Patent ID: 12196942

DETAILED DESCRIPTION

In cooperation with attached drawings, the technical contents and detailed description of the present inventive concept are described thereinafter according to a preferable embodiment, being not used to limit the claimed scope. This inventive concept may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the inventive concept to the skilled person.

FIG.1illustrates a device100for imaging microscopic objects10, according to an embodiment of the inventive concept. The device100is configured to operate in points of operation within a wavelength range of at least 400-1200 nm. It should be realized that the device100may be set to at least two points of operation, but the device100may have many more points of operation, such as 10 points of operation, 50 points of operation or more than 100 points of operation.

The device comprises a light source110, which in the present embodiment is a laser comprising a first point of operation in which the light source110is configured to generate light in a visible part of the wavelength range, and a second point of operation in which the light source110is configured to generate light in an infrared, IR, part of the wavelength range. The device100is further configured to be set in a selected point of operation from the first and second points of operation.

The light source110is configured such that the spectral line-width for each of the wavelengths does not exceed 100 nm, in order to avoid spectral overlap between the wavelengths. It should be realized that other spectral line-widths of the generated wavelengths may be provided, such as spectral line-widths that do not exceed 50 nm or spectral line-widths that do not exceed 10 nm. In particular, when the light source is a laser, a narrow spectral line-width may be provided.

The light from the light source110is guided by an optical fiber112to an output114. The output114is arranged such that the light exiting the output114is directed towards a microscopic object10. In this embodiment, an objective slide20is used on which microscopic objects10are arranged, and the microscopic objects10are thereby illuminated on the objective slide20from a first side of the objective slide20.

It should be understood that, although the light source110is herein described as a laser, it is conceivable that the light source may alternatively be a laser diode or a light emitting diode, LED.

As the microscopic objects10are illuminated by light at a wavelength defined by the selected point of operation, at least part of the light is scattered by the microscopic objects10, forming scattered light, whereas some of the light is non-scattered and passes through the microscopic objects10and the objective slide20. The scattered and non-scattered light is transmitted to a detector120comprising an array of light sensitive areas122, the detector120being arranged on a second side of the objective slide20, opposite to the first side. At the detector120, an interference pattern is formed by interference between light being scattered by the microscopic objects10and non-scattered light from the light source110. By the present arrangement, non-scattered light may be transmitted along a common light path with the light scattered by the microscopic objects10, in a so called in-line holography set-up. In alternative embodiments, however, scattered light and non-scattered light may be transmitted along separate light paths, being combined at the array of light sensitive areas122to form the interference pattern. Also, it should be realized that the array of light sensitive areas122may alternatively be arranged on the side of the microscopic objects10and the objective slide20from which the microscopic objects10are illuminated, with light transmitted through the microscopic objects10being reflected by a mirror towards the array of light sensitive areas122such that a reflective set-up is used instead of a transmissive set-up.

The array of light sensitive areas122is configured to detect the interference pattern, for imaging the microscopic objects10at the wavelength defined by the selected point of operation. Each of the light sensitive areas122are sensitive to detect light spanning the wavelength range of at least 400-1200 nm, thereby being able to detect light in the full range of wavelengths that the light source110may generate.

The array of light sensitive areas122of the detector may selectably be of either InGaAs type or quantum dot image sensor type. However, it should be realized that the array of light sensitive areas122may be formed by another image sensor type that is sensitive to detect light spanning the wavelength range of at least 400-1200 nm.

As the spatial resolution of the detected interference pattern is related to the pitch of the light sensitive areas122in the array of light sensitive areas122, it is preferable to use a detector120with an array of light sensitive areas122having a small pitch, not larger than 100 μm. Provided as non-limiting examples, suitable detectors may have a pitch of 10 μm, 5 μm, or 2.5 μm.

