Patent Application
20240402475
The invention relates to an imaging system for imaging a sample. The invention further relates to a method for imaging a sample.
The imaging of samples, for example biological samples, non-biological specimen or analytes, has important use cases in the life sciences, diagnostics, bioprocessing, and healthcare. In particular, the imaging of biological samples is a key tool for basic and translational research, and an important pillar of diagnostics and healthcare. Further, imaging is an important tool in the quality control and process development in the context of bioprocessing. For example, one important problem is the selection of clones that produce desired biologics, their culture, quality control and quality assurance. Likewise, there is a need for solutions that allow the three-dimensional imaging of fixed specimens such as tissue biopsies or tissue sections or living specimens, so called live imaging. Similarly, three-dimensional cell culture samples, for example spheroids, tumoroids, organoids, cardioids, and brain organoids, are of great interest in basic and translational research. They hold the promise of being of higher physiological relevance, i.e. findings obtained from them are expected to translate well to humans. This is especially true for patient-derived three-dimensional cell culture models. Three-dimensional cell cultures may be generated using scaffolds, which are typically hydrogels of synthetic or natural origin. Cells seeded in these scaffolds and cultivated with the right set of factors and conditions will develop structures that bear stunning resemblance of endogenous tissues.
In many of these applications it is desirable to find ways to image samples at high speeds, with a high throughput, at high spatial resolution, and with a high number of markers. An imaging system suited to image (single) cells for example with (ultra-) high throughput and high spatial resolution (i.e. in the range of 300 to 500 nm lateral resolution or better) may be used to study cellular morphology and behavior, cell division patterns, and growth kinetics, the subcellular location of proteins for a large number of cells or events (e.g. 10,000 s to 100,000 s of cells, 100,000 s to 1,000,000 s of cells, or 1,000,000 s to 10,000,000 s of cells). Such an imaging system is ideally suited to find few cells displaying a desirable phenotype such as a certain behavior (e.g. killing tumor cells) or a certain desirable combination of traits. In the context of cell line development and bioprocessing applications such a system may be used to, for example, identify and isolate single cells or clones that strongly produce a certain biologic, like an antibody, i.e. are suited to reach a high titer of the desired biologic. Further such a system may be used to analyze a large number of single cells or clones to make predictions about relevant parameters in bioprocessing applications like for example expected titer, growth kinetics, or potential aggregation of the produced biologic. In the context of cell therapy applications such a system may be used for deep phenotypic characterization of a large number of clones. This may involve live imaging of the cells to assess their phenotype (e.g. activity, shape, length of filopodia and lamellipodia, number, shape and dynamics of subcellular organelles like mitochondria, mitotic activity, number, shape, duration of cell-to-cell contacts, killing effectiveness in killing assays), and/or their genome, and/or their transcriptome, and/or their proteome, and/or their metabolome.
Fluorescence microscopy is commonly used in such applications to label pre-determined structures which may also be named target molecules, molecular markers or analytes inside biological samples. Some molecular markers may also be referred to as biomarkers, when their presence or absence can be connected to a particular biological phenomenon such as for example the level of blood sugar and insulin.
Typical samples are derived from either field collection, a biopsy or a cell culture. The size of a typical sample may be in the range of a couple microns, for example a single cell, tens or hundreds of μm, for example a three-dimensional cell culture sample like a group of cells, a spheroid, an organoid or a tumoroid, or even in the range of several millimeters, for example large organoids, embryos, or tissue sections and organotypic slices.
For cultivation, sample preparation, and imaging the samples are typically maintained in dedicated labware, for example slides, dishes, microplates, and flasks. Especially for fluorescence microscopy with objectives that have a high numerical aperture it is required that the vessel containing sample has a substantially transparent window.
For this reason, there exist a large number of sample carriers, such as petri dishes, chamber slides, flow cells, microplates comprising one or more vessels for receiving samples, for example wells or fluidic channels, which have substantially transparent bottom that allows the imaging of samples inside the vessel from below, e.g. with an inverse microscopical setup. This bottom window is generally made of glass or of a material with a similar refractive index. Such sample carriers will also be referred to as imaging plates in the following.
