Apparatus and method for simultaneous imaging and execution of contact-free directed hydrodynamic flow

Abstract

An apparatus and method for simultaneous imaging and execution of contact-free directed hydrodynamic flow in a specimen. The apparatus includes a laser adapted to dynamically heat the specimen, a microscope with an objective adapted to image at least a part of the specimen and to guide a light beam of the laser into and/or onto the specimen to heat at least one specified location of the specimen, means for manipulating the specified location, and a sample chamber for the specimen that is accessible for imaging radiation and the light beam to allow simultaneous imaging and manipulation of the sample via the objective.

Claims

1. Apparatus for simultaneous imaging and execution of contact-free directed hydrodynamic flow in a specimen, the apparatus comprising: an infrared laser source adapted to dynamically heat at least one specified location of the specimen, a microscope having a light source which provides visible light and an objective adapted for fluorescence imaging of at least a part of the specimen and to guide or focus a light beam of the infrared laser source into and/or onto the specimen to heat the at least one specified location of the specimen to allow simultaneous imaging and manipulation of the sample via the objective, means for manipulating the specified location of the specimen which is to be heated, means for coupling in light from the infrared laser source into an optical path of the microscope is arranged downstream of the means for manipulating the specified location of the specimen, and a sample chamber for the specimen that is accessible for visible imaging radiation and the light beam to allow simultaneous imaging and manipulation of the sample via the objective, wherein the objective is an immersion objective, and wherein heavy water is located between the objective and the sample.

2. Apparatus according to claim 1, wherein a means for manipulation of a focal volume of the light beam, is provided between the infrared laser source and the microscope in a collimated portion of a light beam path.

3. Apparatus according to claim 1, wherein the means for manipulating the specified location is a scanner.

4. Apparatus according to claim 1, wherein a shutter means is provided and adapted to disrupt coupling of the light beam into an optical path of the microscope.

5. Apparatus according to claim 1, wherein the microscope is at least one of the following: a confocal microscope, a fluorescent laser-scanning microscope, a wide-field-light microscope or a 2-photon fluorescence microscope.

6. Apparatus according to claim 1, wherein the objective has a high numerical aperture of at least 0.5.

7. Apparatus according to claim 1, wherein the infrared laser source has a wavelength in a range of 1000 nanometers and 0.1 millimeters.

8. Apparatus according to claim 1, wherein the sample chamber comprises a specimen holder with a first sample chamber confining surface, at least one spacer on the specimen holder providing a defined sample thickness, and a cooling device with a second chamber confining surface, wherein the spacer is provided between the first and the second confining surfaces.

9. Apparatus according to claim 8, wherein the specimen holder and the second chamber confining surface have a thickness of at least 30 μm.

10. Apparatus according to claim 8, wherein the second chamber confining surface of the cooling device comprises a heat conductive, transparent material.

11. Apparatus according to claim 8, wherein the cooling device further comprises at least one cooling element adapted to cool the heat conductive material.

12. Apparatus according to claim 8, wherein the cooling device comprises a heat management system which is adapted to remove heat from the cooling element.

13. Apparatus according to claim 1, wherein the light source provides a bright or dark field illumination which supports a direct or indirect inspection of the manipulated specimen through the objective of the microscope.

14. Apparatus according to claim 1, wherein a data processing equipment is provided which is connected to the cooling device, the microscope, the shutter, and the light source, whereby a cooling intensity of the cooling device and an intensity of the light beam can be set in dependence of each other and in dependence of an imaging information of the irradiated specimen.

15. Apparatus according to claim 14, wherein the data processing equipment is further connected to the means for manipulating the specified location of the light beam so as to bring about a controlled movement of the light beam through the specimen.

16. Apparatus according to claim 1, wherein the means for manipulating the specified location has at least one spatial light modulator.

17. Apparatus according to claim 1, wherein the infrared laser source has a wavelength in the range of 700 nm and 1 mm.

18. Method for simultaneous imaging and executing contact-free directed hydrodynamic flow in a specimen, the method comprising: fluorescence imaging the specimen via an objective of a microscope using visible light provided by a light source; an infrared laser source dynamically heating at least one of an interior or a surface, of the specimen via a light beam; inducing a hydrodynamic flow in the specimen through changing heat patterns with the specimen by manipulating a specified location of the specimen which is to be heated by variably guiding the light beam to specified locations of the specimen; and coupling the light beam of the infrared laser source into an optical path of the microscope downstream of a means for manipulating the specified location of the specimen which is to be heated, wherein the light beam is directed to the specimen through the objective of the microscope, wherein the objective is an immersion objective, and wherein heavy water is located between the objective and the sample as immersion fluid.

