Patent Application
20150192768
The present invention relates to the field of non-linear imagining, in particular, microscopy applications.
Non-Linear imaging and in particular two-photon imaging systems are a workhouse in today's medical and life science labs.
A non-linear imaging system consists of one or multiple excitation and detection beam paths and a processing unit. The excitation beam path is comprised of a laser, beam forming optics, namely a beam expander, a two dimensional scan unit, a set of optics to relay the beam onto the back aperture of an objective. A microscope objective focuses the beam onto the sample. The scan unit is used to create a 2D scan pattern on the sample to illuminate the region of interest by focus volume. Light scattered backward or forward from the sample is collected by a high NA objective, separated from the excitation light by means of a wavelength selective beam splitter or filters. The light is then detected by one or multiple light detectors. A processing unit reconstructs the image from the individually recorded pixels.
The non-linear excitation commands high peak intensity, which limits the excitation volume to focus of the microscope objective. This allows for depth-resolved measurements. Another advantage of 2 p-microscopy over standard fluorescence or confocal microscopy is that the excitation wavelengths are about twice as long. Long wavelength excitation has two advantages. 1. It allows to image deeper into the sample as longer wavelengths scatter less in dense media like human tissue. 2. Excitation with NIR light reduces photo toxicity and photo bleaching of the specimen.
A great area of interest is to image samples tagged with fluorescence proteins like Green and Yellow florescence's proteins (GFP and YFP, respectively) or mCherry. Often the proteins are genetically encoded in the sample. These fluorophores exhibit strong excitation cross sections in the wavelength region above 950 nm.
Wavelength of up to 1050 nm can be produced using mode-locked Titanium Sapphire Lasers. These lasers, however, are complex and expensive and often present a high barrier of entry into the field. In addition the gain maximum of TiSa is at 800 nm and the gain curve drops quickly when approaching 950 nm limiting the power available at 950 nm and above.
Therefore, there is a need for a cost effective laser system which can produce high output power with short pulses at wavelength above 950 nm.
A fairly new emerging imaging technique deploys three-photon excitation. All the advantages of long wavelength and non-linear excitation mentioned above apply also for three photon imaging. The excitation wavelengths between 1500 nm and 2000 nm are used. The advantage is even less scattering of the excitation light than in the case of two photon excitation. The reduced scattering permits even deeper imaging in highly scattering tissue. The disadvantage of going to higher and higher order non-linear excitation is a drastically reduced excitation cross section. Hence which technique to deploy needs to be carefully decided upon the objectives of the experiment.
Another important technique used in live cell studies is photo activation. One example of photo activation is to release certain substances into the cell upon exposing the cell or part of the cell to intense light. Another term used for this application might be uncaging. A second example of photo activation are light-gated ion channels. Channelrhodopsins are often used to enable light to control electrical excitability, intracellular acidity, calcium influx, and other cellular processes. Channelrhodopsins can be activated with green light (540 nm) in a single photon step or with light above 1 um in a two photon step in case one wants to activate deep in the tissue.
It is often desirable to observe the sample through a 2 p microscope and record certain cell functions time resolved after the photo activation took place. Precise synchronization (<100 ps) between the photo activation and the images taken thereafter is of the essence. Besides it is important to be able to take images at a wavelength different from the activation wavelength immediately after the photo activation without any downtime e.g. caused by tuning of a laser source.
It can therefore be advantageous to have a laser source which emits two wavelengths simultaneously.
Yet another use of the described laser system with a synchronized two wavelength output would be for Stimulated Coherent Raman Imaging or Coherent Anti-Stokes Raman Imaging. Raman imaging provides specificity without the necessity to label the specimens with fluorophores or dyes. Raman imaging in general makes use of the unique rotational and vibrational level structure of molecules to provide specificity in analyzing a sample. Spontaneous Raman Scattering, however, is a low probability event and hence the signal strength is typically low. The Raman signal, however, can be enhanced by several orders of magnitude in the presence of two, intense driving light fields typically provided by two mode-locked lasers. The wavelength difference between the two light fields needs to be tuned to a transition frequency of the inner molecule level structure to get the signal enhancement. In addition it is imperative that both laser pulses overlap in space and time on the sample. It can therefore be advantageous to have a laser source with two tightly synchronized outputs and which allows for a tunable wavelength difference between the two outputs.