Although imaging from below is generally desirable, some geometries preclude the use of high numerical aperture objectives, when trying to image upright, i.e. from above, due to sterical problems as well as reflections, and the meniscus which typically forms at the upper surface of the liquid contained in a well due to surface tension.
For example, a compact light sheet fluorescence microscope has been disclosed DE 10 2019 214 929 A1, which illuminates the sample at angle substantially different from 0° to a reference plane. However, the setup is not compatible with imaging sample from below through the bottom of an imaging plate.
The aforementioned restrictions are the reason why the bulk of light sheet microscopy today is either performed by bringing in the illumination and detection optic into a sample chamber from the top, and at least partially immersing the front lenses of the detection optic. In this case, the sterical requirements necessitate the use of large petri dishes and preclude the use of small vessels, for example the well of a 96-well microplate, since the illumination and detection optic would not fit into these wells. Alternatively, microscope systems are used, which are similar to the original SPIM design as disclosed in WO 2004/053558 A1. In this case, the samples are typically mounted and then brought into the sample chamber from the top to hang in front of the front lens. This may be brought about by using agarose gel rods that are pushed out a few millimeters from a glass capillary or by mounting samples in hydrogels inside fluorinated ethylene propylene (FEP) tubes as disclosed in Kaufmann et al. 2012 Development 139(17):3242-7.
However, the known microscope systems suffer either from having a low throughput or having a low spatial resolution. Many known microscope systems having a high throughput are not capable of volumetric imaging, i.e. three-dimensional imaging.
In an embodiment, the present disclosure provides an imaging system for imaging a sample. The imaging system includes a sample moving unit configured to move the sample in a sample space along a movement direction; at least one detection optic including a detection optical axis that encloses a first angle with the movement direction within a range of 20° to 70°, the detection optical axis of the at least one detection optic and the movement direction defining a first plane; and at least one illumination optic including an illumination optical axis that encloses a second angle with the movement direction within a range of 70° to 110°, and that encloses a third angle with the detection optical axis of the at least one detection optic within a range of 70° to 110°, the illumination optical axis of the at least one illumination optic and the movement direction defining a second plane, the first and second planes intersecting and being different.
Subject matter of the present disclosure will be described in even greater detail below based on the exemplary figures. All features described and/or illustrated herein can be used alone or combined in different combinations. The features and advantages of various embodiments will become apparent by reading the following detailed description with reference to the attached drawings, which illustrate the following:
Embodiments of the present invention can provide an imaging system and a method that allow volumetric imaging of a sample with a high throughput. Embodiments of the present invention can be read in conjunction with EP212009992.2, the contents of which are hereby incorporated by reference herein in full.
In the sense of this document “sample” refers to a biological sample which may also be named a biological specimen including, for example blood, serum, plasma, tissue, bodily fluids (e.g. lymph, saliva, semen, interstitial fluid, cerebrospinal fluid), feces, solid biopsy, liquid biopsy, explants, whole embryos (e.g. zebrafish, Drosophila), entire model organisms (e.g. zebrafish larvae, Drosophila embryos, C. elegans), cells (e.g. prokaryotes, eukaryotes, archea), multicellular organisms (e.g. Volvox), suspension cell cultures, monolayer cell cultures, 3D cell cultures (e.g. spheroids, tumoroids, organoids derived from various organs such as intestine, brain, heart, liver, etc.), a lysate of any of the aforementioned, a virus. In the sense of this document “sample” further refers to a volume surrounding a biological sample. Like for example in assays, where secreted proteins like growth factors, extracellular matrix constituents are being studied the extracellular environment surrounding a cell up to a certain assay-dependent distance, is also referred to as the “sample”. Specifically, affinity reagents brought into this surrounding volume are referred to in the sense of this document as being “introduced into the sample”.
The imaging system for imaging a sample according to an embodiment comprises a sample moving unit configured to move the sample in a sample space along a movement direction. The imaging system has at least one detection optic having an optical axis that encloses an angle with the movement direction within a range of 20° to 70°. The optical axis of the detection optic and the movement direction define a first plane. The imaging system further comprises at least one illumination optic having an optical axis that encloses an angle with the movement direction within a range of 70° to 110°, and that encloses an angle with the optical axis of the detection optic within a range of 70° to 110°. The optical axis of the illumination optic and the movement direction define a second plane. The first and second planes intersect and are different.
