Abstract
The invention relates to a digital holographic microscope, DHM. The DHM comprises a coherent light source (401, 501) for illuminating a sample in a sample holder in a first image plane (101,201,407, 507). The DHM further comprises a detector, e.g. a camera (412, 512), arranged to record images of the sample in the sample holder. The DHM further comprises means for dividing the base light beam into different portions and causing the different portions of the light beam to interfere with each other at the detector and a light beam guiding system for guiding a light beam to the sample and the detector. The DHM further comprises a light reducing arrangement for reducing the intensity of the light in the light beam directed to the sample. The light reducing arrangement includes first lens (102) for collimating the light in the first divided beam scattered by a particle (106) comprised in the sample and a spatial filter (108, 206, 413) arranged at or in the vicinity of the focal plane (103, 203) of said first lens (102) in order to reduce the intensity of the focused light passing through the sample located in the first image plane (101, 201, 307, 407). By this arrangement, the majority of unscattered light passing through the sample is filtered off and the majority of the light scattered by a particle (106) in the sample is guided via a light guiding system to the detector.
Claims
1.-17. (canceled)
18. A digital holographic microscope, DHM, comprising a coherent light source for creating a base light beam for illuminating a sample in a first image plane, a sample holder for holding a sample in the first image plane to be illuminated, a detector arranged to record images of light transmitted through a sample in the sample holder, a means for dividing the base light beam into different portions and causing the different portions of the light beam to interfere with each other at the detector, said means for dividing the base light beam into different portions being located upstream or downstream of the sample holder wherein said digital holographic microscope further comprises a light reducing arrangement for reducing the intensity of the light, said light reducing arrangement comprising at least a first lens for collimating the light scattered by a particle comprised in the sample and focusing the unscattered light passing through the sample in a focal plane, and a partially light transparent spatial filter arranged at or in the vicinity of the focal plane of said first lens in order to reduce the intensity of the focused unscattered light passing through the sample located in the first image plane such that the majority, but not all, of unscattered light passing through the sample is filtered off and the majority of the light scattered by a particle in the sample is guided via a light guiding system to the detector.
19. A digital holographic microscope according to claim 18 wherein the filter is designed to have a shape and size which is adapted to its location relative the focal plane such that the filter filters off at least 50 percent of the focused light from the first lens.
20. A digital holographic microscope according to claim 18, wherein the filter is designed to reduce the intensity of the total light in the object beam by at least 50%, preferably at least 80%, and most preferably at least 95%.
21. A digital holographic microscope according to claim 18, wherein said coherent light source provides light having a coherence length of at least 0.1 mm, preferably at least 0.7 mm.
22. A digital holographic microscope according to claim 18, wherein said light reducing arrangement comprises a second lens and said filter being located between said first lens and second lens.
23. A digital holographic microscope according to claim 22, wherein said first lens and second lens are arranged relative each other such that their respective focal planes are coinciding with each other in the space between the lenses and the filter is located in close vicinity of the coinciding focal points.
24. A method for characterizing particles smaller than the wavelength of the illuminating light by the use of Digital Holographic Microscopy, said method includes the use of a coherent light source for creating a base light beam for illuminating a sample, a sample holder located in a first image plane for holding a sample to be illuminated, a detector such as a camera arranged to record images of light transmitted through a sample in the sample holder, a means for dividing the base light beam into different portions and causing the different portions of the light beam to interfere with each other at the detector, said means for dividing the base light beam into different portions being located upstream or downstream of the sample holder wherein said method further involves the use of a light reducing arrangement located downstream of the sample holder, said light reducing arrangement comprising at least a first lens for collimating the light scattered by a particle comprised in the sample and focusing the unscattered light passing through the sample at a first focal point, and a partially light transparent spatial filter arranged at or in the vicinity of the focal plane of said first lens in order to reduce the intensity of the focused unscattered light from the light beam passing through the sample located in the first image plane such that the majority of the unscattered light, but not all, in the light beam passing through the sample is filtered off and the majority of the light scattered by the particle is guided via the light guiding system to the detector and optical properties of the scattered light originating from the submicron-particles in the sample are detected by recording one or several images and analysing the one or several images.
25. A method according to claim 24 wherein an absolute optical field of the particles in the sample, expressed as a complex number, is quantified by i. determining the optical field of light having passed the particle by determining phase shift and amplitude from analysing the one or several images recorded, ii. normalizing the optical field of the illuminating background light and subtract the same from the optical field of light having passed the particle to isolate the optical field of the particle, e.g. by recording and analyse a video sequence of known calibration particles both with and without the spatial filter iii. dividing or multiplying the optical field of the particle with a predetermined compensation factor, to compensate for the effect of the spatial filter, e.g. by determining a scaling factor r for amplitude by dividing the amplitude with spatial filter by amplitude without spatial filter, and determine a scaling term for phase by subtracting the phase with spatial filter from the phase without spatial filter.