19. Method according to claim 18, wherein a trajectory of the specified location to be heated is chosen such that a symmetry of a focal volume path through the specimen is broken, and wherein every point in the sample is heated equally after one period of the trajectory.

20. Method according to claim 18, wherein a focal volume of the light beam is moved along a path at the specimen by means of an acousto-optical deflector or a galvanometer scanner.

21. Method according to claim 18, wherein a shutter means in the path of the light beam is closed so that a heating of the specimen is interrupted and a hydrodynamic flow is observed at a precise temperature.

22. Method according to claim 18, wherein the specified location is repeatedly moved along a path or trajectory by the light beam.

23. Method according to claim 18, wherein the infrared laser source heats at least one of the specimen, or a heat conductive particle in or on the specimen.

24. Method according to claim 18, wherein no solid-state probe particles are used as probes for heating the specimen.

25. Method according to claim 18, wherein the specimen's response to a flow driving stimulus is recorded.

26. Method according to claim 18, wherein a particle tracking is performed by tracking of a fiducial marker.

27. Method according to claim 26, wherein a fluorescent protein complex is used as the fiducial marker.

28. Method according to claim 18, wherein the specimen is subjected to a series of flow fields.

29. Method according to claim 18, wherein a relative change of a particle position or of particle positions is determined after each application of a flow field.

30. Method according to claim 18, wherein a relative change of a particle position or of particle positions is used for the determination of a suitable further flow field to be applied subsequently.

31. Method according to claim 18, Wherein flow fields are applied such that at least one of a density, spacing, or clustering of particles, in the specimen is changed.

32. Method according to claim 18, wherein flow fields are applied such that for partially or fully positioned particles spatial destinations are changed continuously or in discrete time steps.

33. Method according to claim 18, wherein flow fields are computed and applied to move at least one particle along a desired path.

Description

(1) In the following the invention is further explained by means of the provided drawing.

(2) FIG. 1: A schematic sketch of the inventive apparatus according to a preferred embodiment,

(3) FIG. 2: An example of a time-average equally heated specimen, that experiences directed hydrodynamic flow,

(4) FIG. 3: An example how to induce a rotation as a directed hydrodynamic flow in a specimen.

(5) FIG. 1 shows a preferred embodiment according to the present invention with a setup to provide for coherent light radiation, preferably laser light 26, 28 and a microscope 25 via which manipulation of the specimen 24 may be observed. The person skilled in the art understands that the particular setup of this embodiment is only explanatory and may be adapted according to the experiment conducted.

Manipulation of the Light Beam

(6) The apparatus 30 according to the preferred embodiment comprises a light source 1 which is preferably a laser light source. Between the light source and the microscope 25 several fixtures in particular with the reference signs 2 to 11, 33 and 34 may be provided. The fixtures with the mentioned reference signs may be designed to provide for coherent, collimated radiation with a defined beam diameter. Any additional, supplementary or supersede installation for the same means may be provided.

(7) The light beam between the light source 1 and at the shutter means 11 is referred to as light beam 28. The same light beam but within the microscope is referred to as light beam 26.

(8) As a first step the light beam 28 may be collected from the exit of an optical fibre by the collimator 33 for collimating the radiation of the light source 1. Following the collimator 33 a polarizing beam splitter cube 2 may be provided to select for linear polarized light. The share of the light radiation which is not passed through the polarizing beam splitter cube 2 may be directed to a beam-dump 3 to safely block undesired light which could also be a photo-diode to measure the real output power of the light source 1.

(9) Furthermore, a telescope with at least a first lens 4 and a second lens 5 may be provided as a pre-adjustment for a scanner 8 to eliminate divergences caused by the collimator 33 of the light source 1. Furthermore, a lambda-half plate 6 may be used to rotate the linear polarization state of the light beam to match the optical axis of the scanner 8. Furthermore, a variable optical beam expander 7 (zoom optics, vario optics) may be provided upstream of scanner 8.

(10) The expander 7 is designed to manipulate the beam diameter without changing the size of the scan pattern within the specimen 24 provided by the scanner 8. The resolution of scan patterns can be matched to the size of the sample i.e. small flow fields and subcellular compartments and on larger scales can be generated without changing imaging objective. The objective 16 is adapted to image and/or manipulate at least a part of the specimen 24.