By tilting the optical axis of the detection optic relative to the movement direction, the object plane of the detection optic is non-parallel to the movement direction. The illumination optic is arranged such that the object plane of the detection optic can be illuminated by an illumination provided through the illumination optic. Thus, by moving the sample along the movement direction, i.e. through the object plane, different planes within the sample are illuminated and can be imaged with the detection optic. This is also called optical sectioning. Thereby, it becomes possible to acquire a series of images of the sample, each image corresponding to a different plane within the sample. This series of images is also called an image stack and can form the basis for a volumetric, i.e. three-dimensional, image of the sample. In particular, a large number of samples can be moved past the detection optic in quick succession resulting in a high throughput. Thus, the imaging system according to an embodiment allows volumetric imaging of samples with a high throughput. The angle between the at least one detection optic and the movement direction is preferably within a range of 30° to 60°, 40° to 50°, 43° to 47° or it is 45°. Said angle is such that a field of view of the at least one detection optic and the essential part of the cross section of a sample movement volume at or near the detection optic is covered or is suitable to image essentially the complete sample while the sample passes the detection optics when moving along the movement direction.
In a preferred embodiment, the optical axis of the illumination optic is perpendicular to the optical axis of the detection optic, and perpendicular to the movement direction. An illumination, for example a light sheet, that is focused along the optical axis of the illumination optic will illuminate the object plane of the detection optic, in particular if the light sheet is oriented such that the normal vector of the plane of the illumination light sheet is essentially parallel to the optical axis of the detection optic. Thus, this preferred embodiment does not require additional optical elements for aligning the illumination with the object plane of the detection optic. Thus, the imaging system according to this embodiment has a simple optical design and can be manufactured at low cost.
In another preferred embodiment the imaging system comprises a second detection optic, wherein an object plane of the first detection optic and an object plane of the second detection optic intersect within the sample space. The second detection optic can be used to capture an image complementary to an image captured by the first detection optic. The complementary image may be used to image a different perspective or to create a composite image of the sample. Thereby, the versatility of the imaging system is increased.
The second detection optic may in particular be used to enhance the image captured by the first detection optic. The spatial resolution of a detection optic is described by its point spread function which describes how the detection optic images a point object. For a typical objective this point spread function is an ellipsoid that is elongated along the optical axis of the detection optic, i.e. the z-axis. This means that the z or axial-resolution is lower than the x-y or lateral resolution. In this embodiment, the point spread functions of the first and second detection optics overlap and can therefore be combined into a single effective point spread function by means of registration, deconvolution and fusion of the individual images. The effective point spread function then comprises the intersection of the two individual point spread functions. In the case of two detection optics, being e.g. arranged such that their optical axes are oriented perpendicular to each other, the effective point spread function is approximately cube-shaped. The edge length of this cube is approximately equal to the lateral resolution of the two detection optics. Using even more detection optics, for example six detection optics, will result in an effective point spread function that approximates a sphere. Having a spherical effective point spread function corresponds to an isometric resolution, i.e. a comparable spatial resolution in all directions that is approximately equal to the lateral resolution of a single detection optic. Thereby, in this embodiment significant improvement in resolution can be achieved.
In another preferred embodiment, the optical axis of the second detection optic encloses an angle with the movement direction within a range of 20° to 70°, preferably within a range of 30° to 60°, 40° to 50°, 43° to 47° or it is 45°, and encloses an angle with the optical axis of the first detection optic within a range of 70° to 110°, preferably within a range of 80° to 100°, 85° to 95°, 87° to 93° or it is 90°, and encloses an angle with the optical axis of the illumination optic within a range of 70° to 110°, preferably within a range of 30° to 60°, 40° to 50°, 43° to 47° or it is 45°. In this embodiment, the optical axes of the first and second detection optic are preferably nearly perpendicular. Thereby, the arrangement allows the sample to be imaged such that features of the sample hidden in the image captured by the first detection optic are visible in the image captured by the second detection optic and vice versa. Thus, within this range the second detection optic might complement the first detection optic best.
In another preferred embodiment the imaging system comprises a second illumination optic. The optical axis of the second illumination optic encloses an angle with the movement direction within a range of 70° to 110°, preferably within a range of 80° to 100°, 85° to 95°, 87° to 93° or it is 90°, and encloses an angle with the optical axis of the first illumination optic being smaller than 20°. The second illumination optic can be used to provide a complementing illumination in order to improve the image quality of the imaging system.
