WO2022070173A1

WO2022070173A1 - An integrated optical arrangement for combinatorial microscopy

Info

Publication number: WO2022070173A1
Application number: PCT/IB2021/060503
Authority: WO
Inventors: Ashwin Lal; Mohanasundaram S M; Abhay Sane; Ravi Roshan
Original assignee: Shilps Sciences Private Limited
Priority date: 2020-10-01
Filing date: 2021-11-12
Publication date: 2022-04-07

Abstract

The invention provides an integrated optical arrangement for combinatorial microscopy. The arrangement includes a base having three distinct motorized mounts. Each of the mount is configured to have movement along at least one of the six degrees of freedom. A cantilever mounted on the first mount is configured for a planar motion parallel to the base. A sample surface is mounted on the second mount and positioned below the cantilever. The second mount is provided with a positioner that is configured for both a planar motion parallel to the base and a vertical motion perpendicular to the base. An illumination setup is mounted on the third mount and is configured for a vertical motion with respect to the base. The mounting of the illumination setup at an inclination with respect to the cantilever enables the user to perform combinatorial microscopy without the need to disturb a sample under observation.

Description

AN INTEGRATED OPTICAL ARRANGEMENT FOR COMBINATORIAL MICROSCOPY

FIELD OF INVENTION

The invention generally relates to the field of microscopy and particularly to an integrated optical arrangement for combinatorial microscopy.

BACKGROUND

The Atomic Force Microscope, AFM is a very high resolution microscope to visualize and characterize surfaces at the nanometer scale, well below the diffraction limits of optical microscopes. The AFM generates an image point-by-point by scanning the sample surface. The AFM is capable of generating an image in the range of micrometers. The process of obtaining the image is typically time consuming. Also, AFM is advantageous to scan isolated objects. Applying AFM to probe objects dispersed in a medium can be challenging. Examples of such objects include but are not limited to micro-crystals, nanoparticles, bacteria and microfabricated structures. Typically, samples are dispensed on a substrate for imaging the objects. Optical microscopy is often applied to image objects dispersed in a medium. However, the resolution obtained is limited. It is often desirous to combine various imaging techniques to obtain a detailed analysis of the sample. Performing independent measurements can be time consuming. Further, the image information can change if the objects dispersed in the medium undergo physical transportation. There are methods and apparatuses known in the art that combine different microscopic techniques to obtain a detailed analysis of the objects dispersed in the medium.

One such technique is disclosed in US Patent No.7473894, assigned to JPK Instruments AG, the details of which are incorporated herein by reference. The ‘894 patent discloses an apparatus and a method for a scanning probe microscope, comprising a measuring assembly which includes a lateral shifting unit to displace a probe in a plane, a vertical shifting unit to displace the probe in a direction perpendicular to the plane, and a specimen support to receive a specimen. A condenser light path is formed through the measuring assembly so that the specimen support is located in the area of an end of the condenser light path. One advantage of the apparatus is that the technique provides a common platform for performing two distinct imaging techniques. However, the disadvantage of the apparatus is that only transparent samples can be imaged with the optical microscope.

An optical beam positioning unit for atomic force microscope is disclosed in US Patent no. 10054612 assigned to Oxford Instruments Asylum Research Inc, wherein an optical light beam positioning system that enables the combination of two or more light beams of different wavelengths to be focused onto a probe or sample of a scientific instrument, such as an atomic force microscope, for a number of specific uses typical to AFMs, like measuring the deflection or oscillation of the probe and illuminating an object for optical imaging, and less traditional ones like photothermal excitation of the probe, photothermal activated changes in the sample, photothermal cleaning of the probe and photochemical, photovoltaic, photothermal and other light beam induced changes in the sample. The focused light beams may be independently positioned relative to each other. A significant disadvantage of the system is that there is a requirement of an elaborate arrangement of optics to provide two distinct beams of light. There is a need for a system that provides for performing two different microscopic technique without the need for any complex arrangement of the system.

BRIEF DESCRIPTION OF DRAWINGS

So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

FIG.1 shows an integrated optical arrangement for combinatorial microscopy, according to an embodiment of the invention.

FIG.2a shows an exploded view of the integrated optical arrangement during atomic force microscopy mode, according to an embodiment of the invention.

FIG.2b shows an exploded view of the integrated optical arrangement during laser scanning microscopy mode, according to an embodiment of the invention. FIG.3a shows the optical path in the atomic force microscopy mode for AFM imaging, according to an embodiment of the invention.