(11) The scanner 8 might be an acousto-optical deflector (AOD), a galvanometric scanner, in particular a quasi-static galvanometric scanner or an SLM. Its purpose (8) may be described as directing the light beam 26, 28 to different locations within the specimen 24 and thereby moving a focal volume of the light beam 26, 28 along a trajectory or path at the specimen 24. The scanner 8 is a well-known tool in manipulation of light, in particular laser light. The scanner 8 may be provided with at least one mirror or a light refracting entity which may provide an adjustable angle of reflection/refraction with respect to the light beam 28. Consequently, the light beam 28 may be dynamically guided by the scanner 8 along the trajectory at least along the x- and y-axis within the specimen 24. These axes are preferably orthogonal to the light beam 26. Preferably the scanner 8 is used to repeatedly irradiate a chosen trajectory at the specimen 24 to keep an initially induced flow up and running.

(12) The repetition leads to a pumping effect that provides a flow, which may be maintained from hundreds of nanometers up to even over a longer distance of millimeters. When reaching the endpoint of the trajectory with the light beam 26 the scanner 8 jumps to the starting point of the trajectory. Preferably, the light source 1 does not need to be switched off.

(13) In the direction of propagation of the light beam after or downstream of the scanner 8 a second telescope with at least a first lens 9 the second lens 10 may be provided. The second telescope might be provided to precisely translate the scanner-induced beam movement into the back focal plane of the microscope objective.

(14) Also, mirrors 34 may be provided between each of the installations between reference sign 1 and 11 for redirecting the light beam 28.

(15) Further in the direction of propagation of the light beam after the scanner 8, preferably after the second telescope, a shutter means 11 is provided and designed to decouple the light beam from the imaging optics. This might be required for quantitative (fluorescent) imaging. For example, when a fluorescent protein is provided as a dye for imaging the manipulation of the specimen 24 the emitting activity of the dye is temperature dependent. By closing the shutter means 11 the temperature may be adjusted to a precisely defined temperature which allows imaging of the specimen at a well-defined concentration and at a well-defined temperature of the dye/the fluid.

(16) With beforehand imaging experiments the temperature dependence of the behaviour of the dye, in particular its emitting activity and/or absorption activity, may be analysed. Subsequently, when manipulating and imaging the specimen 24 in which the dye is provided simultaneously, the actual temperature of a certain area within the specimen 24 may be determined in real time owing to the imaging information on the dye.

(17) Furthermore, after inducing a flow within the specimen 24 the shutter means 11 may be closed for interruption of light beam intake. Therefore, the specimen 24 cools down to a temperature that equals about the temperature that is provided by the cooling device 18.

Structure of the Microscope

(18) Following the manipulation of the light beam by any of the means to 2 to 11, the microscope 25 is provided for both imaging and manipulation of the specimen 24. At least one element 12 is provided for coupling in of the light beam into the optical path a) of the microscope 25. Preferably, such element 12 is a dichroic mirror that preferably reflects the light beam but transmits in the visible wavelength regime used for fluorescent imaging. Thus, the light beam entering the microscope 25 might be redirected by the element 12 but the radiation that is needed for imaging the specimen passes through the element 12 to reach the objective 16 of the microscope 25.

(19) The microscope 25 may be a standard microscope, with preferably any of at least one (fluorescent) filter cube 13 consisting of respective excitation, dichroic, and emission filters, a light source for imaging (e.g. a fluorescent light source or a bright field illumination) 14, a detector for imaging radiation 15, in particular a high-speed and high-sensitivity camera providing quantitative (fluorescent) imaging and a microscope objective 16. The imaging radiation source 14 and/or a detector for imaging radiation 15 (e.g. camera) may be provided within the microscope beam path a) and/or may be projected into the same, for example via the emission filter 13. FIG. 1 shows a beam path of imaging radiation 27 that is projected into the irradiation beam path a).

(20) By the continuous arrows the direction of the detection beam path b) as well as the irradiation beam path c) is indicated along which the light beam is provided. The detection beam path b) starts at the specimen 24 and goes through the objective 16 to the detector 15 for imaging radiation. The irradiation beam path a) for imaging radiation starts at the source of imaging radiation 14 and goes through the objective 16 to the specimen 24.

(21) The microscope objective 16 preferably comprises a high numerical aperture providing simultaneous high-resolution (fluorescent) imaging and precise light beam (e.g. infrared laser) scanning. Preferably heavy water or silicone oil is provided as an immersion liquid 29 for the respective objectives. Also, a high NA air objective may be used. The immersion liquid 29 is preferably placed between the sample chamber 17 and the objective 16. This eliminates additional phase transitions with a gaseous phase (air) which would lower resolution of the imaged specimen 24.