In another preferred embodiment, the first and second illumination optics are arranged opposite each other. In this embodiment, the arrangement of the first and second illumination optics allows the sample to be illuminated from opposite sides. Thereby, shadowing effects can be reduced.
In another preferred embodiment the imaging system comprises a light sheet illumination unit configured to generate at least one light sheet by directing illumination light through the at least one illumination optic into the sample space for essentially only illuminating the object plane of the detection optic. The light sheet is used to illuminate a thin stripe of the sample. Thereby optical sectioning of the sample is achieved, e.g. by the sampler movement through the illuminated stripe or plane. The light sheet illumination unit allows the imaging system to be used for light sheet microscopy. Light sheet microscopy has many advantages. In particular, light sheet fluorescence microscopy allows imaging of planes deep within samples that would not be accessible otherwise and/or smaller phototoxic illumination compared to for example white-field microscopic illumination. Thus, the imaging system according to this preferred embodiment may in particular be used to generate volumetric images of thick samples. The generation of the at least one light sheet can be made at the object plane of the detection optic parallel to the object plane of the detection optic, however in the depth of focus of the detection optic.
The light sheet illumination unit may comprise at least one of the following light sources a continuous wavelength laser, a pulsed laser, a white light laser, a semiconductor laser, or an LED illumination. The illumination unit may further be configured for multi-photon excitation.
In particular, (inverted) selective plane of illumination microscopy, SCAPE—as described in e.g. U.S. Pat. No. 8,619,237 B2—and/or OPM—as described e.g. in WO 2010/012980 A1—, or related setups may be used. Several beam profiles including but not limited to Gaussian, sinc2, Bessel beam as well as multi-beam configurations like for example lattice light sheet may be deployed to illuminate a thin stripe of the sample, which overlaps at least partially with the object plane of at least one detection optic.
In another preferred embodiment, the light sheet illumination unit is configured to generate a second light sheet by directing illumination light through the at least one illumination optic into the sample space, and to alternatingly illuminate the object plane of the first detection optic with the first light sheet and illuminate the object plane of the second detection optic with the second light sheet. By sequentially illuminating the object planes of the first and second detection optics the image quality of images of samples that have more out-of-focus blur, for example large samples, samples with more autofluorescence or more densely labeled samples can be substantially improved. It is possible to illuminate the first and second object plane not in an alternative fashion but simultaneously. However perpendicular parts of the generated illumination light distribution might cause out of focus blur or a detected higher light intensity at local parts of the acquired images, if the first and second illumination light sheets intersect in the sample space. That might result in cross talk contributions of the two images. These artifacts can be removed or corrected by respective computational algorithms.
In another preferred embodiment, the light sheet illumination unit is configured alternatingly direct illumination light through the first illumination optic into the sample space for illuminating the object plane of the first or second detection optic with the first or second light sheet, and through the second illumination optic into the sample space for illuminating the object plane of the first or second detection optic with the first or second light sheet. This preferred embodiment combines the advantages of sequentially illuminating the object planes of the first and second detection optics with the advantages of an illumination from opposite sides. Thereby, large samples, samples with more autofluorescence or more densely labeled samples can be imaged with reduced shadowing effects. Thus, the image quality is further improved. The concept of not alternatively illuminating but simultaneous illuminating the first and second illumination light sheet might apply for this embodiment as well.
In another preferred embodiment, the at least one detection optic comprises an immersion objective. Immersion objectives have a higher numerical aperture compared to dry objectives. Thus, it is possible to achieve a high spatial resolution with an immersion objective compared to an objective being calculated for air as the immersion medium.
In another preferred embodiment, the first plane and the second plane intersect within the sample space. At the intersection of the first and second planes, the object plane of the detection optic is illuminated by the illumination optic. Further, the intersection of the first and second planes preferably is parallel to the movement direction.
In another preferred embodiment, the sample moving unit comprises a flow cell defining the sample space. In this embodiment, the sample is suspended in a flow medium which may be in a liquid or a gas phase. By moving the flow medium through the flow cell, the sample is also moved. Both (micro)fluidic and pneumatic systems may be used to move the flow medium. More than one sample may be suspended in the flow medium. Thereby, flow cells can be used to image a large number samples in quick succession. Thus, this embodiment allows to generate three-dimensional images of a large number of samples in a short time.