FIG.3b shows the optical path in the laser scanning microscopy mode for LSM imaging, according to an embodiment of the invention.

FIG.4 shows the optical path in an additional configuration of the laser scanning microscopy mode for fluorescence imaging, according to an embodiment of the invention.

FIG.5a and FIG.5b show the respective LSM image and AFM image of a hair sample, according to an embodiment of the invention.

FIG.6a and FIG.6b shows the respective LSM image and AFM image of a gold electrode, according to an embodiment of the invention.

FIG.7a and FIG.7b shows the LSM images of bacteria on a substrate, according to an embodiment of the invention.

SUMMARY OF THE INVENTION

One aspect of the invention provides an integrated optical arrangement for combinatorial microscopy. The arrangement includes a base having three distinct motorized mounts. Each of the mount is configured to have movement along at least one of the six degrees of freedom. A cantilever mounted on the first mount is configured for a linear motion parallel and perpendicular to the base. A sample surface is mounted on the second mount and positioned below the cantilever. The second mount is provided with a positioner that is configured for both a planar motion parallel to the base and a vertical motion perpendicular to the base. An illumination setup is mounted on the third mount and is configured for a vertical motion with respect to the base. The mounting of the illumination setup at an inclination with respect to the cantilever enables the user to perform combinatorial microscopy without the need to disturb a sample under observation. This concurrent observation of the sample through combinatorial microscopy enables the user to understand and infer different aspects of the sample simultaneously.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention provide an integrated optical arrangement for combinatorial microscopy. The combinatorial microscopy can be two distinct microscopic techniques. The distinct microscopy described herein includes but is not limited to an atomic force microscopy, a laser scanning microscopy, an emission microscopy, a fluorescence microscopy and an optical microscopy. In one example of the invention, the combinatorial microscopy includes a combination of atomic force microscopy, hereinafter referred to as AFM, laser scanning microscopy, hereinafter referred to as LSM and optical microscopy.

FIG.1 shows an integrated optical arrangement for combinatorial microscopy, according to an embodiment of the invention. The arrangement includes a base 1 having three distinct motorized mounts 2, 3 and 4. Each of the mounts is configured to have movement along at least one of the six degrees of freedom. A cantilever 5 for the AFM probe mounted on the first mount 2 is configured for a motion along at least one of the three linear motion axes with respect to the base 1. The planar motion is enabled by an XY positioner 6 for positioning the probe in the X and Y planes and a cantilever positioner 7 for setting vertical position of the cantilever 5. A sample surface 8 is mounted on the second mount 3 and positioned below the cantilever 5. The second mount 3 is provided with a positioner 9 that is configured for both a planar motion parallel to the base 1 and a vertical motion perpendicular to the base 1. The positoner 9 is categorised into two types, one is sample XY positioner 9a for positioning the sample in X and Y planes, and a sample Z positioner 9b for positioning the sample in Z plane. Further, second mount 3 includes a scanner 10 for scanning the sample with finer resolution in the different planes. An illumination setup is mounted on the third mount 4 and is configured for a vertical motion with respect to the base 1. The mounting of the illumination setup at an inclination with respect to the cantilever enables the user to perform combinatorial microscopy without the need to disturb a sample under observation. The illumination setup includes a laser source 11 for illuminating the sample surface 8 with a laser beam during LSM mode, a LED illumination 12 for illuminating the sample surface 8 during optical microscopy mode, an objective lens 13 and at least two distinct detection apparatus 14a and 14b. The objective 13 is configured for a dual mode of operation. The dual mode of operation includes directing the light from the laser source 1 1 onto the cantilever and/or the sample surface and collecting the scattered light and reflected light from the sample surface 8. The detection apparatus described herein includes but is not limited to a digital camera, and a photosensitive detector. In one example of the invention, two distinct apparatus, a digital camera 14a and a photosensitive detector 14b, are utilized for imaging the sample surface 8 in high resolution. Further, illumination setup includes an optical assembly 15 for guiding a beam of light, a lens, a pinhole assembly 16 for directing the beam of light into the photosensitive detector 14b, a camera lens 17 and a positioner 18.

FIG.2a shows an exploded view of the integrated optical arrangement during atomic force microscopy mode, according to an embodiment of the invention. A laser beam 11 a from the laser source 11 is focused on the cantilever 5 for AFM imaging. The sample surface 8 is approached from below with closed- loop feedback control to achieve a precise interaction with a tip 5a of the cantilever 5.