(22) The specimen 24 may be provided within a sample chamber 17. The sample chamber 17 may be regarded as the place in which a defined volume for the specimen 24 is provided. The specimen 24 may be a single- or multicellular life form, a fluid with at least one particle within or any other liquid sample, cell lysates or embryonic extracts, viscous-elastic materials that are hydrodynamically or thermally movable. The sample chamber 17 may be provided with a first cover providing a first chamber confining surface that preferably comprises sapphire or diamond or any other high heat conducting and/or transparent material. Transparency may be provided at least for visible light and/or any other radiation used according to the invention for imaging, manipulation and/or backlight. Also, the first cover surface may be a standard microscope slip.

(23) The sample chamber 17 is preferably restricted by a second chamber confining surface that may be part of the cooling device 18 or an open multi-well plate for high-throughput experiments. The second chamber confining surface preferably comprises sapphire or diamond or any other high heat conducting and transparent material. Alternatively, a glass surface like a coverslip/cover glass may also be provided as the second confining surface. Transparency may be provided at least for visible light and/or any other radiation used according to the invention for imaging, manipulation and/or backlight. Below the second chamber confining surface of the cooling device 18 a cooling element 31, preferably at least one Peltier element, may be provided. The person skilled in the art understands that every cooling element also outputs heat, particularly in the form of waste heat that needs to be dissipated. Therefore, a heat management system 32 may be provided that removes heat from the cooling element 31.

(24) Preferably, the heat management system 32 comprises a metal or a different heat conducting surface wherein an exchanging cooling liquid, preferably water, may be provided as a heat dump. Also, passive cooling may be provided. Preferably behind the cooling device 18, a bright- or dark-field illumination 19 is provided for the specimen 24. The heat management system 32 is preferably provided with an opening or transparent layer to allow the bright- or dark-field illumination 19 to irradiate the specimen 24. Additionally or alternatively, the bright- or dark-field illumination 19 may be provided at the location of the imaging sensor 15 and vice versa according to FIG. 1.

(25) Additionally, a computer 20 may be provided that is preferably connected to the light source 1, the scanner 8, the microscope 25, the imaging sensor 15, the shutter 11 and/or the cooling element 31. Between the computer and the cooling element 31 a proportional integral differential (PID) control 23 may be provided which preferably helps avoiding oscillations of the temperature of the cooling element 31 when readjustment of the cooling temperature is required. The installations 21 and 22 are preferably provided between the scanner 8 and the computer 20 and may comprise a PCI controller card 21 providing an analogue signal for the scanner control box 22 and the scanner control box 22 may include electronic oscillators and electronic amplifiers for operation of the scanner 8.

(26) The computer 20 is preferably set-up with and according to a computer program product that provides a coordination of application of light radiation to the specimen 24 (light beam, laser beam), adjusted cooling by the cooling element 31 with respect to the power output of the light source 1, movement of the scanner 8 according to a predetermined trajectory so as a focal volume of the light beam 26 is provided with in the specimen 24 that moves along the predetermined trajectory and/or real-time imaging of the specimen 24, when the program is executed by a computer.

(27) Thus, the imaging information at each point in time may be brought into context with the output power of the light source 1 and the actual point or area at the specimen 24 that is irradiated by the light beam 26 or the focal volume respectively at a more or less constant temperature since the change in output energy by the light source 1 may be compensated by additional cooling power of the cooling element 31.

Technique

(28) The actual movement of the hydrodynamic flow may be observed by the movement of the particle that is carried with the flow of the fluid. The flow speed of the fluid according to the present invention may be the speed of travel of the particle within the flow. Preferably the direction of the flow is opposite to the trajectory of the light beam movement.

(29) Furthermore, the flow may be observed by usage of a dye, whereby a temperature gradient and/or the concentration gradient may be recordable from the imaging detector 15 and therefore allows a determination of the flow speed of the dye representative for the flow of the liquid/fluid.

(30) In case that only compartments of the specimen for example a surface area of the specimen is to be dynamically heated by the light source 1 without heating additional fluid above and/or below that compartment in an optical path direction, a particle or layer in that area of desired dynamical heating may be provided to absorb light of suitable wavelength and consequently be heated by the light beam or the focal volume respectively.

(31) The wavelength and the particle/layer material may be chosen to absorb a wavelength that is preferably not absorbed by the fluid of the specimen. Such particle may be for example a gold particle that is irradiated and is therefore heated up by the light beam. The introduced energy is then transferred to the surrounding liquid which ultimately leads to the hydrodynamic flow in the liquid when particles along the trajectory are dynamically heated. The person skilled in the art understands that several of these particles need to be provided along a trajectory of hydrodynamic flow within the fluid or specimen 24. Additionally or in lieu of particles within the fluid, a layer may be provided on the specimen 24 or within the specimen 24 that is irradiated by the light beam and consequently heated.