In another preferred embodiment, the sample moving unit comprises a movable microscope table, in particular a motorized microscope table. The sample space is defined by a sample carrier arranged on or at the microscope table. In this embodiment, the movement of the sample that results in the sample being optically sectioned during the image acquisition is accomplished by the movement of the microscope table. Thereby, an easy way of generating a three-dimensional image of the sample is provided. The microscope table may in particular comprise one or more stepper motors, linear motors or Piezo drives. Further, the microscope table may allow a translation in x-, y- and z-direction as well tilting and/or rotating the sample, i.e. having up to 6 axes.
In another preferred embodiment, the sample carrier or the flow cell comprises an optical medium in which the sample is received, the optical medium having a first refractive index. The sample carrier or the flow cell further comprises at least one window portion defining two parallel surfaces that comprises an optically transparent material having a second refractive index, and that is arranged at a bottom side of the sample carrier or the flow cell. The first and second refractive indices do not deviate by more than 2.5%. The sample carrier or the flow cell may further contain a second or third window portion through which the sample may be illuminated and at a side where an illumination optic and or a detection optic is located. The second or third window portion may be index-matched, i.e. may be of the second refractive index (“index-matched”) when the angle between the axis of the illumination objective(s) and the surface of the second and/or third window portion is substantially non-orthogonal. Alternatively, the second or third window portion may have a different third refractive index, when the angle between the axis of the illumination objective(s) and the surface of the second and/or third window portion is substantially orthogonal.
An optical interface is formed where the optically transparent material of the window portion meats with the optical medium in which the sample is received. Such an optical interface typically results in aberrations, in particular spherical aberrations. The negative effects of the optical interface are even stronger when the sample is imaged at an oblique angle, i.e. when the sample is imaged with a detection optic having an optical axis that encloses an angle with the normal of the (inner and the outer, e.g. the) two parallel surfaces of the window portion that is substantially different from 0°. Typically, this tilt of the optical axis of the detection optic will lead to the rapid degradation of the image, i.e. loss of intensity, coma, spherical aberration and chromatic aberrations.
However, the afore-mentioned negative effects of the optical interface are mainly caused by a difference in refractive indices between the two mediums forming the optical interface. Since the refractive indices of the window portion and the optical medium do not deviate e.g. by more than 2.5%, the negative effects of the optical interface are greatly reduced in this preferred embodiment.
The optical medium may be any optically transparent medium configured to receive the sample. In particular, the optical medium is an embedding medium configured to embed the sample, or a cell culture medium, or a cell culture matrix like for example a hydrogel, or a flow medium configured for use in a flow cell.
In another preferred embodiment, the imaging system is a microscope or an imaging cytometer. Microscopes and imaging cytometers have many important uses, in particular in the life sciences, diagnostics, bioprocessing, and healthcare. The imaging system may therefore be used in a wide range of applications, making it more versatile.
Embodiments of the present invention further relate to a method. The method comprises the following steps: Moving the sample in a sample space along a movement direction. Illuminating the sample with at least one illumination optic having an optical axis that encloses an angle with the movement direction within the range of 70° to 110°, and in a preferred embodiment, within a range of 80° to 100°, 85° to 95°, 87° to 93° or it is 90°, and that encloses an angle with the optical axis of at least one detection optic within the range of 70° to 110°, preferably within a range of 80° to 100°, 85° to 95°, 87° to 93° or it is 90°. Capturing at least one image of the sample with the at least one detection optic having an optical axis that encloses an angle with the movement direction within the range of 20° to 70°. The optical axis of the detection optic and the movement direction define a first plane. The optical axis of the illumination optic and the movement direction define a second plane. The first and second planes intersect and are different.
The method has the same advantages as the sample carrier and the imaging system described above.