26. A method according to claim 24 wherein the hydrodynamic diameter or size of the particle is estimated by analysis of its Brownian motion.
27. A method according to claim 26 wherein the hydrodynamic diameter is used in combination with an optical property such as phase shift to estimate a Refractive Index (RI) of the detected particle.
28. A method according to claim 24, wherein the size of the particle is estimated from an absolute optical signal of the particle in relation to Mie theory.
29. A method according to claim 24, wherein different particle populations in the same sample are identified through their respective relationship between two independent variables where one variable is the hydrodynamic diameter or diffusivity or any variable derived therefrom, and the other variable is an optical property such as integrated phase shift.
30. A method according to claim 24, wherein different particle populations in the same sample are identified through their respective relationship between two independent optical variables such as the integrated phase shift and optical extinction cross section, or related variables.
31. A method according to claim 24, wherein the effect of the filter is quantified by imaging a sample of particles both with and without the filter and numerically comparing the optical signal from the two measurements.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0061] FIG. 1A discloses path of unscattered light in a double lens arrangement to be used in a digital holographic microscope (DHM) according to prior art
[0062] FIG. 1B discloses flow path of scattered light in a double lens arrangement to be used in a digital holographic microscope (DHM) according to prior art
[0063] FIG. 1C discloses a stand-alone double lens arrangement provided with a spatial filter
[0064] FIG. 1D discloses a stand-alone double lens arrangement provided with a spatial filter placed at an angle to avoid back reflections
[0065] FIG. 2A discloses a built-in lens arrangement provided with a spatial filter
[0066] FIG. 2B discloses a built-in lens arrangement provided with a mirror filter
[0067] FIG. 3 discloses a digital holographic microscope (DHM) set up according to prior art
[0068] FIG. 4 discloses a digital holographic microscope (DHM) set up comprising a stand-alone double lens arrangement with a spatial filter
[0069] FIG. 5 discloses a digital holographic microscope (DHM) set up comprising a stand-alone double lens arrangement with a spatial filter and a means for separating a reference beam downstream of the sample
[0070] FIG. 6 discloses a digital holographic microscope (DHM) set up comprising a stand-alone double lens arrangement with a spatial filter and a diffraction grating in the focal plane as a means for separating different beams downstream of the sample.
[0071] FIG. 7 discloses a digital holographic microscope (DHM) set up comprising a stand-alone double lens arrangement with a spatial filter and a diffraction grating near the camera as a means for separating different beams downstream of the sample.
[0072] FIG. 8A illustrates a small area of a captured image where a particle is visible in the middle and diagonal lines of the interference pattern generated by the off-axis configuration.
[0073] FIG. 8B illustrates a Fourier transform of the entire captured image, showing the three separate Fourier peaks.
[0074] FIG. 8C illustrates the phase contrast image generated from the image in FIG. 6A.
[0075] FIG. 9A discloses the optical field of 240 nm diameter polystyrene particles plotted in a complex plane, as measured without filter, with filter, and with filter and numerical compensation.
[0076] FIG. 9B discloses the optical field of 240 nm and 190 nm diameter polystyrene particles plotted in a complex plane, as measured with filter and numerical compensation.
DETAILED DESCRIPTION OF THE INVENTION
[0077] FIGS. 1A and 1B show a double lens arrangement for creating a focal plane 103 according to prior art. The double lens arrangement comprises a first lens 102 and a second lens 104 having a focal length f. FIG. 1A. shows the path of unscattered light, represented by arrows indicating light flowing from right to left, from a first image plane 101 which propagates in parallel with the optical axis of the first lens 102, which focuses the light at the focal plane 103. The light will continue to reach the second lens 104 where the light will be caused to propagate in parallel once again and an image may be recorded at a second image plane 105.
[0078] In FIG. 1B, the path of scattered light is shown. Light scattered by a small particle 106, e.g. a solid substance or bubble, in the sample in the first image plane 101 is collimated by the first lens 102 to cover the entire object beam cross-section at the focal plane 103. The double lens arrangement in FIG. 1A-C is known as a 4f-arrangement, where f is the focal length of the lenses. In this arrangement both lenses 102, 104 have the same focal length. It is however not strictly necessary for the first and second lenses 102, 104 to have the same focal length for the arrangement to fulfil its function, using different focal lengths will merely magnify or reduce the image recorded at the second image plane 105.
