Abstract
A method of controlling a position at which laser beams used in flow cytometry impinge on a flow cell. The method includes directing each of the laser beams through a respective prism pair including a first and a second prism and. controlling a temperature of at least one of the first and second prisms. The first and second prisms are oriented such that an ellipticity of a laser beam passing through the prism pair is changed, and such that controlling the temperature of at least one of said first and second prisms results in a displacement of the laser beam along a direction corresponding to a minor transversal axis of the beam at the flow cell, wherein a position at which each of the plurality of laser beams impinges on the flow cell is controlled by controlling a temperature of at least one of the first and second prisms.
Claims
1. A method of controlling a position at which laser beams used in flow cytometry impinge on a flow cell, comprising directing each of said laser beams through a respective prism pair comprising a first and a second prism, and controlling a temperature of at least one of said first and second prisms of each prism pair, wherein said first and second prisms of each prism pair are oriented such that an ellipticity of a laser beam passing through the prism pair is changed, and such that controlling the temperature of at least one of said first and second prisms results in a displacement of the laser beam passing through the prism pair along a direction corresponding to a minor transversal axis of said beam at the flow cell, and wherein a position at which each of the plurality of laser beams impinges on the flow cell is controlled by controlling a temperature of at least one of said first and second prisms of each corresponding prism pair.
2. The method according to claim 1, wherein controlling a temperature of at least one of said first and second prisms includes controlling the temperature of both said first and second prisms.
3. The method according to claim 2, wherein said first prism and said second prism have a respective refractive index temperature dependency (dn/dT) of the same sign, and wherein controlling the temperature of both said first and second prism includes controlling the temperature of the first and second prisms in opposite directions.
4. The method according to claim 2, wherein said first prism and said second prism have a respective refractive index temperature dependency (dn/dT) of opposite sign, and wherein controlling the temperature of both the first and second prism includes controlling the temperature of the first and second prisms in the same direction.
5. An arrangement for providing excitation light to a flow cell in a cytometry system, comprising a plurality of laser modules, each laser module comprising a laser source, a prism pair comprising a first prism arranged in a radiation beam path of said laser source, configured to transmit a laser beam of said laser source, and a second prism arranged in said radiation beam path of said laser source, configured to transmit said laser beam transmitted through said first prism, and a first temperature controller for controlling a first temperature of at least one of said first prism and said second prism, wherein said first and second prisms are oriented such that an ellipticity of the laser beam is changed by passing through the prism pair, and such that controlling the temperature of at least one of said first and second prisms results in a displacement of the laser beam passing through the prism pair along a direction corresponding to a minor transversal axis of said laser beam at the flow cell.
6. The arrangement according to claim 5, wherein at least one of said laser modules further comprises a second temperature controller for controlling a second temperature of at least one of said first prism and said second prism, wherein the first temperature controller and the second temperature controller correspond to different prisms.
7. The arrangement according to claim 6, wherein said first prism and said second prism have a respective refractive index temperature dependency (dn/dT) of the same sign, and wherein the first and second temperature controllers are configured to control the temperature of the first and second prisms in opposite directions.
8. The arrangement according to claim 7, wherein said first prism and said second prism are made from the same material.
9. The arrangement according to claim 6, wherein said first prism and said second prism have a respective refractive index temperature dependency (dn/dT) of opposite sign, and wherein the first and second temperature controllers are configured to control the temperature of the first and second prisms in the same direction.
10. The arrangement according to claim 5, further comprising one or more cylindrical lenses positioned along the beam path, and configured to further shape the laser beam into desired dimensions at the flow cell.
11. The arrangement according to claim 5, wherein the first and second prisms of each prism pair are arranged at angles that result in a desired thickness of the laser beam, along the minor axis, at the flow cell.
12. The arrangement according to claim 11, wherein the angles are determined based on a wavelength of the laser beam.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The above object, as well as additional object, features and advantages of the present invention, will be more fully appreciated by reference to the following illustrative and non-limiting detailed description of preferred embodiments of the present invention, when taken in conjunction with the accompanying drawings, wherein:
[0026] FIG. 1 is a block diagram of an exemplary method according to the invention.
[0027] FIG. 2 is a block diagram of an exemplary method according to the invention.
[0028] FIG. 3 shows a schematic view of an exemplary laser module according to the invention.
[0029] FIG. 4 shows a schematic view of an exemplary laser module according to the invention.
[0030] FIG. 5 shows a schematic view of an exemplary laser module according to the invention.
[0031] FIG. 6 shows a schematic view of an arrangement including a plurality of laser modules according to the invention.
[0032] FIG. 7 shows a schematic drawing of light sheet separation in accordance with the invention.
[0033] FIG. 8 shows graphs of test results from an embodiment of the invention.