The imaging system 100 comprises a flow cell 104 that defines a sample space 106. The flow cell 104 is filled with a flow medium in which several samples 102 are suspended. A flow direction of the flow medium is from left to right in
An optical detection system 112 of the imaging system 100 is arranged below the flow cell 104. The optical detection system 112 comprises a detection optic 114, e.g. a microscope objective, configured to capture detection light emitted by the samples 102. The detection optic 114 is directed at the first window portion 108 of the flow cell 104. The optical axis O1 of the detection optic 114 and the movement direction M of the samples 102 enclose an angle of about 45°. Accordingly, the object plane 116 of the detection optic 114 and the movement direction M enclose an angle of about 45° as well. The optical detection system 112 further comprises a tube lens 118 that directs the detection light captured by the detection optic 114 onto a detector element 120. The optical detection system 112 may comprise at least one of the following as the detector element 120 a CMOS camera, a CCD/EM-CCD camera, a spectral camera, a hyperspectral camera, a FDFLIM camera or another time-sensitive detector, a light-field camera, and a (multi) point-/line-scanning unit for confocal imaging. The refractive index of the immersion medium between the detection optic 114 and the first window portion 108 is matched to the refractive index of the first window portion 108 of the flow medium.
The imaging system 100 further comprises an illumination optic 122 arranged at a back side of the flow cell 104 and directed at the second window portion 110. The optical axis O2 (c.f.
As can be seen in
In the present embodiment the detection optic 114 is exemplary arranged below and the illumination optic 122 is arranged at a back side of the flow cell 104. However, their positions may as well be interchanged. It is also possible to design the two optics such, that each can be used as a detection optic 114 as well as an illumination optic 122.
As can be seen in
The imaging system 300 according to
Further, the optical axes O1, O3, O3 of the first and second detection optics 114, 304 and of the illumination optic 122 intersect in one point in this embodiment. They might intersect in volume being defined by the field of view of the first and second detection optics 114, 304 and of the illumination optic 122. Thus, the object planes 116, 306 of the first and second detection optics 114, 304 can both be illuminated by the illumination optic 122, in particular with a light sheet 400, 402 (c.f.
A first light sheet 400 illuminates the object plane 116 of the first detection optic 114 and is shown in
The light sheet illumination unit 500 comprises a light source 502. The light source 502 exemplary comprises four beam splitters 504 that are arranged such that they combine illumination light from four light sources shown as four hatched rectangles into a single beam 506. Alternatively, the light source 502 may be a white light laser or any other—preferably coherent—light source 502. The single beam 506 is directed at a chopper wheel 508 via a stationary mirror or a scanning mirror 510. A scanning mirror or a light direction alternating unit, e.g. an AOD (acousto optical deflector), a digital mirror device, or tunable lens, may be used for Illuminating a sample 102 out of slightly different illumination directions, for instance +/−5 degrees relative to the optical axis, in order to destrip or to reduce stripe artefacts, which are a common problem in light sheet illumination, or to generate a scanned light sheet. The chopper wheel 508 comprises holes 700 and mirrors 702 arranged alternately (c.f.
Regarding the possibility mentioned above to simultaneously generate both, the first and second light sheets 400, 402, the chopper wheel 508 could be omitted and a beam splitter 518 could be arranged e.g. as a 50:50 beam splitter directing light to the first and second light sheet forming unit 512, 514.
In the illustration according to
In the illustration according to
The chopper wheel 508 comprises mirrors 702 and holes 700 that are arranged in alternating fashion circumferentially along the outer rim of the chopper wheel 508. The beam 506 is directed at the outer rim such that it alternatingly hits a mirror 702 and a hole 700 of the chopper wheel 508, when the chopper wheel 508 is rotating. When the incoming beam 506 hits a hole 700 of the chopper wheel 508, the beam 506 passes the chopper wheel 508 unhindered. This situation is depicted in a first view of the chopper wheel 508 in the top left of
The imaging system 800 according to
The imaging system 900 according to
The flow cell 104 in
The single beam 506 generated by the light source 502 is directed at a chopper wheel 508 via a scanning mirror 510 or a stationary mirror 702. The chopper wheel 508 comprises holes 700 and mirrors 702 arranged alternately (c.f.