[0079] In FIG. 1C, a double lens arrangement as disclosed in FIGS. 1A and 1B is disclosed but in this case, there is a transparent plate 107 made by e.g. glass with a disk filter of semi- or non-transparent material in its centre placed at the focal plane to function as a spatial filter 108. The focused light is then obscured to a large extent (for example 90%) whereas the very most of the scattered light which is spread out over the entire object beam cross-section is passing unobstructed to be further guided to the detector.
[0080] In FIG. 1D, a similar arrangement as disclosed in FIG. 1C is disclosed having a transparent plate 107 located in the double lens arrangement. The transparent plate is provided with a disk of semi-transparent and reflective material such as a thin metal film to function as a spatial filter 108. The arrangement in this figure differs from the one disclosed in FIG. 1C in that the transparent plate 107 is placed at an angle to avoid back reflections within the beam path. The reflected light 109 follows a cone shaped path similarly as it would if it had not been reflected. When using a metallic filter of transmission type, a possible problem is that the metal reflects light backwards, which is subsequently reflected forwards by upstream components, causing reflection patterns in the image. This may be remediated by placing the filter at a certain angle such that the reflected light 109 exits the beam path as described in this figure. Alternative methods could also be used to avoid backwards reflection, e.g. covering the metal with antireflection coating.
[0081] In FIG. 2A is disclosed an alternative option of a lens arrangement having a focal plane 203 where unscattered light from a sample holder in a first image plane 201 is focused by one or several lenses in a microscope objective. In case there is a sample comprising a particle or bubble, the light scattered by the particle or bubble will be collimated by the first lens in a similar way as disclosed in FIG. 1B. Instead of adding two lenses in the object beam line to create a focal plane, another possibility is to use the focal plane 203 already present in the microscope. The focal plane 203 is in general present somewhere downstream the lenses in a microscope objective 202. In particular for higher magnifications, the focal plane 203 is usually placed inside a barrel of the objective 202 and is thus somewhat technically difficult to place a filter at. However, a filter can be placed slightly downstream the focal plane 203 as shown in FIG. 2A. In this case, the semi- or non-transparent disk forming the spatial filter 206 located on the transparent plate 205 will need to cover a larger area in order to obscure the same amount or portion of the focused background light compared to if the spatial filter 206 should have been placed in the focal plane 203. Consequently, a larger share of the light scattered by the sample will be lost. A second lens 207, e.g. a tube lens, will collimate the background light which passes through the filter. The second lens will also focus the light scattered by a particle in the sample holder, which was collimated by the first lens in the microscope, at a second image plane (not shown) in a similar manner as disclosed in FIG. 1B.
[0082] FIG. 2B demonstrates that instead of using a transmission type of filter as disclosed in FIGS. 1C and 2A, it is also possible to use a reflection type of filter. In this example, the set-up is similar to the setup in FIG. 2A with the sample holder in the first image plane 201 and the focal plane 203 inside the barrel of the objective 202 and the first lens forming part of the microscope. However, in this set up a mirror 208 of which a central area is semi-transparent, is placed behind the microscope objective 202. The mirror 208 reflects the very most of the scattered light, whereas the majority of the focused background light passes through the centre of the mirror 208. The reflected light is directed to the second lens 207 and continues downstream through the optical beam path.
[0083] FIG. 3 shows an example of a digital holographic microscope (DHM) set-up according to prior art. The set-up is in this case built around a commercial microscope body of the inverted type, which means that an objective 308 is under the sample. The light beam from a coherent light source 301, such as a laser, is expanded by lenses and directed towards a first beam splitter 304. The first beam splitter 304 is of a polarizing type, which splits the light into two orthogonally polarized beams. The laser beam is already partially polarized. By rotating a first half-wave plate 302 between the laser and the first beam splitter 304, the direction of polarization can be adjusted and thereby the relative intensity of the two outgoing beams can be adjusted. A second half-wave plate 303 in the reference beam line adjusts that beam to have the same polarization as the object beam when they meet again at a second beam splitter 311. Hence, a beam splitter is used both as a device for splitting a beam as well as for unifying light beams. The object beam is collected into an optical fibre 305 which connects to a collimator lens 306 which illuminates the sample at the first image plane 307 from above. The liquid sample can for example be placed between a microscope slide and a cover slip, or in a microfluidic channel. The latter option is advantageous in that a controlled liquid flow can be achieved and the thickness of the liquid sample is well defined. After the sample, the beam passes through a microscope objective 308, a tube lens 309, and via a mirror exits the microscope body 310. The object beam is subsequently recombined with the reference beam at the second beam splitter 311. The second beam splitter is slightly rotated compared to the direction of the two beams, which causes the two beams to reach the detector with a slight angle relative to each other, which creates an interference pattern in the image recorded by a camera 312, e.g. a CCD camera.