[0034] FIG. 9 shows graphs of test results from an embodiment of the invention.
DETAILED DESCRIPTION
[0035] With reference to FIG. 1, an exemplary method of controlling a position at which a laser beam used in flow cytometry impinges on a flow cell will be explained. In step S1, a laser beam is directed through a first prism. The laser beam may have been transmitted from a laser source of any type, for example selected from the group: diode laser sources, diode-pumped solid state laser sources, and solid state laser sources. In step S2, the laser beam is directed through a second prism. The first and the second prism may be of the same material and have the same properties. The first and the second prism may also not be of the same material, for example one of the first and the second prism may have a refractive index that increases with temperature while the other has a refractive index that decreases with temperature.
[0036] In step S3, a temperature of one of said first prism or said second prism is controlled. A change in temperature leads to a change in refractive index of the prism for which the temperature is controlled. The first and second prisms are oriented such that controlling the temperature of at least one of said prisms results in a displacement of said laser beam along a minor transversal axis of the laser beam.
[0037] FIG. 2 shows an exemplary method of controlling a position at which a laser beam used in flow cytometry impinges on a flow cell. Steps S1, S2 and S3 are the same as those illustrated in FIG. 1. In step S4 a temperature of the other one of said first prism and said second prism is controlled. So, for example, if the temperature of the first prism is controlled in step S3, then the temperature of the second prism is controlled in step S4, and vice versa. Controlling the temperatures may be done in the same temperature direction, meaning for example raising the temperature of both the prisms. Or it may be done in opposite directions, thus lowering the temperature of one prism and raising the temperature of the other prism. Depending on the materials from which the prisms are made, the skilled person will be able to select a suitable control scheme. Controlling the temperature of both prisms may have multiple advantages. A first advantage is that a smaller temperature change may be needed to each respective prism if a smaller adjustment to the position is wanted. This will make the adjustment process quicker and the thermal lag will be smaller, meaning that the system will stabilize within a shorter time. Further, if a maximum temperature adjustment to both prisms is made, a larger span of adjustment positions is available compared to only adjusting a temperature of one prism with a maximum temperature adjustment.
[0038] FIG. 3 shows a laser module 100, which can be a sub-component of embodiments of the present invention, comprising a laser source 110, a first prism 120 and a second prism 130. The laser module 100 further comprises a first temperature controller 140. The first prism 120 and the second prism 130 are arranged in a radiation beam path of the laser source 110 and configured to transmit a laser beam of the laser source 110. The first temperature controller 140 is in the exemplary embodiment configured to control the temperature of the first prism 120. When the temperature of the first prism 120 is adjusted, the refractive index of the prism is changed. This effect causes the laser beam to refract at a larger, or smaller, angle depending on the properties of the prism and the change in temperature. The first prism 120 and the second prism 130 are oriented such that controlling the temperature of at least one of said prisms results in an angular displacement of said laser beam. For an elliptical laser beam (i.e. a light sheet), the displacement will typically be along a minor transversal axis of said beam. Such angular displacement upstream from the final focusing lens results in a displacement of the beam position at the focus (where the flow cell is located).
[0039] The laser source 110 may for example be selected from the group: diode laser sources, diode-pumped solid state laser sources, and solid state laser sources. For practical reasons, it may be preferred in embodiments to let the first prism 120 and the second prism 130 may be similar in terms of size and material. The prisms 120, 130 may be for example made of plastic, glass or other transparent materials. The prisms 120, 130 may be cut at a 45-degree angle, but they may also be cut at a different angle. It should be understood, however, that the prisms may very well be different in both size and material in embodiments of the invention.
[0040] The first temperature controller 140 may for example be a thermo-electric controller (TEC). The prism can be mounted directly on the surface of the TEC, or on a thermally conductive mount which in turn is mounted on the TEC. When a voltage is applied over the TEC, heat is transferred from one side to the other, and the component (prism) mounted on the TEC will be heated or cooled depending on the polarity of the voltage.
[0041] FIG. 4 shows an exemplary laser module 100 similar to that of FIG. 3. The laser module 100 comprises a laser source 110, a first prism 120 and a second prism 130. The laser module 100 further comprises a first temperature controller 140, and a second temperature controller 150. The first and the second temperature controllers 140, 150 are configured to adjust the temperature of the prisms 120, 130 respectively. In this exemplary embodiment the temperature controllers 140, 150 may adjust the temperature of the prisms 120, 130 in the same direction, for example raising the temperature of the prisms 120, 130, or in different directions, i.e. raising the temperature of for example the first prism 120 and lowering the temperature of the second prism 130. Depending on the material of the prisms 120, 130 one of these examples may be more beneficial. If the prisms 120, 130 are made of materials that have a respective refractive index temperature dependency (dn/dT) of opposite sign, the first and second temperature controllers may be configured to control the temperature of the respective prisms in the same direction. This could lead to a larger angular displacement of the laser beam emitted from the laser source compared to embodiments in which only one of the prisms is temperature controlled. For the same reason, if the prisms 120, 130 are made of materials having a respective refractive index temperature dependency (dn/dT) of the same sign, the first and second temperature controllers may be configured to control the temperature of the respective prisms in opposite directions.