The device100may optionally be configured to set the selected point of operation to the first and second points of operation sequentially, whereby the light source110may sequentially illuminate the microscopic objects10with the generated light at the wavelengths defined by the first and second points of operation. The device100is further configured to, by the array of light sensitive areas122, sequentially detect the interference patterns for imaging the microscopic objects10at each of the wavelengths defined by the first and second points of operation. More specifically, the device100is configured such that each illumination event of the light source110is synchronized with a corresponding detection event of the array of light sensitive areas122. The interference pattern detected at the first point of operation may originate from a different part of the microscopic objects10than the interference pattern detected at the second point of operation. By way of example, this may be applied for investigations of pollen. Light in the visible range in the first point of operation may be used for imaging the exine of the pollen, since light in the visible range is largely scattered by the exine of the pollen, whereas in the second point of operation the nucleus of the pollen may be images as light in the SWIR range may penetrate the exine of the pollen.

Application in which nanoparticles are used as holographic imaging labels may be another example. The shape and dimension of the nanoparticle determines spectral wavelength range at which the scattering cross-section of the particles is the highest. Thus, by using more than one type of differently shaped particles, different bio structures may be labeled. Thanks to the first and second points of operation being provided in a visible and an infrared part of the wavelength range, the spectral range in which nanoparticle labeling can be used may be extended. Such arrangement may find application in biosensing and invitro tissue imaging.

In an alternative embodiment, the device100may be configured to illuminate the microscopic objects10by light at the wavelengths defined by the first and second points of operation simultaneously, and the interference patterns of light at the two wavelengths may be detected simultaneously. Given as a non-limiting example, the interference patterns at the two wavelengths may be separated by means of an array of filters arranged in front of the array of light sensitive area122, thereby allowing simultaneous detection of the individual interference patterns by the same detector120. An example of such an array of filters is described in more detail in relation toFIG.3, allowing simultaneous detection of up to three different wavelengths.

In the present embodiment, the light source110generates light at the two wavelengths defined by the first and second points of operation, and the light at the two wavelengths exit the light source through a common exit and the two wavelengths are pre-aligned on a common light path. The illumination therefore follows the same path for each of the two wavelengths. Further, no objective is used for imaging that may cause chromatic aberrations, and the interference patterns for each of the two wavelengths are detected by the same detector120with the array of light sensitive areas122. Hence, the device100is configured to detect the interference patterns at the two wavelengths such that spatial information of the microscopic objects10in the interference patterns for each of the two wavelengths are aligned with each other on the array of light sensitive areas122. The present arrangement enables combination of the information from each of the two wavelengths into chromatic information of the microscopic objects10.

However, it should be realized that a microscopic object10may be imaged only at a single wavelength in a single point of operation. Thus, it is not necessary to use more than one point of operation when imaging a particular microscopic object10. Nevertheless, the device100is versatile in that it may be used in different points of operation for different instances of imaging such that microscopic objects10that are to be imaged in vastly different wavelengths ranging from visible to SWIR part of the wavelength range may be imaged using the same device100without requiring any change in the set-up of the device100between different imaging instances.

The device100further comprises a processor130configured to perform digital holographic reconstruction on the interference patterns detected by the array of light sensitive areas122. The digital holographic reconstruction for each wavelength detected generates a three-dimensional monochromatic image of the microscopic objects10on the objective slide20. For the digital holographic reconstruction, the processor130may utilize any suitable algorithm as known to the person skilled in the art, including a Gerchberg-Saxton algorithm or multi-acquisition (multi-depth and/or multi-wavelength) for phase retrieval, or reconstruction based on angular spectrum diffraction by means of Gabor wavelet transform.

The processor130is further configured to optionally combine the monochromatic images of the microscopic objects10generated for each of the wavelengths, to form an aligned chromatic image of the microscopic objects10. Since the spatial information of the microscopic objects10in the interference patterns for each of the wavelengths are aligned with each other on the array of light sensitive areas122, combination of the monochromatic images may be performed without further intermediate image transformation such as resampling, rescaling, or dewarping, that may otherwise be required to align the image views of the different wavelengths.