FIG.2b shows an exploded view of the integrated optical arrangement during laser scanning microscopy mode, according to an embodiment of the invention. The laser beam 1 1a is focused on the sample surface 8 for LSM imaging. The upward movement of the cantilever 5 by using the cantilever positioner 7 results in movement of the tip 5a of the cantilever 5 away from the laser path due to the angle of the optical setup. When the sample is moved in the vertical direction using the sample positioner 9, the laser beam comes into focus on the sample surface 8, thus enabling LSM mode imaging.

FIG.3a shows the optical path in the atomic force microscopy mode for AFM imaging, according to an embodiment of the invention.The cantilever 5 is positioned perpendicular to the laser beam 11 a from the objective lens 13. The objective Iens13 collects the reflected light 1 1 b from back surface of the cantilever 5. The optical assembly 15 guides the reflected light 1 1 b to the photosensitive detector 14b through the lens 16a and pinhole16b. The photosensitive detector 14b is positioned such that the reflected light 11 b falls partly outside of photosensitive area of the photosensitive detector 14b. The deflection of the cantilever tip 5a due to interaction with the sample surface 8, results in changes in the amount of overlap between the reflected light and the photosensitive area and causes change in the photocurrent.

FIG.3b shows the optical path in the laser scanning microscopy mode for LSM imaging, according to an embodiment of the invention. The laser beam 11 a is focused on the sample surface 8. The reflected light 1 1 b heads off in a direction away from the objective lens 13. The objective lens 13 collects scattered light 1 1c. The optical assembly 15 guides the scattered light 11 c to the photosensitive detector 14b through the lens 16a and the pinhole 16b. The photosensitive detector 14b is positioned to capture all the scattered light 1 1 c coming through the pinhole16b. The laser beam 1 1a can also be focused below the surface of a semitransparent sample. The changing of position of the sample surface 8 into different planes by using the positioner 9 or the scanner 10, enables the construction of 3- dimensional images of the sample surface 8. In the optical microscopy mode, the setup is similar to LSM mode. The laser source 1 1 is turned off and the LED illumination 12 is turned on. Correspondingly the signal from the photosensitive detector 14b is not used, but the image from the digital camera 14a is acquired.

FIG.4 shows the optical path in an additional configuration of the laser scanning microscopy mode for fluorescence imaging, according to an embodiment of the invention. In this configuration, an optical filter 19 is introduced in front of the photosensitive detector 14b for blocking the wavelength of the laser source 1 1. Consequently, higher wavelengths of light emitted by the sample surface 8 due to the laser excitation can be measured by the detector 14b. In this case, the sample is prepared or augmented with fluorescence producing materials.

Industrial Application:

The LSM image, AFM image or bright field image of a sample can be obtained through the single combinatorial microscopy. Initially, the cantilever tip is moved sufficiently away in z direction from the sample surface with the help of cantilever positioner, to allow the light from the light source in optical microscopy mode or the laser in LSM mode to fall on the sample surface. The sample surface is brought in focus by moving the Z positioner. Lateral surface features in the range of 1-100 microns are detected by moving the sample surface in XY plane with the help of XY positioner. Once a feature of interest is identified, positions of the XY positioner and sample bright field images are recorded. Subsequent to recording of the bright field images, laser source 1 1 is switched on. The laser spot size is minimized on the sample surface by repositioning the Z positioner, while inspecting visually through the camera. The LED illumination is switched off and the Laser Scanning Microscope mode is enabled. The sample surface is scanned in x and y either by the scanner or by XY positioner. For each point in the scan, the scattered light from the surface is collected by the objective lens and is directed to the photosensitive detector via the optical assembly and pinhole assembly. The LSM image is constructed point by point from the photo detector signal. Multiple images are created by shifting the sample in z direction by 100 nanometers to a few microns and rescanning the surface. The z positioning is done by either the scanner or the z positioner. The multiple z sections of the sample surface are combined to create a high contrast 3D map of the sample features. In the case of an opaque sample, the 3D map depicts the surface topography. In the case of a semi-transparent sample, such as most biological samples, the 3D map depicts sub-surface features.

The analysis of surface topography of different samples using the combinatorial microscopy is explained below.