In the illustration according to
In the illustration according to
The light sheet illumination unit 1200 according to
In the illustration according to
The schematic view of the light sheet illumination unit 1200 as shown in
In the illustration according to
In the illustration according to
In the illustration according to
The light sheet illumination unit 1600 according to
The light sheet illumination unit 1600 according to
The light sheet illumination unit 1700 according to
The diagram comprises 8 graphs 1800 to 1814. The abscissa of each graph 1800 to 1814 denotes time. A first graph 1800 shows whether a sample 102 is passing the object planes 116, 306 of the first and/or second detection optics 114, 304. This could be e.g. determined by a light gate sensor unit which might be a simple device being arranged upstream of an optical detection system with regard to the movement direction M. The graph 1800 has the value 1 when a sample 102 is passing the object planes 116, 306 and 0 when no sample 102 is passing the object plane 116, 306.
A second and third graph 1802, 1804 show the exposure time of the first and second optical detection systems 112, 302, respectively. When the graphs 1802, 1804 have the value 0, no image is captured. When the graphs 1802, 1804 has the value 1, an image is or a plurality of images are captured by the first and/or second optical detection systems 112, 302, respectively. As can be seen by comparing the first, second and third graphs 1800 to 1804, the exposure is synchronized with the detection of a sample 102. In other words, when a sample 102 is passing the object planes 116, 306 of the first and/or second detection optics 114, 304, an image is or a plurality of images are captured by the first and second optical detection systems 112, 302. However, the exposure time may also be set much shorter. In particular, the exposure time is set such that only a certain fraction of the depth of field of the first and second detection optics 114, 304 is traversed within the exposure time. Thus, no motion blur is visible in the resulting image.
A fourth graph 1806 shows whether the first or second light sheet 402 is guided into the sample space 106. When the graph 1806 has the value 0, no illumination takes place. When the graph 1806 has the value 1, the first light sheet 400 is guided into the sample space 106, i.e. the object plane 116 of the first detection optic 114 is illuminated. When the graph 1806 has the value −1, the second light sheet 402 is guided into the sample space 106, i.e. the object plane 306 of the second detection optic 304 is illuminated. As can be seen in
A fifth and sixth graph 1808, 1810 show a shutter position of the first and second optical detection systems 112, 302, respectively. A shutter could be implemented in the form of a physical shutter, e.g. a chopper wheel having at least one opening, or by a “virtual shutter”, i.e. an electronic shutter of the camera of the detector element 120. When the graphs 1808, 1810 have the value 0, the shutters are closed and no light enters or no image is acquired by the first or second optical detection system 112, 302. When the graphs 1808, 1810 have the value 1, the shutters are open and detection light may enter the first or second optical detection system 112, 302. As can be seen in
A seventh graph 1812 shows whether the first or second illumination optic 902 is used to is generate the two light sheets 400, 402 into the sample space 106. When the graph 1802 has the value 0, no illumination takes place. When the graph 1812 has the value 1, the two light sheets are guided through the first illumination optic 122 into the sample space 106. When the graph 1812 has the value −1, the two light sheets are guided through the second illumination optic 902 into the sample space 106. While the first or second light sheet 400, 402 are generated, the illumination side is switched multiple times between opposite sides. This is optional, since a particular imaging unit may not have a second illumination optic 902, or a dual-side illumination may not be desirable in a given experimental situation. Even in imaging units comprising a second illumination optic 902 the dual side illumination may therefore be switched to a single side illumination by stopping the second chopper wheel 1202 at the appropriate position.
An eighth graph 1814 shows whether the first or second orientation of the light sheet is used being generated by chopper wheels 1602, 1604 and the spatial light modulators 1606 and 1608. As evident from a comparison of graphs 1812 and 1814 the orientation may be changed multiple times within the time, that is used to illuminate the sample from one side.
Alternatively, the switching of illumination sides may be faster than the switching of the illumination orientation. This may be controlled by the user to adapt to the specific needs of the respective application.