The processor130may be implemented as a general-purpose processor, which may be provided with instructions, e.g. through computer programs for performing digital holographic reconstruction and for providing any other functionality of the processor130. Thus, the processor130may for instance be a central processing unit (CPU).

The processor130may alternatively be implemented as firmware arranged e.g. in an embedded system, or as a specifically designed processor, such as an Application-Specific Integrated Circuit (ASIC) or a Field-Programmable Gate Array (FPGA).

It should be understood that the signal-to-noise ratio, SNR, of the detected interference patterns may decrease as the distance increases between the microscopic objects10and the array of light sensitive areas122of the detector120. Thus, in order to ensure a good SNR, it may be preferable to have the objective slide20with the microscopic objects10arranged at a minimum distance from the array of light sensitive areas122of the detector120. Thus, in a practical set-up it would be advantageous to have the objective slide20arranged in immediate proximity to the detector120. In this respect, the illustrations inFIG.1as well as in subsequent figures are to be interpreted as schematic illustrations, wherein the objective slide20is illustrated at a distance away from the detector120for clear illustrational purposes only.

In the present embodiment, the microscopic objects10have been placed on a transparent objective slide20for easy handling of the microscopic objects10into and out from the device100. However, it is conceivable that also other solutions for introducing and removing microscopic objects10to and from the location at which imaging of the microscopic objects10are performed. By way of example, the microscopic objects10can be placed on rotating disks, rolls of tape, or in flow channels through which liquid or gaseous flow may pass.

FIG.2Aillustrates a device200for imaging microscopic objects10, according to an embodiment of the inventive concept. The device200is configured to operate in a wavelength range of at least 400-1200 nm. The device200comprises a set of three light sources210a,210b,210d, each of which is a laser configured to generate light at an individual wavelength or at several different wavelengths in an individual wavelength interval within a wavelength range of at least 400-1200 nm, such that the set of light sources210a,210b,210ccomprises a first point of operation in which the set of light sources210a,210b,210cis configured to generate light in a visible part of the wavelength range, a second point of operation in which the set of light sources210a,210b,210cis configured to generate light in a short-wave infrared, SWIR, part of the wavelength range, and a third point of operation in which the set of light sources210a,210b,210cis configured to generate light in a near infrared, NIR, part of the wavelength range.

The number of light sources210a,210b,210cis illustrated as being three inFIGS.2A and2B. However, it should be understood that the number of light sources, as well as the number of modes of operation, may vary between different embodiments, and therefore may be two or more.

Each of the light sources210a,210b,210cis configured such that the spectral line-width of the generated wavelength does not exceed 100 nm, in order to avoid spectral overlap between the generated wavelengths. It should be realized that other spectral line-widths of the generated wavelengths may be provided, such as spectral line-widths that do not exceed 50 nm or spectral line-widths that do not exceed 10 nm. In particular, when the light source is a laser, a narrow spectral line-width may be provided.

The light from each of the light sources210a,210b,210cis guided by respective optical fibers212a,212b,212cto respective outputs214a,214b,214c. The outputs214a,214b,214care arranged such that the light exiting the outputs214a,214b,214cis directed towards a microscopic object10. In this embodiment, an objective slide20is used on which microscopic objects10are arranged, and the microscopic objects10are thereby illuminated on the objective slide20, at slightly different angles from a first side of the objective slide20.

The device200is configured to set a selected point of operation to the first, second, and third points of operation sequentially, whereby the light sources210a,210b,210cmay sequentially illuminate the microscopic objects10with the generated light at the wavelengths defined by the first, second, and third points of operation.

At least part of the light is scattered by the microscopic objects10, whereas non-scattered light passes through the microscopic objects10and the objective slide20. The scattered and non-scattered light is transmitted to a detector220on the opposite side of the objective slide20, the detector220comprising an array of light sensitive areas222. At the detector220, an interference pattern is formed for each of the wavelengths between light being scattered by the microscopic objects10and non-scattered light from the light sources210a,210b,210c.

