3D LASER ABLATION TOMOGRAPHY AND SPECTROGRAPHIC ANALYSIS
A laser ablation tomography system includes a specimen stage for supporting a specimen. A specimen axis is defined such that a specimen disposed generally on the axis may be imaged. A laser system is operable to produce a laser sheet in a plane intersecting the specimen axis and generally perpendicular thereto. An imaging system is operable to image the area where the laser sheet intersects the specimen axis.
This application is a continuation of U.S. patent application Ser. No. 14/646,250, filed May 20, 2015 which is a U.S. National Stage of Application No. PCT/US2013/067438, filed Oct. 30, 2013, which claims priority from Provisional Application No. 61/819,882, filed May 6, 2013 and Provisional Application No. 61/720,144, filed Oct. 30, 2012. Application Ser. No. 14/646,250 is also a Continuation-in-part of application Ser. No. 14/067,502, filed Oct. 30, 2013, now U.S. Pat. No. 9,437,041, which claims priority from Provisional Application No. 61/720,144, filed Oct. 30, 2012. The entire content of each application is incorporated herein by reference.
STATEMENT OF GOVERNMENT SUPPORTThis invention was made with government support under Grant No. W00014-05-C-0241 awarded by the Office of Naval Research. The government has certain rights in the invention.
FIELD OF THE INVENTIONThe present invention relates generally to imaging methods and apparatus and to spectrographic analysis methods and apparatus.
BACKGROUND OF THE INVENTIONA commonly practiced non-destructive technique, confocal microscopy, allows for the imaging of thin planes of focus without background noise interference. These images can be stacked to form a three dimensional model of a subject, but is restricted to sub-millimeter regimes and translucent or transparent matter. Another newly developed method of laser tomography described in M. P. Echlin et al., “A New Femtosecond Laser-Based Tomography Technique for Multiphase Materials,” Adv. Mater., 23:2339-2342 (2011), uses a beam oriented perpendicularly to the imaged surface, and removes material at known rates. This method is used for metals and would not work well for samples with varying consistency, density, porosity, and absorption coefficients, as the penetration depth would vary depending on these characteristics.
Other means to image specimens include a microtome method and X-ray microtomography. A microtome (similar in concept to a meat slicer) is used to cut thin sections of a specimen embedded in a paraffin wax substrate which then are transferred to a solution bath to remove excess substrate, then transferred to an imaging device, aligned, acquired, and digitally stacked into a 3D model. X-ray tomography is nondestructive and uses virtual slices taken by a thin X-ray beam and computationally reconstructed. However, this method does not yield compositional data or color, and instead produces a map of the materials' X-ray absorptivity (related to a density profile).
There are a variety of methods for the analysis of composition or molecular analysis of materials or specimens. For example, spectroscopic techniques include Laser Induced Fluorescence (LIFs), Laser Induced Breakdown (LIBs), Hyperspectral imaging and other techniques. However, these techniques are typically practiced as a standalone process with a dedicated apparatus.
SUMMARY OF THE INVENTIONThe present invention provides a method for imaging or analysis of a specimen, including its interior, using a laser sheet or beam. The invention incrementally ablates only the desired amount of the specimen and creates a stack of images.
The present invention provides a method and apparatus for tomography. It is noted that the term “tomography” often refers to the reconstruction of a specimen through interpretation of the interaction of a “penetrating wave” like X-rays or sound waves within a body. The term “tomography” is used herein to more broadly mean “analysis of three dimensional structure through stacking cross-sectional images”, and will refer to the reconstruction of a selected specimen to produce data similar to conventional tomographic techniques.
Embodiments of the present invention provide a high-speed, automated, high resolution, and inexpensive tomographic technique. A working system has been reduced to practice for the specific case of sectioning and 3D reconstruction of plant roots, among other biological specimens. However, the present invention is useful for analysis of other types of materials and specimens, and the herein described embodiments are not intended to limit the scope of the invention.