[0042] Although it is preferred to use a pair of prisms for each laser beam, it is also conceivable to have a single prism for each beam. The beam control may then not be as versatile, but may in some applications still be sufficient.
[0043] When a single prism is used, there will also be some change of the beam ellipticity. For example, with a right angled glass prism having a do/dT around 9×10.sup.−6 K.sup.−1 a temperature change of about 30° C. may give a change in propagation angle for the laser beam of about 500 μrad, while the transversal cross section will change by a factor of about 1.00015. Such small change in the transversal cross section of the beam is, however, entirely negligible in the context. When a single prism is used for changing the propagation direction of the laser beam, it is preferred to also have one or more mirrors downstream from the prism to correct for the change in direction.
[0044] Alternatively, the optical set-up can be adapted to the expected change in direction.
[0045] FIG. 5 shows an exemplary embodiment of a laser module 100 similar to those disclosed in FIG. 3 and FIG. 4. The laser module 100 comprises a laser source 110, a first prism 120, a second prism 130 and a first temperature controller 140. The first temperature controller 140 is in this exemplary embodiment configured to control the temperature of both the first prism 120 and the second prism 130. In this embodiment where both the first prism 120 and the second prism 130 are mounted on the same temperature controller, the prisms will have the same temperature and should thus be made of materials having a respective refractive index temperature dependency (dn/dT) of opposite sign.
[0046] FIG. 6 shows an exemplary arrangement 600 for providing excitation light to a flow cell in a cytometry system. The arrangement 600 comprises laser sources 610, 610′, 610″ emitting one laser beam each. The laser sources 610, 610′, 610″ may all emit beams of different frequencies. The arrangement further comprises a first prism 620, 620′, 620″ and a second prism 630, 630′, 630″ for each respective laser source 610, 610′, 610″. A first temperature controller 640, 640′, 640″ is coupled to each respective first prism 620, 620′, 620″. The first temperature controllers 640, 640′, 640″ are configured to individually control the temperature of each respective first prism 620, 620′, 620″. Each laser source 610, 610′, 610″ is further associated with respective beam shaping optics 605, 605′, 605″. Further associated with each laser source 610, 610′, 610″ are lenses 635, 645, 660, 670 to shape and direct each laser beam. The last two lenses 660, 670 are shared for all laser sources 610, 610′, 610″ and provide for the final shape and direction of the composite output beam. To direct the laser beams, a folding mirror 625 and beam splitters 625′, 625″ are used as shown in the figure. There may also be provided an optical filter 655, 655′, 655″, such as a band pass filter, in the path of one or more of the laser beams. A final focusing lens 680 is also provided for focusing the laser beams onto the cytometry flow cell. Lens 680 may preferably be an achromatic lens having a focal length of about 10-150 mm, such as 60-100 mm.
[0047] It is to be understood that an arrangement according to FIG. 6 could also include other optical components and lenses, or include less components than disclosed in the figure. Other embodiments could also include a number of second temperature controllers for each second prism 630, 630′, 630″ in accordance with the laser module disclosed in FIG. 4. It is also understood that the first temperature controllers 640, 640′, 640″ could be configured to control the temperature of both the first prisms 620, 620′, 620″ and the second prisms 630, 630′, 630″ in other embodiments. The optical components may be similar for each laser source 610, 610′, 610″ but they may also include different characteristics such as focal lengths or lenses of different materials. Depending on the type of laser source 610, 610′, 610″ used or the wavelength, different optical components might be beneficial to the use with the individual laser source 610, 610′, 610″. For example, the optical components are typically provided with surface coatings designed for a particular wavelength or wavelength range. A person skilled in the art will understand that there are many different possible ways to construct an arrangement according to the invention.