(32) The light beam is preferably moved along the trajectory heating a particular path. The liquid in close proximity below and/or above that layer absorbs the heat from the layer which yields a localized hydrodynamic flow in proximity to that layer. The layer might be a thin carbon layer or a comparable material that provides absorption of the light beam that is preferably an infrared laser but allows transmission of visible light so that fluorescent imaging of the specimen may be conducted. However, the light beam may comprise other non-infrared wavelengths as well.

(33) The vertical and horizontal lines according to FIG. 2 depict trajectories along which a focal volume of a laser beam can been moved in a specimen. The lines according to FIG. 2 may be of a small distance such that the specimen is regarded as evenly heated. Along each line the focal volume is moved twice. The arrows on the lines indicate a direction of the focal volume movement.

(34) In the lines 1 and 3 the movement is one from left to the right and from right to the left.

(35) Consequently, the induced hydrodynamic flow in these lines is neutralized, in particular due to the almost simultaneous induced flow in two opposite directions. In line 2 the focal volume is moved twice from the left side to the right side (or the other way around), whereby the induced flow from both focal volume movement is in the same direction.

(36) Consequently, a hydrodynamic flow is induced although the average temperature is about evenly distributed. The flow in line 2 is induced in the opposite direction as the movement direction of the focal volume.

(37) The laser may also be moved through the specimen according to the scheme in FIG. 3.

(38) The depicted squares 1 to 8 represent at least an excerpt of a specimen as defined according to the invention.

(39) Each square 1 to 8 represents about the same area within the specimen at different points in time.

(40) The eight squares represent eight different focal volume light beam patterns the specimen is exposed to one after another, preferably according to an ascending numbering.

(41) The manipulated specimen can be smaller, bigger or equal in size compared to the squares.

(42) By an according change in viscosity over time in that square due to the local change in temperature, a spinning movement of a fluid in the specimen may be generated.

(43) Each dashed area within the squares stands for a heated area within the specimen. The area may comprise a number of heated locations that a discretely heated and/or a trajectory that is moved along to provide a heated area in time average. Preferably, each heated area within a particular square is induced into the specimen one after another however, faster than a change of the pattern from one square to another is provided. Consequently, in a time average, a heated area according to the dashed lines in each square is provided.

(44) Preferably after one full cycle during which the pattern according to all eight squares has been introduced, each dashed area is equally heated.

(45) The number of heated locations/trajectories or squares, i.e. parts of a cycle, can be adapted and be of any number bigger than or equal to 2.

(46) The invention enables an active and probe-free micro-rheology based on oscillatory hydrodynamic flows. More specifically, the invention, in an effort to actively measure the rheological material properties of intracellular components such as the cytoplasm or chromatin of living cells, developed an active and probe-free micro-rheology.

(47) For embodiments of the invention key ideas in this regard comprise inducing weak oscillatory hydrodynamic flow stimuli and simultaneously measuring the response of the material by tracking a cellular fiducial marker. A fiducial marker can be any expressed fluorescent protein complex i.e. μNS tracer particles or other cellular organelles like small granules that can be detected with standard microscopy techniques.

(48) To induce the oscillatory hydrodynamic flows, flows can e.g. be applied in alternating, opposing directions for typically half periods of 80 ms, before reversing the flow (160 ms for a full period). This will lead to an effective oscillation frequency of the cellular material of 6.25 Hz. The technical details i.e. laser scan frequency and number of scans are by way of example described in Table 1. By changing the number of scans at a given laser scan frequency the oscillation frequency can be tuned over at least two orders of magnitude.

(49) The response of the material to the induced oscillatory hydrodynamic flows can typically performed by tracking a fiducial marker followed by Fourier analysis of the particle trajectory. Here, it is preferable that each flow period was recorded with 2{circumflex over ( )}n camera images. Typically, 8 images per period can be used and in total 128 periods per measurement. The details are shown in Table 1.

(50) The technological advantage of the invention over existing micro-rheology techniques, such as magnetic tweezers or optical tweezers, is that the inventive apparatus and the inventive method do not require any sample modification or probe injection. This feature is particularly important for working in closed geometries such as in living embryos and cells that are often severely affected by the particle incorporation.

(51) TABLE-US-00001 TABLE 1 Possible settings for flow-driven micro-rheology Oscillation Scan Scans Camera Camera Number of frequency frequency per half frame rate Exposure recorded (Hz) (kHz) period (Hz) time (s) frames 0.5 2 2000 4 0.05 256 6.25 2 160 50 0.018 1024 50 2 20 400 0.0017 8196