An embodiment of the invention provides a femtosecond fiber laser at the telecommunications band around 1550 nm and a tunable wavelength shifting method that converts the pulse wavelength to the amplification band of Thulium or Holmium doped optical fibers (around 2000 nm). This approach offers two advantages: (a) the femtosecond fiber lasers at 1550 nm have been developed into reliable and stable systems in the recent years and are commercially available from several companies, and (b) the amount of wavelength shift in the system can be tuned, offering the capability to adjust the ultimate output wavelength of the source. The output average power can be scaled up using a fiber amplifier in the 1800 nm to 2100 nm wavelength range. The output from said amplifier is then frequency-doubled in a non-linear medium to cover the biological interesting wavelength range from 950 nm to 1050 nm.
Another embodiment of the present invention provides a method for operating the imaging system that includes a mode-locked fiber laser configured to output a pulse having a center wavelength; a first nonlinear waveguide configured to shift the wavelength of the pulse from the mode-locked fiber laser; a first fiber amplifier configured to amplify the output from the first mode-locked fiber laser; a first fiber amplifier configured to amplify the output from the first nonlinear waveguide; and a nonlinear medium configured to frequency-double the output from the first fiber amplifier, the method including: receiving a feedback from the output of the first nonlinear waveguide, the output of the first fiber amplifier or the output of the nonlinear medium; and adjusting a gain of the first fiber amplifier, the light polarization, or the amount of wavelength shift in the first nonlinear waveguide to optimize the image brightness and quality.
In another embodiment the light from the 1550 mode locked laser is split into two arms. In one arm the light is shifted to the amplification band of Thulium or Holmium doped optical fibers (around 2000 nm) and then frequency doubled. The other arm is amplified in an Erbium doped fiber amplifier (EDFA) before frequency doubling. This embodiment produces two precisely synced laser pluses at two different wavelengths in the two photon excitation window from 760 nm to 1050 nm.
Another embodiment of the present invention provides a three-photon microscopy system, including: a mode-locked fiber laser configured to output a pulse having a center wavelength; a nonlinear waveguide configured to shift the wavelength of the pulse from the mode-locked fiber laser; a fiber amplifier configured to amplify the output from the first nonlinear waveguide; and a microscopy imaging system.
The description of illustrative embodiments according to principles of the present invention is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. In the description of embodiments of the invention disclosed herein, any reference to direction or orientation is merely intended for convenience of description and is not intended in any way to limit the scope of the present invention. Relative terms such as “lower,” “upper,” “horizontal,” “vertical,” “above,” “below,” “up,” “down,” “top” and “bottom” as well as derivative thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description only and do not require that the apparatus be constructed or operated in a particular orientation unless explicitly indicated as such. Terms such as “attached,” “affixed,” “connected,” “coupled,” “interconnected,” and similar refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. Moreover, the features and benefits of the invention are illustrated by reference to the exemplified embodiments. Accordingly, the invention expressly should not be limited to such exemplary embodiments illustrating some possible non-limiting combination of features that may exist alone or in other combinations of features; the scope of the invention being defined by the claims appended hereto.
This disclosure describes the best mode or modes of practicing the invention as presently contemplated. This description is not intended to be understood in a limiting sense, but provides an example of the invention presented solely for illustrative purposes by reference to the accompanying drawings to advise one of ordinary skill in the art of the advantages and construction of the invention. In the various views of the drawings, like reference characters designate like or similar parts.
Multi-Photon Imaging
An embodiment of the invention is a system that comprises four key components, as shown in
In one embodiment the nonlinear medium could be a bulk nonlinear crystal like BBO.
In another embodiment the nonlinear medium could be a periodically poled nonlinear crystal.
The generated pulses have center wavelengths between 900 nm and 1350 nm and can be used to excite e.g. fluorescence markers or dyes with excitation wavelengths within this range. The pulses are sent into a microscopy system (150)
In other embodiments of the invention, one or more of the following components can be added to the system to improve its performance, as shown in
Fiber amplifier 2 (260): A fiber amplifier can be included between the MLFL (210) and Nonlinear Waveguide 1 (220). The amplifier has a gain in the wavelength region from 1500 nm to 1650 nm, for example, an Er-doped fiber amplifier. The amplifier has three functions. First, it boosts the power from a low-power MLFL to the level needed for the Raman self-frequency shifting process. Second, it compresses the pulses from the mode-locked oscillator, which improves the efficiency of the frequency-shifting process, leading to a pulse energy increase or a pulse width decrease for the frequency-shifted pulses. Third, by adjusting the amplifier gain, it provides means for adjusting the amount of wavelength shift. The wavelength adjustment is used to tune the output of the frequency doubled light.