The array of light sensitive areas222is configured to sequentially detect the interference pattern for each wavelength. Each of the light sensitive areas222are sensitive to detect light spanning the wavelength range of at least 400-1200 nm, thereby being able to detect light in the full range of wavelengths produced by the light sources210a,210b,210c.

The array of light sensitive areas222may selectably be of either InGaAs type or quantum dot image sensor type or another type that is sensitive to detect light spanning the wavelength range of at least 400-1200 nm, similarly as was described in relation to the embodiment illustrated inFIG.1.

The device200further comprises a processor230configured to perform digital holographic reconstruction on the interference patterns detected by the array of light sensitive areas222, thereby generating a three-dimensional monochromatic image of the microscopic objects10on the objective slide20, for each of the wavelengths.

Since the light from the different light sources210a,210b,210creaches the detector220with slightly different angles of incidence, the interference patters may not necessarily be detected such that spatial information of the microscopic objects10for each of the wavelengths are aligned with each other. The processor230may therefore be further configured to perform intermediate image transformation such as resampling, rescaling, and/or dewarping. Image transformation may facilitate subsequent optional combination of the monochromatic images of the microscopic objects10, to form an aligned chromatic image of the microscopic objects10. Alternatively, it is conceivable that the acquired data be transferred to an external unit such as a computer where processing such as image transformation and/or image combination may be performed.

However, it should also be realized that a particular microscopic object10may be imaged using only one of the light sources210a,210b,210c, such that there is no need of aligning spatial information acquired for different wavelengths.

FIG.2Billustrates a device300for imaging microscopic objects10, according to an embodiment of the inventive concept. The device300shares a number of features with device200illustrated inFIG.2B, all of which will not be explicitly repeated in this section. The device300comprises a set of three light sources310a,310b,310c, of the same type as described for device200.

The light from each of the light sources310a,310b,310cis guided by optical fibers312a,312b,312c, which are combined to a common output314, from which light from the light sources310a,310b,310cmay exit, following a common aligned light path. The output314is arranged such that the light exiting the output314illuminates the microscopic objects10on the objective slide20.

Scattered and non-scattered light is transmitted to a detector320comprising an array of light sensitive areas322, of the same type as described for device200. The array of light sensitive areas322is configured to detect the interference pattern for each wavelength.

In the present embodiment, the light from the light sources310a,310b,310care combined onto a common, aligned light path, prior to exiting the output314. The illumination therefore follows the same path for each of the wavelengths. Hence, the device300is configured to detect the interference patterns at the three wavelengths such that spatial information of the microscopic objects10in the interference patterns for each of the wavelengths are aligned with each other on the array of light sensitive areas322. The present arrangement facilitates combination of the information from each of the wavelengths into aligned chromatic images of the microscopic objects10.

The device300further comprises a processor330configured to perform digital holographic reconstruction on the interference patterns detected by the array of light sensitive areas322, thereby generating a three-dimensional monochromatic image of the microscopic objects10on the objective slide20, for each of the three wavelengths.

The processor330may further be configured to combine the monochromatic images of the microscopic objects10for each of the wavelengths, to form an aligned chromatic image of the microscopic objects10. Such combination may be performed without further intermediate image transformation, since the spatial information of the microscopic objects10in the interference patterns for each of the wavelengths are aligned with each other.

FIG.3illustrates a detector420and an array440of filters444, according to an embodiment of the inventive concept. The array440of filters444is arranged above the array of light sensitive areas422such that light passes the array440of filters444before reaching the array of light sensitive areas422. The array440of filters444may be arranged close to the array of light sensitive areas422, such as being arranged directly on the array of light sensitive areas422or being monolithically integrated with the array of light sensitive areas422.