According to some embodiments, a specimen is moved through a laser “sheet” which ablates a cross section of the specimen. This cross section is imaged using an imaging system. The movement of the specimen, the laser ablation and the imaging may be done in a continuous process or in incremental steps, as will be described in more detail.
Referring to
In the illustrated embodiment, the stage moves such that the specimen is moved along the specimen axis. Additional movement may also be provided, such as vertical, side-to-side, and or tilting in various ways. A movement system 16 is used for moving the stage 14. This movement may take a variety of forms. While the stage 14 is illustrated as having a flat surface, the stage may take any form useful for supporting a specimen, and may be considered a specimen support. The specimen may also be partially or completely embedded in a supporting substrate, such as a foam.
A laser “sheet” is provided by a laser system. In this embodiment, a short pulsed laser (<10−6 seconds, e.g. a nanosecond, picosecond or femtosecond laser) 18 produces a beam 24 that passes through beam shaping optics 20. A beam director 22 rapidly scans a laser beam along a line to create a “plane” of light, thereby providing the laser sheet 26. The beam director may also take the form of an optical element that splits or reflects the laser beam to create a sheet or sheet-like laser. Such an optical element may be a cylindrical optic. As shown, the laser sheet 26 is perpendicular to the specimen axis A-A. As such, the laser sheet ablates a “slice” of the specimen as the specimen is moved into the sheet.
Short-pulsed lasers are well-suited for a laser system of the present invention because of their high peak-power (irradiance, measured in W/cm2). This high peak power and short pulse duration induce rapid vaporization, or ablation, of the impinged target material with minimal heat effects. This characteristic allows this method to be used on a wide range of materials including ones of porous or inconsistent density.
An imaging system 30 is positioned in alignment with the axis A-A so that the system can image the cross-section of the specimen as it is being ablated. It may also image the specimen before, after, or during ablation, depending on setup of the system. In this embodiment, the imaging system is a magnified Charged Coupled Device (CCD) type camera system, such as a digital SLR camera with a lens focused on the imaging plane defined by the sheet 26. Other types of imaging systems may also be used.
A control system is provided for controlling the imaging device, the stage movement mechanism and the laser system. In the illustrated embodiment, the control system takes the form of a general-purpose computer, represented at 32, modified to perform the control functions.
In this example, reconstruction of a specimen occurs in the following 5 steps:
1. Articulated by mirrors and focusing optics, a rapidly scanned, pulsed laser beam repeatedly sweeps along a line, and creates a planar “sheet of light”. The pulse repetition rate of the laser is sufficiently high to allow each subsequent pulse to overlap the prior as the beam sweeps along the line, ensuring a smooth surface. Alternatively, the laser beam may be optically split into a sheet, if the beam has sufficient power.
2. The imaging device with its focus co-planar with the laser sheet records visual data from a specimen on the opposite side of the laser sheet.
3. The stage holding the specimen moves the specimen into the laser sheet, either incrementally or continuously.
4. As the specimen intersects the laser sheet, its camera-facing surface is ablated, exposing the next layer.
5. This laser-sheet may have a dual purpose-providing a mechanism by way the surface is both ablated and illuminated. This exposure is useful in that the specimen is illuminated only in a plane, and illuminates evenly regardless of porosity, homogeneity, or material type. Alternatively, the system is not limited to using the beam as a source of illumination and in some cases the auto-fluorescence may not be desirable. In such a case , an external beam may be used to excite the sample. This could be necessary when attempting to fluoresce particular molecules.
This method rapidly scans or splits a laser beam along a line to create a “plane” of light. A high magnification camera has its focus set to this plane. A mechanical stage holding the object to be processed slowly pushes the specimen through this focal plane, with its camera-facing surface continuously being ablated. Or, alternatively, the specimen can be moved through the focal plane in discrete steps. The camera captures this sequence of surface removal in HD video or still images. Because the laser beam is irradiating and illuminating just the surface, it is the surface that is imaged. This characteristic enables subsequent post-processing and stacking of images with a computer program with minimal re-editing.
