Abstract
Atomic force microscope measuring device comprising a micro-cantilever and an intensity modulated laser exciting the cantilever, wherein the measuring device comprises an optical microscope, in particular a fluorescence microscope, a confocal microscope, a fluorescence energy transfer (FRET) microscope, a DIC and/or phase contrast microscope, all of those in particular construed as an inverted microscope.
Claims
1. Art atomic force microscope measuring device comprising a micro-cantilever and an intensity modulated light source exciting the cantilever, wherein the measuring device comprises an optical microscope.
2. The measuring device according to claim 1, wherein the cantilever is transparent for a wavelength of the visual spectrum.
3. The measuring device according to claim 1, wherein the cantilever has a length in the range of 10 m to 1000 m, and/or a fundamental resonance frequency in a range of 1 Hz to 10 MHz when immersed in water and/or an oscillation amplitude when excited at resonance in the range of 0.01 nm to 500 nm and/or the exciting light has a wavelength in the range of 350 nm to 750 nm.
4. The measuring device according to claim 1, wherein the exciting light spot is focused on the base of the cantilever.
5. The measuring device according to claim 1, wherein the exciting light spot is smaller than 100 m in diameter.
6. The measuring device according to claim 1, wherein said optical microscope is at least one selected from the group consisting of a fluorescence microscope, a confocal microscope, a fluorescence energy transfer (FRET) microscope, a DIC and/or phase contrast microscope or a Raman spectrometer all of those in particular construed as an inverted microscope.
7. The measuring device according to claim 3, wherein the cantilever has a length in the size of 10 m to 500 m and/or a fundamental resonance frequency in the range of 1 kHz to 2000 kHz, when immersed in water and/or an oscillation amplitude when excited at resonance smaller than 100 nm and/or the exciting light has a wavelength in a range of 350 nm to 450 nm.
8. The measuring device according to claim 5, wherein the exciting light spot is smaller than 50 m in diameter in diameter.
9. The measuring device according to claim 1, wherein the exciting light spot is smaller than 10 m in diameter.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The invention will be described in more detail hereinafter with reference to an exemplary embodiment. In the drawing,:
[0026] FIG. 1 shows a schematic sketch of a head scanner,
[0027] FIG. 2 shows schematic three-dimensional cross sectional drawing o a head scanner,
[0028] FIG. 3 shows schematic three-dimensional top view drawing of a head scanner,
[0029] FIG. 4 shows a sketch of a sample scanner,
[0030] FIG. 5 shows schematic three-dimensional cross sectional drawing of a sample scanner,
[0031] FIG. 6 shows schematic three-dimensional top view drawing of a sample scanner and
[0032] FIG. 7 shows schematic two-dimensional top view drawing of a head and sample scanner (since they are identical in this perspective) with arrows that indicate the directions of movement.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0033] As can be seen the FIGS. 1 and 4, a 405 nm laser (7) is focused on the base of a microcantilever using a set of optical elements represented as (5) and the dichroic mirror (4) in order to photothermally excite the cantilever. The movement of the cantilever is detected using a beam deflection scheme but other methods can be also applied. The laser beam (8) is focused at the end of the cantilever using a set of optical elements represented as (9) and the dichroic mirror (3). The laser beam (8) reflects partially on the cantilever (20) and hits a gold mirror (12) that addresses the beam into a four quadrants photodetector (13) to be analyzed. Thanks to the optical properties of the dichroic mirrors (3) and (4) together with the cantilever holder (19), the cantilever itself which is totally or partially transparent and the sample holder (18), special light generated in (2) can go through the sample and be collected with an inverted microscope (21) for DIC imaging or similar techniques. In addition, the microscope (21) allows to illuminate the sample with suitable light to excite fluorescent and collect back the fluorescent emitted light from the sample according to an inverted microscope set-up. An XYZ scanner enables to locate the cantilever in different positions of the sample by either moving the sample holder (18) or the box (11) with subnanometer resolution. The actuators (15) carry out a coarse vertical movement to approach or withdraw the cantilever to the sample. In addition these actuators can tilt the box (11) with respect to the sample holder (18).