The array440of filters444comprises a plurality of subsets442a,442b,442cof filters444, each of the filters444in the subset442a,442b,442cof filters444being arranged in front of a light sensitive area422. Each of the subsets442a,442b,442cof filters444is configured to transmit light at one of the plurality of wavelengths, so that each of the subsets442a,442b,442cof filters444transmit light at a different wavelength than other subsets442a,442b,442c. By the present arrangement, each of the wavelengths of the plurality of wavelengths is transmitted through a corresponding subset442a,442b,442cof filters444, such that some of the light sensitive areas422detect the interference pattern for light at a first wavelength, some other light sensitive areas422detect the interference pattern for light as a second wavelength, and so on. In the manner described above, a detector assembly may be provided with which light at different wavelengths may be detected by different light sensitive areas422, thereby allowing simultaneous detection of the individual interference patterns by the same detector420.

The number of subsets442a,442b,442cof filters444is illustrated as being three inFIG.3. However, it should be understood that the number of subsets of filters may vary between different embodiments, and therefore may be two or more. The number of subsets of filters typically correspond to the number of light wavelengths generated and the number of modes of operation of the device.

Each of the embodiments illustrated inFIGS.1,2A, and2Bmay optionally be provided with the present array440of filters444, thereby enabling simultaneous detection of the generated wavelengths.

FIG.4illustrates a schematic block diagram shortly summarizing the method for imaging a microscopic object as previously described in relation to the operation of the devices100,200,300. It should be understood that the steps of the method, although listed in a specific order herein, may be performed in any order suitable.

The method may comprise selecting S502a point of operation from at least a first point of operation in which at least one light source is configured to generate light in a visible part of a wavelength range of at least 400-1200 nm, and a second point of operation in which the at least one light source is configured to generate light in an infrared part of the wavelength range. It should be understood that there may be implementations of the method in which the selecting S502a point of operation may be performed to set the selected point of operation to a single one of the at least first and second points of operation. It should be understood that there may be implementations of the method in which the selecting S502a point of operation may be performed to set the selected point of operation to the at least first and second points of operation sequentially. It should be understood that there may be implementations of the method in which the selecting S502a point of operation may be performed to set the selected point of operation to the at least first and second points of operation simultaneously.

The method may further comprise generating S504, by the at least one light source, light at a wavelength defined by the selected point of operation.

The method may further comprise illuminating S506the microscopic object with the generated light such that at least part of the light is scattered by the microscopic object, forming scattered light. It should be understood that there may be implementations of the method in which the illuminating S506the microscopic objects may be performed at a single one of the at least first and second points of operation. It should be understood that there may be implementations of the method in which the illuminating S506the microscopic objects may be performed at the at least first and second points of operation sequentially. It should be understood that there may be implementations of the method in which the illuminating S506the microscopic objects may be performed at the at least first and second points of operation simultaneously.

The method may further comprise transmitting S508the scattered light and non-scattered light, from the same light source, to an array of light sensitive areas, such that an interference pattern is formed by interference between the scattered light and the non-scattered light.

The method may further comprise detecting S510, by an array of light sensitive areas sensitive to detect light spanning the wavelength range, the interference pattern for imaging the microscopic object at the wavelength defined by the selected point of operation. It should be understood that there may be implementations of the method in which the detecting S510interference patterns may be performed at a single one of the least first and second points of operation. It should be understood that there may be implementations of the method in which the detecting S510interference patterns may be performed at least first and second points of operation sequentially. It should be understood that there may be implementations of the method in which the detecting S510interference patterns may be performed at least first and second points of operation simultaneously.

In the above the inventive concept has mainly been described with reference to a limited number of examples. However, as is readily appreciated by a person skilled in the art, other examples than the ones disclosed above are equally possible within the scope of the inventive concept, as defined by the appended claims.

Although the light sources are mainly described herein as being laser light sources, it should be realized that the light source(s) may alternatively be implemented as one or more light emitting diodes (LEDs). The light output by a LED may be guided through a pinhole for generating at least partially coherent light such that an interference pattern may be detected that allows digital holographic reconstruction.