Identifying suitable locations for AFM imaging on a large structure:

The combinatorial microscopy allows a user to identify potential regions on the sample surface with a large slope using the laser io scanning or optical microscopy mode, thereby avoiding problems associated with the AFM. One such example is depicted in the FIG.5a and FIG.5b. The FIG.5a shows the LSM image of a hair sample and FIG.5b shows the AFM image of the hair sample. The LSM image enables identifying the crest of the curvature of the hair surface. The AFM scans the surface of the hair sample in high resolution.

Imaging of thin metal films:

Metallic thin films are often part of micro and nanofabricated devices and are part of various sensors, micro fabricated electromechanical systems (MEMS), micro fluidics chips, etc. It is often a requirement to conduct AFM imaging of the metallic thin films to characterize properties such as surface roughness, presence of foreign material, defects, etc. Because of the difference in optical properties of the thin film, it is advantageous to conduct LSM to identify the region of the thin film. FIG.6a and FIG.6b show one such example of a gold electrode layer used in a micro fluidic chip. The FIG.6a represents LSM image of two parallel gold electrodes (dark bands) on a heavily used micro fluidic chip. Round particles and scratches on the electrode surface are easily visible, and the gold electrode is easily distinguished from the surrounding glass substrate. The FIG.6b represents AFM images of two different regions on the gold electrode. Scratches and particulate matter also seen in LSM images are clearly visible on the gold film.

Distinguishing single objects from a cluster: n Typically, materials such as nanoparticles, liposomes, bacteria, etc. are suspended in a solution which is deposited on an AFM substrate and the solvent dried for AFM imaging. In such cases, it is possible that the suspended objects of interest form clusters that are difficult to spot by simple optical methods and whose AFM images are difficult to interpret. An LSM image can distinguish between a cluster of objects from isolated objects that are more amenable to AFM imaging and analysis. One such example is shown in the figures FIG.7a and FIG.7b. Bacteria exposed to a surface-active agent were suspended in a solution and deposited on a substrate. The LSM scan enables accurate location of objects of interest and the invention disclosed above enables rapid and accurate positioning of the AFM probe above the individual objects of interest and simplifies the AFM imaging process. The FIG.7a represents an LSM image of bacteria on a substrate. A less densely occupied region (square A) is easily identified from a dense cluster (square B). The LSM image FIG.7b (left) enables easy identification of objects of interest on the sample where the AFM probe can be brought in a deterministic manner for imaging (right).

Hence, the invention provides an integrated optical arrangement for combinatorial microscopy. The concurrent observation of the sample through combinatorial microscopy enables the user to understand and infer different aspects of the sample simultaneously. The arrangement can operate in AFM mode or LSM mode. The arrangement is compact and a user can switch from AFM mode to LSM mode or vice versa within a common graphical user Interface. The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.

Claims

WE CLAIM:

1. An integrated optical arrangement for combinatorial microscopy, the arrangement comprising of: a base having three distinct motorized mounts, wherein each mount is configured to have movement along at least one dimension; a cantilever mounted on the first mount and configured for a motion along at least one of the three linear motion axes with respect to the base; a sample surface mounted below the cantilever, on the second mount having a positioner configured for both a planar motion parallel to the base and a vertical motion perpendicular to the base; and an illumination setup mounted on the third mount configured for a vertical motion with respect to the base, wherein the illumination setup is mounted at an inclination with respect to an axis perpendicular to the sample surface.

2. The integrated optical arrangement as claimed in claim 1 , further wherein the illumination setup comprises of two distinct sources of illumination; an objective coupled to the sources of illumination, the objective configured for a dual mode operation; and at least two distinct detection apparatus.

3. The integrated optical arrangement as claimed in claim 1 , wherein the detection apparatus can be a digital camera and a photosensitive detector. The integrated optical arrangement as claimed in claim 1 , wherein the dual mode of operation of the microscope objective are directing the light from one of the sources of illumination onto the cantilever and/or the sample surface and collecting the light scattered, emitted and/or reflected from the cantilever or the sample surface. The integrated optical arrangement as claimed in claim 1 , wherein the combinatorial microscopy can be two distinct microscopic techniques selected from a list comprising of atomic force microscopy, light scanning microscopy, confocal microscopy, emission microscopy, fluorescence microscopy. The integrated optical arrangement as claimed in claim 1 , wherein the movement of the sample surface along at least one dimension is selected from translational and/or rotational degrees of freedom. The integrated optical arrangement as claimed in claim 1 , wherein the inclination of the illumination setup enables combinatorial microscopy, as stated in Claim 4, by obtaining at least two distinct images of any location on the sample.