[0034] In order to ensure a very high stability of the excitation of the cantilever, the exciting laser diode is kept at a fixed temperature by using a peltier element. This thermoelectric system ensures that wavelength and intensity of the laser will not shift due to temperature changes. By modulating the current that feeds the diode, the intensity of the laser can be modulated at a certain frequency. However other ways to modulate the light intensity can be applied as for instance by using acousto-optical or electro-optical modulators. The light emitted by the laser diode is then introduced into an optical fiber in order to reach an optical collimator which is located in the head of the instrument. The optical collimator is focusable and allows to get a laser beam with parallel rays. However, in order to modify the laser waist (minimum spot diameter) the collimator can be set to produce non parallel rays as well. At the end of the collimator and attached to it there is a micro focus optics which ensures that the laser light will be focus at a certain distance, usually the distance or close to the distance the cantilever is at.
[0035] The end of the optical fiber, together with the collimator and the micro focus optics (46 in FIG. 2) are mounted on a nanopositioner system (40 in FIG. 3) which is able to travel several millimeters with nanometric resolution. This nanopositioner system allows to modify the distance between the cantilever (51 in FIG. 2) and the optical system (46). Doing so, the system has a very precise and comfortable way to modify the laser spot size on the cantilever, just by changing the position where the beam waist is. This adjustment can be very important in order to optimize the cantilever excitation for the following reasons: [0036] 1.It enables to find the optimum laser spot diameter to excite the cantilever using the minimum power. This fact, for instance, can be very important when a biological sample is sitting close the end of the cantilever, since that part of the cantilever might be required to be at a physiological temperature and hence to excite the cantilever with very low power can be a need. [0037] 2. It brings the possibility of modifying the length and/or material of the cantilever holder (50 in FIG. 2) making the system very versatile to work using for instance petri dishes of different heights or dealing with a special sample holder in order to keep the sample under incubation conditions by using for instance air with 5% CO2 and high humidity.
[0038] In addition the nanopositioner system (40 in FIG. 3) is held by a set of nanopositioners (41 in FIG. 3) which enable to move in XY directions the optical system (46) in order to ensure that the laser hits the cantilever on the desired location. This set of nanopositioners hold as well a dichroic mirror (39 in FIG. 2) which deviates in 90 degrees the laser light coming from the optical system (46) but not other wavelengths of the visual spectrum. This way, the laser (44) can hit on the cantilever with almost normal incidence and simultaneously other types of illumination such as DIC, phase contrast or related techniques can be utilized from the top.
[0039] A laser diode can be used in the device as a source of light to excite the cantilever, however the use of laser light is not a requirement and other types of light can be used.
[0040] The advantage of our invention over the state of the art is that our device allows using a photothermal based AFM in filly combination with modern optical techniques such as DIC, fluorescence, FRET, confocal microscopy and in general all those based on an inverted microscope.
[0041] Reference numbers for FIGS. 1 and 4: [0042] 1. Atomic force microscope measuring device [0043] 2. DIC, phase contrast or other type of illumination to characterize the morphology of biological samples [0044] 3. Dichroic mirror that reflects the 850 nm wavelength allowing other wavelengths in the visual spectrum to go through [0045] 4. Dichroic minor that reflects the 405 nm wavelength allowing other wavelengths in the visual spectrum and the 850 nm wavelength to go through [0046] 5. Optical system to correct and focus the 405 nm laser beam (6) [0047] 6. 405 nm laser beam [0048] 7. 405 nm laser diode and optical fiber to drive the laser into the optical system (5) [0049] 8. 850 nm laser beam [0050] 9. Optical system to correct and focus the 850 nm laser (8) [0051] 10. 