distance from the relay lens.

It should be understood that the image is actually formed by the telan lens unit

18. As for the objective lens unit 14, although it is not intended to operate as a collimator, since it operates at/near its focal length, it can also collimate the illumination. Generally, the functions of the lenses used in the system changes depending on the direction of the light propagating through. For example, the objective lens performs collimation function for the illumination. It should further be understood that in any of the above described not limiting examples of the optical system, the configuration of the optical elements may be such that illuminating light is focused by the objective onto the sample, while the collected emission undergoes collimation.

Thus, the present invention provides a simple and effective solution for spatial separation between the illuminating light and emitted light in the collection/detection channel of an optical system. This eliminates or at least significantly reduces requirements for spectral filtering of light propagating from a sample under inspection, and provides unprecedented flexibility in multi-color imaging or spectral analysis on the emitted light.

Claims

CLAIMS:

1. An optical system for use in inspection of a sample by detecting optically excited emission from the sample, the optical system comprising:

a lens arrangement defining an illumination path for directing illuminating light of a first wavelength range onto an illumination region on a sample plane and a collection path for directing reflected illuminating light of said first wavelength range and emitted light of a second wavelength range propagating from the sample plane towards a detection plane, said lens arrangement comprising a plurality of lenses configured for focusing the reflected illumination light onto a conjugate aperture plane and imaging the illumination region by the collected emitted light onto a conjugate sample plane corresponding to the detection plane, thereby spatially separating optical paths of the collected reflected illumination light and emitted light in the vicinity of a focal location of the reflected light; and

a spatial filter in the collection path in the conjugate aperture plane at a focal location of said reflected illumination light, thereby substantially preventing the illuminating light from reaching the detection plane.

2. The system of Claim 1, configured for operation in an epi-illumination mode.

3. The system of Claim 2, wherein the lens arrangement comprises an objective lens unit for directing a collimated beam of the illuminating light onto the region on the sample plane, and collimating the collected emitted light.

4. The system of Claim 2, wherein the lens arrangement comprises an objective lens unit for directing the illuminating light to the sample and collecting reflected illumination light and the emitted light from the sample, and a focusing and imaging lens unit for focusing the collected emitted light onto the detection plane and focusing the reflected illumination light onto the conjugate aperture plane.

5. The system of any one of Claims 1 to 3, wherein the lens arrangement defines the conjugate sample and aperture planes which are arranged such that focusing of either one of the reflected illumination light and the emitted light is aligned with collimation of other one of the reflected illumination light and emitted light, thereby providing spatial separation between them and enabling said spatial filtering of the reflected illumination light.

6. The system of any one of Claims 1 to 5, wherein the spatial filter comprises said light blocking region placed in or near a Fourier plane of a relay lens of said lens arrangement.

7. A method for inspection of a sample by detecting optically excited emission from the sample, the method comprising: illuminating a region on the sample with light in a first wavelength range to cause emission from the sample in a second wavelength range; collecting light reflected from the sample and light emitted by the sample propagating substantially along the same optical path and directing the collected light towards a detection plane and spatially separating between said reflected light of the first wavelength range and said emitted light of the second wavelength range in the vicinity of a focal location of the reflected light, thereby substantially spectrally separating between the reflected illuminating light and the emitted light at the detection plane.

8. The method of Claim 7, wherein said illuminating and collecting comprises directing a collimated beam of the illuminating light onto the region on the sample plane, and collimating the collected emitted light propagating towards the detection plane.

9. The method of Claim 7, wherein said spatially separating between the reflected light and the emitted light comprises focusing the collected emitted light onto the detection plane and focusing the reflected illumination light onto a conjugate aperture plane.

10. The method of any one of Claims 7 to 9, wherein said illuminating and collecting comprises: providing conjugate sample and aperture planes arranged such that focusing of either one of the reflected light and the emitted light is aligned with collimation of other one of the reflected light and emitted light, thereby providing said spatial separation between them and enabling said spatial filtering of the reflected light.