[0048] FIG. 7 shows an exemplary output from the arrangement disclosed in FIG. 6. FIG. 7 shows a cross section of three light sheets 710, 720, 730 that may be directed to impinge on a flow cell in flow cytometry to excite fluorophores. The light sheets 710, 720, 730 are individually adjustable along a minor transversal axis (i.e. up/down in the drawing) of the respective beam by means of a respective prism pair in accordance with the present invention. Using light sheets, i.e. laser beams of elliptical cross section as shown in the figure, a width of a flow cell transverse to the sample movement direction may be fully illuminated at an almost uniform intensity, and therefore alignment of each light sheet may be required only along the minor transversal axis of a beam, i.e. along the flow cell. In FIG. 7, a flow cell is schematically indicated by a pair of dashed lines. The wavelengths of the light sheets are typically different in order to excite different fluorophores in the samples. In an exemplary embodiment, the minor transversal axis of each light sheet (the “height”), indicated by h in FIG. 7, can be about 10 μm and the major transversal axis (the “width”), indicated by w in FIG. 7, can be about 50-150 μm. The separation d between the light sheets (laser beams) in the flow direction of the flow cell depends on the implementation, and can be several hundreds of micrometers or less than a few tens of micrometers. In fact, the separation d may be similar to the minor transversal axis of the light sheets, such that there is no gap between the light sheets (i.e. that the light sheets are just “touching”). It is preferred, however, that any overlap between neighboring light sheets does not exceed 5% in terms of beam power. In some implementations, however, it is even conceivable that the separation d is zero (i.e. some or all light sheets overlap), which may be preferred, for example, if the laser beams can be modulated such that only one laser beam at the time illuminates the flow cell.
[0049] FIG. 8 shows results from a test performed for an embodiment of the invention. The test setup was a laser module according to an embodiment of the invention as described in relation FIG. 3, i.e. only the temperature of one prism of each pair was controlled. The prisms used in these tests were made from glass having an absolute refractive index temperature dependence (dn/dT) around 9×10.sup.−6 K.sup.−1. The test setup further comprised a focusing lens, focusing the output laser beam from the laser module onto a focus point. Further, a camera/detector consisting of an imaging lens and a camera was focused on the same focus point. In the focus point, a light sheet similar to one of those shown in relation to FIG. 7 was detected by the detector. If a similar setup would be used in flow cytometry, the focus point would correspond to the position of the flow cell. The position along a minor and a major axis of the light sheet was then measured when a temperature of one prism in the laser module was adjusted. In this particular test illustrated in FIG. 8, a laser with a wavelength of 405 nm was used. The results can be seen in the graphs 810 and 820 and will now be explained.
[0050] Graph 810 shows the change in position of a center point of a light sheet along a minor axis of the laser beam in relation to temperature. The temperature is varied along a first curve 830 (right axis) and the data points measured are plotted in a second curve 840 (left axis). As can be seen, when varying the temperature according to a sinusoidal pattern, the position of the center point of the light sheet also changes according to a sinusoidal pattern. As seen from graph 810, a change in temperature between approximately 15 and 65 degrees Celsius resulted in an adjustment range of around 11 μm for the position of the center point of the light sheet along the minor axis of the laser beam. This result shows that the invention as disclosed in the present application achieves the goal of adjusting a light sheet in a flow cell by varying a temperature of a prism. The result of 11 μm is considered a fair result that is sufficient for most flow cytometry systems, and it is plausible for the adjustment range to double if the temperature of both prisms were to be controlled.
[0051] Graph 820 shows the varying in position of the center point of the light sheet along a major axis of the laser beam in relation to temperature. A third curve 850 represent the data points measured and a fourth curve 860 represents the temperature. The temperature is varied along the fourth curve 860 approximately between 15 and 65 degrees Celsius, and as can be seen by the third curve 850 the position of the center point of the light sheet along a major axis of the light sheet varies around 2 μm from peak to peak. In these tests, the prisms were not intentionally oriented in order to introduce any adjustment along this direction, and it is thus positive that the shift along this direction is small. The shift of 2 μm is expected to be entirely negligible in view of the large extension of the light sheet in this direction. The change in position for the center point along the major axis of the light sheet is mentioned here as an observation, and to illustrate that it is sufficiently small to be negligible. The underlying reasons for the adjustment of the light sheet in this direction was not investigated, but it may be due to some thermal effect, or due to non-parallel surfaces or slight misalignment within the test setup.
[0052] FIG. 9 shows similar graphs 910, 920 to those disclosed in FIG. 8. The test setup used is the same as for FIG. 8 except the laser source used has a wavelength of 638 nm. Graph 910 shows the relation between the position along a minor axis of a laser sheet of the center point of the laser sheet and temperature. The first curve 930 shows the data points and the second curve 940 the temperature. When varying the temperature between approximately 15 and 65 degrees Celsius, an adjustment range of 7.5 μm was observed. Graph 920 shows the relation between the position along a major axis of a laser sheet of the center point of the laser sheet and temperature. The third curve 950 shows the data points and the fourth curve 960 shows the temperature. Here a peak to peak distance of 4 μm was observed.
[0053] While the invention has been disclosed with reference to specific implementations and embodiments thereof, it will be understood that various variants are conceivable within the scope of the appended claims.