850 nm laser diode and optical fiber to drive the laser into the optical system (9) [0052] 11. Base of the box to hold the apparatus [0053] 12. Mirror that reflects the 850 nm laser (8) to hit the photodiode (13) [0054] 13. Four quadrant photodiode [0055] 14. Band-pass filter center at 850 nm. It allows the 850 nm laser beam to reach the photodiode (13) but blocks other wavelengths [0056] 15. Actuators to move the box and hence the cantilever along the vertical direction. These actuators for instance allow approaching and withdrawing the cantilever to the sample [0057] 16. Platform that holds the box (11). The platform contains XY positioner that allow to do a coarse movement of the box (11) with respect to the sample holder (18) [0058] 17. Hollow scanner system that holds the sample holder (18). It allows the move the sample holder (18) in three orthogonal directions (X, Y and Z) with respect to the box (11). The system contains in addition XY positioners that allow to do a coarse movement of the sample holder (18) with respect to the box (11) [0059] 18. Sample holder [0060] 19. Cantilever holder [0061] 20. Cantilever chip [0062] 21. Inverted microscope. It is compatible with modern optical techniques such as fluorescence, DIC, phase contrast, confocal microscopy, FRET, raman spectroscopy [0063] 22. Hollow scanner system that allows to move the box (11) with respect to the sample holder (18) in three orthogonal directions (X, Y and Z). In addition the scanner contains two positioners to do a coarse movement of the box (11) with respect to the sample holder (18) Reference numbers for FIGS. 2 to 3 and 5 to 7: [0064] 30. Atomic force microscope measuring device [0065] 31. Base of the box to hold the apparatus [0066] 32. Actuators to move the box and hence the cantilever along the vertical direction. These actuators for instance allow approaching and withdrawing the cantilever to the sample [0067] 33. Actuator that moves the mirror (43) by spinning the minor's holder. This actuator allows addressing (reflecting) the 850 nm laser (45) on the photodiode (49) [0068] 34. Dichroic mirror that reflects the 850 nm wavelength allowing other wavelengths in the visual spectrum to go through [0069] 35. Positioner system that holds the optical system to focus the 850 nm laser (45). This system can move as shown by the arrows, which for instance allows modifying the laser spot diameter on the cantilever (51) [0070] 36. Positioner system that holds the positioner system (35) and the dichroic mirror (34). The system can move as shown by the arrows, which allows positioning the 850 nm laser beam (45) to hit on a certain place as for instance the cantilever (51) [0071] 37. Window to allow DIC, phase contrast or other illumination types [0072] 38. Positioner system that allows moving the photodiode as shown by the arrows. This system for instance can be used in combination with (33) to make the 850 nm laser hit on a certain part of the photodiode [0073] 39. Dichroic mirror that reflects the 405 nm wavelength allowing other wavelengths in the visual spectrum and the 850 nm wavelength to go through [0074] 40. Positioner system that holds the optical system to focus the 405 nm laser (44). This system can move as shown by the arrows, which for instance allows modifying the laser spot diameter on the cantilever (51) [0075] 41. Positioner system that holds the positioner system (40) and the dichroic mirror (39). The system can move as shown by the arrows, which allows positioning the 405 nm laser beam (44) to hit on a certain place as for instance the cantilever (51) [0076] 42. Hollow scanner system that allows to move the box (31) with respect to the sample holder (53) in three orthogonal directions (X, Y and Z). In addition the scanner contains XV positioners to do a coarse movement of the box (31) with respect to the sample holder (53) [0077] 43. Mirror that reflects the 850 nm laser (45) to hit the photodiode (49) [0078] 44. 405 nm laser beam [0079] 45. 850 nm laser beam [0080] 46. Optical system to correct and focus the 405 nm laser (44) [0081] 47. Optical system to correct and focus the 850 nm laser (45) [0082] 48. Band-pass filter center at 850 nm. It allows the 850 nm laser beam to reach the photodiode (49) but blocks other wavelengths [0083] 49. Four quadrant photodiode [0084] 50. Cantilever holder [0085] 51. Cantilever chip [0086] 52. Inverted microscope. It is compatible with modem optical techniques such as fluorescence, DIC, phase contrast, confocal microscopy, FRET, Raman spectroscopy [0087] 53. Sample holder. It contains XY positioners to do a coarse movement of the sample holder (53) with respect to the box (31) [0088] 54. Platform where the box (31) is sitting. The platform contains XY positioner for a coarse movement of the box (31) with respect to the sample holder (53) [0089] 55. Hollow scanner system that holds the sample holder (53). It allows to move and scan the sample holder (53) with respect to the box (31) in three orthogonal directions (XYZ). In addition contains XY positioners for a coarse movement of the sample holder (53) with respect to the box (31)
[0090] Having described preferred embodiments of the invention, it will be apparent to those skilled in the art to which this invention relates, that modifications and amendments to various features and items can be effected and yet still come within the general concept of the invention. It is to be understood that all such modifications and amendments are intended to be included within the scope of the present invention.