Example System: In one example, a system is used for horticulture specimens using the following components:
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- A Q-switched, 3rd harmonic, Coherent Avia laser with 30 nanosecond pulses and 355 nanometer wavelength
- GSI Lumonics scanner
- Aerotech stages
- Canon T3i DSLR with a 65 millimeter MP-E 1-5×macro lens
- ImageJ—software from the National Institute of Health
A method in accordance with the present invention can produce high-resolution 3D models of nearly any small specimen that can be ablated with a laser. Laser tomography removes small predefined increments of a surface continuously. By adjusting laser parameters, specimens ranging from extremely delicate to much harder materials can be processed. Both a feather and a silicon wafer have been demonstrated on a prototype system.
Using a picosecond or femtosecond laser may be preferred for the sectioning and 3D reconstruction method of the present invention, as it is well understood that the duration of the pulse is on the same order as the thermal diffusion time for many materials, allowing for ablation with minimal thermal effects. This means that specimens that are delicate, fragile, flammable, or highly sensitive to thermal damage can be cleanly sectioned using this process. Additionally, the high irradiance of these pulses means that nearly any material can be ablated and imaged.
The use of a femtosecond or picosecond laser may be preferred for the inventive method. Both investigations and research have shown that there is superior edge quality, reduced thermal impact, increased allowable specimen dimensions (due to higher irradiance and more obtainable ablation thresholds) and wider range of materials that can be processed using ultrafast lasers. Additionally, using shorter wavelengths causes the photons to have bond-breaking energies, which is an athermal process. The use of components like axicon and Fresnel lenses in conjunction with beam-shaping optics can increase the effective depth of field (DOF) and further enhance the process quality.
Referring again to
While the system was described as having the stage 14 move the specimen 12 into the laser sheet 26, alternatively the laser sheet may move while the stage remains stationary, or both may move. Further, the stage and/or laser may tilt, move side to side, or make other adjustments, including ablating the specimen 14 at an angle to the specimen axis A-A. In one example, the stage 14 may rotate such that an outer portion of a specimen is ablated and then the stage is rotated so as to oblate another outer portion of the specimen, such as the opposite end or a side portion. The imaging system may include additional image capturing elements, such as additional cameras imaging from different angles.
Referring now to
In a second mode of operation, the system 110 provides for analysis of the specimen, such as analysis by spectrometry. A beam splitter 134 is disposed between the imaging system 130 and the specimen 112. As known to those of skill in the art, a beam splitter is an optical element that reflects a portion of the light hitting the splitter and allows a portion to pass directly therethrough. The beam director 122 directs a laser beam 136 downwardly to this beam splitter 134 and the splitter reflects at least a portion of the laser beam to the end of the specimen 112. This reflected beam 138 is a very narrow beam that hits and ablates a small area of the end of the specimen. This creates a “flash” for spectrographic analysis.
An additional portion of the light 142 is reflected by the splitter 134 and becomes beam 144. Optical elements may be provided at any point in the system, such as optical element or lens 145. The optical element is illustrated as also in the path of the laser beam 136 in
It is noted that the beam splitter 148 is also in the path of the laser beam 124 in
In this embodiment, the laser 118 is preferably of the same type discussed for the embodiment of
As will be clear to those of skill in the art, depending on the type and settings of the laser 118 and second laser 160, the system may be used for a variety of spectroscopic techniques. These include, but are not limited to, Laser Induced Fluorescence (LIF), Laser Induced Breakdown (LIB), Ultrafast Transient Absorption, Two-Photon Excitation Spectroscopy, Nonlinear Optical methods, Coherent anti-Stokes Raman Scattering (CARS) and Fourier Transform Spectroscopy. As will also be clear, the spectroscopy may collect light when the laser is actually ablating a small area of the specimen as well as thereafter, allowing time-resolved spectroscopic approaches. Raman spectroscopy measures variations is reflected photon frequencies caused by interference between lattice vibrations and incident monochromatic light. This technique is useful in identifying molecular composition and typically utilizes lasers with wavelengths in the near infrared to ultraviolet. Laser induced breakdown spectroscopy uses short pulse duration lasers typically 200 microjoules or less to ablate a small volume of material. The ablated ions emits light relevant to their elemental composition. Laser induced fluorescent microscopy uses pulsed, continuous, or tunable lasers to excite molecules that reemit light at characteristic wavelengths. Tunable lasers allow scanning through excitation wavelengths to get stronger signals, while pulsed lasers allow time resolved data adding another dimension of identification. All the above use a spectrometer to look and the emitted wavelengths arbitrarily in discrete steps. Any of these approaches may be used with the present invention.