[0084] FIG. 4. shows a DHM set-up according to an embodiment of the invention. The overall set up is similar to the set-up of FIG. 3. FIG. 4 discloses a DHM comprising a coherent light source 401 from which light is guided via first half wave plate 402 to a first beam splitter 404 which divides the light into a first divided beam which will serve as an object beam guided to the sample holder in order to illuminate a sample and a second beam functioning as a reference beam being guided to bypass the sample holder and sample. The reference beam is guided via a second half-wave plate 403 and suitable light guiding means such as mirrors and optical fibres to a second beam splitter 411. The object beam is guided via similar light guiding means including mirrors and an optical fibre 405 to a microscope body 410. In the microscope body, the light is directed to a collimator lens 406 illuminating a sample at the first image plane 407. After the sample, the beam passes through a microscope objective 408, a tube lens 409, and via a mirror exits the microscope body 410. Hence, these parts correspond to the set-up in FIG. 3. However, the set-up in FIG. 4 further comprises two lenses and a spatial filter 413, e.g. a disk filter, in the focal plane between them as disclosed in the stand-alone double lens arrangement depicted in FIG. 1C. The light passes through the double lens arrangement and the spatial filter 413 before it enters the second beam splitter 411 to be reunited with the reference beam before being directed to the camera 412. In the microscope body, the objective 408, tube lens 409 and mirror are positioned to create an image plane at the port where the object beam exits the microscope as disclosed in FIG. 3. The 4f-arrangement therefore begins at this plane. Note that FIG. 4. is not drawn to scale and the distance from the first image plane to the first lens is in reality the same as from the second lens to the camera.
[0085] It is obvious that the stand-alone double lens arrangement of FIG. 1C or 1B could be replaced with a built-in lens arrangement as disclosed in FIG. 2A or 2B to be used in the embodiment disclosed in FIG. 4. This arrangement may for example be achieved by placing a disk filter at or in the vicinity of a focal plane between the objective lens 407 and the camera 412 and thus replace the stand-alone arrangement.
[0086] FIG. 5 discloses a DHM setup similar to FIG. 4, but where the first divided beam to be used as the object beam being guided to the sample holder in order to illuminate the sample and the second divided beam to be used as reference beam bypassing the sample are formed from splitting the base light beam in a first beam splitter 511 located downstream of the sample. In this arrangement, the first beam splitter 511 is placed downstream of the objective lens 508 to split up the base light beam in at least a first and a second divided beam to be used as an object beam and a reference beam. To make use of this second divided beam as a reference beam in an off-axis DHM arrangement, it should preferably contain mostly unscattered background light. This is achieved by focusing the reference beam similarly as the object beam and placing a pinhole or transparent area 514 at the centre of the focal plane (instead of using an obstructive a filter as for the first divided beam functioning as an object beam) through which the focused background light can pass while scattered light is obstructed by the otherwise opaque plate preventing light from passing through. Note that unlike in the figure, the two beam paths will need to be of similar length to enable good interference, unless the laser light has very long coherence length.
[0087] FIG. 6 discloses a DHM setup similar to FIGS. 4 and 5, but where all light shares a common path. At the focal plane 613 between the lenses, where the filter is located, also a diffraction grating is placed which separates the light into different diffraction orders. Preferably three orders (1,0,1) are used. As the orders interfere with each other they give rise to three separate images on the image sensor, phase shifted relative to each other and containing holographic information. The spatial filter may be placed on the grating or on a separate substrate. Preferably a mask 614 is placed at the image plane upstream the focal plane, comprising one or several apertures. The aperture or apertures need to transmit light from both an area where the sample is located and an area where the sample is not located which acts as a reference. Alternatively, it is also possible to use a separate reference beam as reference in combination with this type of grating-based method.
[0088] FIG. 7 discloses a DHM setup similar to FIG. 6 where all light shares a common path. The filter is placed in the focal plane 713 between the two lenses as in FIGS. 4 to 6. A grating is placed in close proximity to the camera which is used as detector. The grating may comprise a 2D pattern where different fields provide a different phase shift of the light, these fields may be separated by opaque lines. Light portions having passed different fields interfere with each other at the detector, generating an interference pattern based on four different diffraction orders.