Polarization controller 1 (250): This device is a manual or an automated polarization controller inserted between the MLFL (210) and Nonlinear Waveguide 1 (220). The polarization controller is used as a second adjustment mechanism for controlling the amount of wavelength shift through the self-frequency shifting process. An automated controller can be used to dynamically tune the wavelength to a desired point in the spectrum for added stability.
In some embodiments, the MLFL (210) and Fiber Amplifier 2 (260) are built using polarization maintaining fibers. In these cases, the wavelength shift is adjusted only using the gain of Fiber Amplifier 2 (260).
Note that in one embodiment, polarization controller 1 (250) can be placed directly after the Mode-Locked Fiber Laser (210) or in between Fiber Amplifier 2 (260) and Nonlinear Waveguide 1 (220).
Dispersive Element 1 (270): This component is included after Nonlinear Waveguide 1 (220) in order to create a desired amount of chirp on the pulse entering Fiber Amplifier 1 (230). The component comprises a dispersive device, including but not limited to optical waveguides, chirped Bragg gratings, prism pairs, and diffraction grating pairs. In some embodiments, the dispersion value is designed to compress the output pulse from Fiber Amplifier 1 (230) to the shortest duration through the interplay between the dispersion and the nonlinearity in the amplifier. In other embodiments, Dispersive Element 1 is designed to increase the pulse duration in order to reduce the nonlinear effects in the amplifier. In such cases, the pulses are re-compressed using the Dispersive Element 2 (see below).
Polarization controller 2 (290): This component adjusts the polarization state of the pulses before entering Fiber Amplifier 1. By controlling this polarization state, the effective nonlinearity in Fiber Amplifier 1 can be adjusted, which is used to optimize the nonlinear pulse compression in Fiber Amplifier 1.
In some embodiments, Fiber Amplifier 1 (230) is built using polarization maintaining fibers. In these cases, the nonlinearity in Fiber Amplifier 1 is adjusted using the gain of Fiber Amplifier 1 (230).
Note that in one embodiment, polarization controller 2 (290) can be placed directly after Nonlinear Waveguide 1 (220) or in between Dispersive Element 1 (270) and Fiber Amplifier 1 (230).
Dispersive Element 2 (280): This component is included before the Nonlinear medium as means to adjust the amount of chirp on the pulse entering the nonlinear medium (240). The component comprises a dispersive device, including but not limited to optical waveguides, chirped Bragg gratings, prism pairs, and diffraction grating pairs.
Polarization controller 3 (291): This component is included before the nonlinear medium (240) as means to adjust the state of polarization of the pulse entering the nonlinear medium (240) to optimize the efficiency of the frequency doubling process.
In some embodiments, Fiber Amplifier 1 (230) is built using polarization maintaining fibers and the light polarization entering the nonlinear medium (240) is linear. In such cases, the frequency doubling efficiency can be simply adjusted by rotating the orientation of the output fiber from Fiber Amplifier 1 (230).
An embodiment of the invention provides a system and method for stabilizing and tuning the pump wavelength and pulse shape and consequently optimizing the parameters of the two-photon imaging by adjusting the gains of Fiber Amplifiers 1 or 2 (330 or 360), or the polarization controllers 1 or 2 or 3 (350, 390, or 391), as shown in
Three-Photon Fluorescence Microscopy
In another embodiment, the output from Amplifier 1 (530) can be sent directly into a microscopy system (550) for three-photon imaging, as shown in
The various embodiments discussed in above section also apply to this embodiment as well.
Furthermore, there are various possible applications of some of the embodiments discussed in this document, such as photo activation combined with three photon imaging.
Dual-wavelength microscopy
In another embodiment, the light from the 1550 mode locked laser (410) is split into two arms using a splitter (440), as shown in
While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention. Furthermore, the foregoing describes the invention in terms of embodiments foreseen by the inventor for which an enabling description was available, notwithstanding that insubstantial modifications of the invention, not presently foreseen, may nonetheless represent equivalents thereto.
This application claims the benefit of U.S. Provisional Application No. 61/924,629, filed on Jan. 7, 2014, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61924629 | Jan 2014 | US |