In further embodiments, additional lasers may be provided, with additional optical elements to allow a shared beam path. Alternatively, different lasers may be moved into the position of the second laser 160, depending on the analysis being done. As a further alternative, the analysis light beam 140 may take a different path than the laser beam 138, such as being non-coaxial and analyzing light at an angle to the beam 138. However, this is not preferred.
In some versions, the imaging system 130 is used only for visible light image capture. The imaging system captures images of the specimen as layers are ablated by the laser sheet, as was described with respect to
The imaging system may provide additional functionality. For example, it may perform multi-spectral imaging by collecting light both in and outside the visible range. The system may include filters, such as a liquid crystal tunable filter (CLTF) to image the specimen at multiple wavelengths during the ablation of the analysis stage, giving additional information on each point sampled.
The system of the present invention may operate in a variety of ways. In one approach, a layer of the specimen is ablated, as was discussed with respect to
As will be clear to those of skill in the art, the illustrated and discussed embodiment of the present invention may be altered in various ways without departing from the scope or teaching of the present invention. As one non-limiting example, mirrors may be used to move the imaging device off axis, for packaging or other reasons. As another non-limiting example, fiber optics may be used to route light, such as to the spectrometer. It is the following claims, including all equivalents, which define the scope of the invention
Claims
1. A laser ablation tomography system, comprising:
- a specimen stage for supporting a specimen, a specimen axis being defined such that a specimen disposed generally on the axis may be imaged;
- a laser system operable to produce a laser sheet in a plane intersecting the specimen axis; and
- an imaging system operable to image the area where the laser sheet intersects the specimen axis.
2. A laser ablation tomography system in accordance with claim 1, further comprising:
- a spectrometer;
- the laser system being further operable to produce a laser beam to ablate a small area of the specimen, the spectrometer capturing a portion of the light created when the small area is ablated.
3. A laser ablation tomography system in accordance with claim 1, wherein the laser system comprises a laser operable to produce a laser beam and a beam director operable to rapidly scan the laser beam so as to create the laser sheet.
4. A laser ablation tomography system in accordance with claim 3, wherein the laser system further comprises beam shaping optics disposed such that the beam from the laser passes therethrough.
5. A laser ablation tomography system in accordance with claim 1, wherein the laser sheet intersects the specimen axis generally perpendicular thereto.
6. A laser ablation tomography system in accordance with claim 1, further comprising:
- a movement system operable to move the specimen stage along the specimen axis.
7. A laser ablation tomography system in accordance with claim 6, further comprising:
- a control system operable to control the imaging system and the movement system.
8. A laser ablation tomography system in accordance with claim 1, wherein the imaging system comprises a camera with a Charged Coupled Device (CCD).
9. A laser ablation tomography system in accordance with claim 1, wherein the laser system includes a laser, the laser being a short pulsed laser having pulses with a duration less than 10−6 seconds.
10. A method for laser ablation tomography, comprising the steps of:
- providing the laser ablation tomography system of any of claims 1-9;
- positioning a portion of a specimen where the laser sheet intersects the specimen axis;
- ablating the specimen with the laser sheet;
- imaging the specimen where it was ablated; and
- repeating the positioning, ablating and imaging steps.
11. A method in accordance with claim 10, wherein:
- the imaging step is performed during the ablating step.
12. A method in accordance with claim 10, wherein when the positioning step is repeated, the specimen is moved with respect to the laser sheet.