From the synchronization signals shown in
A first column, in
The point spread function describes how an optical system images a point object and determines the optical systems spatial resolution. As can be seen in a first row, the point spread functions 1900, 1904 of a typical confocal laser scanning microscope and a respective detection optic are ellipsoid and elongated along the optical axis of the detection optic, i.e. the z-axis. This means that the Z or axial-resolution is lower than X-Y or lateral resolution. A second row shows an illumination light distribution 1902 in comparison to the detection point spread function 1904 of a single detection optic 114 in case of a light sheet illumination. As can be seen in
A third row shows the point spread functions 1904, 1906 of two detection optics whose object planes 116, 306 intersect within the sample 102. The point spread functions 1904, 1906 of the two detection optics 114, 304 overlap and can therefore be combined into a single effective point spread function 1918 by means of registration, deconvolution and fusion of the individual images. The effective point spread function 1918 then comprises the intersection of the two individual point spread functions 1904, 1906. A can be seen in
Using even more detection optics, for example six detection optics as shown in the fourth row, will result in an effective point spread function 1920 that approximates a sphere. Having a spherical effective point spread function corresponds to an isometric resolution, i.e. a comparable spatial resolution in all directions that is approximately equal to the lateral resolution of a single detection optic 114.
The imaging system 2000 according to
The imaging system 2100 according to
The sample carrier 2102 is received on a motorized microscope table 2114 that is exemplary formed as a x-y table. In the present embodiment, the microscope table 2114 forms the sample moving unit. By moving the sample carrier 2102 by means of the microscope table 2114, the samples 102 are moved relative to the first and second detection optics 114, 304. Thus, the movement of the microscope table 2114 defines the movement direction M of the samples 102 that is shown in
In the present embodiment, the sample carrier 2102 is exemplary formed as a microplate. The sample carrier 2102 may also be formed as a petri-dish, a microscope slide, a chamber slide or any other suitable sample carrier geometry.
The samples 102 are embedded in a first optical medium 2200, for example an embedding medium or a flow medium, that is arranged above a window portion 2202 in
Optical interfaces 2206, 2208 are formed at the boundary where the window portion 2202 made from the index-matched material touches the first optical medium 2200, and the second optical medium 2204 respectively. In
Two parallel light rays propagate from the top through the first optical medium 2200, the window portion 2202, and the second optical medium 2204 in that order. Since the refractive indices Rlm1, Rlm2 of the first and second optical medium 2204, are essentially equal to the refractive index Rlw of the window portion 2202, the light rays are not or only slightly refracted at the first and second optical interfaces. In the case the refractive indices Rlm1, Rlm2, Rlw do not deviate by more than 2.5%, the resulting spherical aberrations and coma are so mild that they can still be corrected computationally. Preferably, the first optical medium 2200, the window portion 2202, and/or the second optical medium 2204 are selected such that the dispersion of these items are similar or (almost) identical to each other as well.
Typically, optical interfaces result in aberrations, in particular spherical aberrations. The negative effects of the optical interface are even stronger when the sample 102 is imaged at an oblique angle, as is the case with the proposed imaging system 100. Typically, a tilt of the optical axis O1 of the detection optic 114 will lead to the rapid degradation of the image, in particular loss of intensity, coma, spherical aberration and chromatic aberrations. However, the afore-mentioned negative effects of the optical interface are mainly caused by a difference in refractive indices between the two mediums forming the optical interface. Since the refractive indices of the window portion 2202 and the optical medium 2106 do not deviate by more than e.g. 2.5%, the negative effects of the optical interface are greatly reduced. Thus, the sample carrier 2102 or the flow cell 104 according to the present embodiment is well suited for use with the imaging system 100 described above.
Identical or similarly acting elements are designated with the same reference signs in all Figures. As used herein the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”.
Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.
While subject matter of the present disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. Any statement made herein characterizing the invention is also to be considered illustrative or exemplary and not restrictive as the invention is defined by the claims. It will be understood that changes and modifications may be made, by those of ordinary skill in the art, within the scope of the following claims, which may include any combination of features from different embodiments described above.
The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article “a” or “the” in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of “or” should be interpreted as being inclusive, such that the recitation of “A or B” is not exclusive of “A and B,” unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of “at least one of A, B and C” should be interpreted as one or more of a group of elements consisting of A, B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. Moreover, the recitation of “A, B and/or C” or “at least one of A, B or C” should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g., A and B, or the entire list of elements A, B and C.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21201008.6 | Oct 2021 | EP | regional |
This application is a U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2022/077379, filed on Sep. 30, 2022, and claims benefit to European Patent Application No. EP 21201008.6, filed on Oct. 5, 2021. The International Application was published in English on Apr. 13, 2023 as WO 2023/057349 A1 under PCT Article 21(2).
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/077379 | 9/30/2022 | WO |