[0089] FIG. 8A. discloses an example of an interference pattern caused by a beam splitterbeing slightly rotated, e.g. the second beam splitters 311, 411, 515 as disclosed in FIG. 3, FIG. 4 and FIG. 5, respectively. A rotated beam splitter will cause the first and second beam to be slightly off-set relative each other and an interference pattern can be seen as diagonal lines. FIG. 8A. shows a magnified area of a recorded image, with a dispersed particle. Due to the interference pattern, a Fourier transform of the image will produce three peaks/images as disclosed in FIG. 8B. Field information can be extracted from the two side peaks. By numerically shifting one side peak to the centre and applying a low pass filter, the optical information of interest is separated. By inverse Fourier transform, the optical field can be determined at different planes along the optical axis in the sample. This can be used to generate bright field as well as phase contrast images at different planes, an example of a phase contrast image is seen in FIG. 8C which corresponds to the recorded image in FIG. 8A. In this 3-dimensional volume of optical field information, detection and tracking of particles are subsequently performed.
[0090] In order to achieve such an interference pattern as can be seen in FIG. 8A, the light intensities of the object and reference beams should preferably be of similar magnitude. Since the optical information of interest is extracted from irregularities in the parallel interference lines, these lines need to be sharp and clearly visible in the image. The intensity loss is however much larger in the object beam than in the reference beam since the light passes more optical components in the object beam, such as the sample and the many lenses in the microscope objective. When adding a filter of the transmission type as described in some versions of the present invention, the intensity loss increases considerably. The intensity of the two beams must therefore be adjusted so that much more light intensity is being directed to the object beam than to the reference beam, in order for the light intensity to be of similar magnitude at the camera. In the optical set-up described in FIGS. 3 and 4, the intensities of the object and reference beams are adjusted with the help of the first half-wave plates 302, 402 and the second half-wave plates 303, 403 in the respective set-ups in combination with the polarizing first beam splitters 304, 404. The first half-wave plates 302, 402 adjust the polarization angle of the light directed to the beam splitters 304, 404 in the respective systems, which splits the light in the respective set-ups into two orthogonally polarized beams. The relative intensity of the object and reference beams in the microscope set ups in FIGS. 3 and 4 will depend on the polarization angle of the incoming light. In order for the two beams to have the same polarization angle when they reunite at the non-polarizing second beam splitters 311, 411 in the respective set-ups, the polarization angle of the reference beam is adjusted with the second half-wave plates 303, 403. Several other means of adjusting the relative intensity of the object and reference beams are possible. What is important is that the relative intensity of the two beams should be of similar magnitude at the camera. The same reasoning also applies to the optical set up in FIG. 5.
[0091] In an alternative embodiment, a laser directly coupled to a first optical fibre is used and the light in the first fibre is directly split into two different fibres which are directed to the reference and object beam respectively. The relative intensity of light in the two fibres can be adjusted using fibre-coupled components in several different ways, for example by using a fixed ratio fibre-splitter in combination with fibre-coupled attenuators, by using a fibre-switch to switch between different fixed ratio fibre beam splitters or by using a variable ratio fibre-splitter.
Example 1
[0092] A spatial filter was used which consisted of a circular disk of gold, 0.5 mm in diameter and 35 nm in thickness, sputtered onto a transparent glass plate. This filter was placed in the Fourier plane (focal plane) between two lenses such as in FIG. 4, at an angle to avoid back reflections such as in FIG. 1D. The relative beam intensities were adjusted so that much more of the light was directed to the object beam than when not using the spatial filter, and an interference pattern was clearly visible in the image. A sample of 240 nm diameter Polystyrene latex (PSL) was used for calibration since these particles were detectable both with and without the spatial filter. FIG. 9A shows the optical field of these particles with (stars) and without (circles) spatial filter in the complex plane. The optical field of the particles depicted here is relative to the illumination background, and with the filter the field amplitude is several times larger as the illumination background is weaker. The scaling factor for amplitude and phase of the optical field of the particles were determined to be r=2.9 and =1.1 respectively. The 240 nm PSL particles measured with the filter in place and subsequently numerically compensated are plotted as diamonds in the complex plane and it can be seen that they overlap well with the particles measured without the filter. Subsequently, a sample of 190 nm diameter PSL particles was measured with the filter and subsequently numerically compensated. FIG. 9B shows numerically compensated field of 190 nm (circles) and 240 nm (stars) PSL particles. Following compensation, both particle populations display optical characteristics expected from Mie theory.