13. A laser ablation tomography and spectroscopic analysis system, comprising:
- a specimen stage for supporting a specimen, a specimen axis being defined such that a specimen disposed generally on the axis may be imaged and analyzed;
- a laser system operable to produce a laser sheet in a plane intersecting the specimen axis, the laser system further operable to produce a laser beam to ablate a portion of the specimen;
- an imaging system operable to image the area where the laser sheet intersects the specimen axis, and
- a spectrometer operable to capture light from ablation of the portion of the specimen.
14. A laser ablation tomography and spectroscopic analysis system in accordance with claim 13, wherein:
- the laser system has a first mode of operation wherein the laser system produces the laser sheet and a second mode of operation wherein the laser system produces a laser beam for ablating a small area of the specimen;
- the spectrometer capturing light from the ablation when the laser system is in the second mode of operation.
15. A laser ablation tomography and spectroscopic analysis system in accordance with claim 14, wherein:
- the laser beam from the laser system in the second mode of operation is generally perpendicular to the plane defined by the laser sheet.
16. A laser ablation tomography and spectroscopic analysis system in accordance with claim 14, wherein:
- the laser system comprises a laser operable to produce a laser beam and a beam director operable in the first mode of operation to rapidly scan the laser beam so as to create the laser sheet and in the second mode of operation to position the laser beam for ablating the small area.
17. A laser ablation tomography and spectroscopic analysis system in accordance with claim 16, wherein:
- the laser system further comprises at least one beam splitter disposed between the specimen stage and the imaging system, the laser beam in the second mode of operation hitting the beam splitter.
18. A laser ablation tomography and spectroscopic analysis system in accordance with claim 14, wherein:
- the imaging system is further operable to capture a location of the ablation when the laser system is in the second mode of operation.
19. A laser ablation tomography and spectroscopic analysis system in accordance with claim 14, wherein:
- the laser system comprises a first laser operable to produce a first laser beam and a second laser operable to produce a second laser beam;
- the laser system further comprising a beam director operable in the first mode of operation to rapidly scan the first laser beam so as to create the laser sheet and in the second mode of operation to position the second laser beam for ablating the small area.
20. A laser ablation tomography and spectroscopic analysis system in accordance with claim 19, further comprising:
- a beam splitter positioned where the laser beams from the lasers intersect so that the laser beams share a beam path after the splitter.
21. A laser ablation tomography and spectroscopic analysis system in accordance with claim 19, wherein:
- the first laser is a femtosecond or picosecond laser.
22. A laser ablation tomography and spectroscopic analysis system in accordance with claim 21, wherein:
- the second laser is selected from the group consisting of a short pulsed tunable laser and a continuous laser with narrow bandwidth.
23. A method for laser ablation tomography and spectroscopic analysis, comprising the steps of:
- providing the laser ablation tomography and spectroscopic analysis system of any of claims 13-22;
- positioning a specimen on the specimen stage such that a portion of the specimen may be ablated;
- ablating the specimen with the laser sheet;
- imaging the specimen where it was ablated;
- ablating a portion of the specimen with the laser beam;
- analyzing the ablated portion of the specimen with the spectrometer; and
- repeating the positioning, ablating, imaging and analysing steps.
24. A laser ablation spectroscopic analysis system, comprising:
- a specimen stage for supporting a specimen, a specimen axis being defined such that a specimen disposed generally on the axis may be imaged and analyzed;
- a laser system operable to produce a laser beam to ablate a portion of the specimen;
- an imaging system operable to image the area where the laser beam ablates the portion of the specimen; and
- a spectrometer operable to capture light from ablation of the portion of the specimen.
Type: Application
Filed: Dec 4, 2017
Publication Date: Apr 12, 2018
Inventors: Benjamin Hall (State College, PA), Jonathan Lynch (Boalsburg, PA), Edward W. Reutzel (State College, PA), Galen Lynch (Cambridge, MA), Brian T. Reinhardt (Lemont, PA)
Application Number: 15/830,513