WO2014076445A1

WO2014076445A1 - Tunable laser for fluorescence microscopy

Info

Publication number: WO2014076445A1
Application number: PCT/GB2013/000487
Authority: WO
Inventors: Ksenia FEDOROVA; Edik Rafailov; Grigorili SOLOLOVSKII
Original assignee: The University Of Dundee
Priority date: 2012-11-14
Filing date: 2013-11-14
Publication date: 2014-05-22
Also published as: GB201220478D0

Abstract

A compact broadly tunable visible spectrum laser source which can provide a continuous wave (CW) output or a pulsed output with a picosecond pulse length for use in fluorescence microscopy. The laser source has a quantum-dot external-cavity diode laser optically coupled with a periodically poled nonlinear crystal waveguide in which, two or more tunable wavelengths may be generated substantially simultaneously in the visible spectral range. By using an one or more additional diffraction grating and a beam splitter in the external cavity setup to provide the coverage of a broad visible spectral range. Multi-wavelength emission is enabled using a coupled resonator with a beam splitter (6) and at least two diffraction gratings (3,5) and a Quantum-Dot active media (9) to realize the external cavity laser diode. The broad tunable emission is frequency- converted into the visible with a periodically poled waveguide SHG PPKTP (19).

Description

Tunable Laser for Fluorescence Microscopy Introduction The present invention relates to a laser for Fluorescence-based microscopy in particular confocal microscopy.

Background Fluorescence-based microscopy techniques, rely on the use of at least one light source, to excite (or photoactivate, photobleach, photoswitch, etc.) in the linear regime, the different fluorescent markers. This type of microscopy uses fluorescence (and phosphorescence) to generate an image through an imaging system (normally a microscope). Spectral filtering is used to ensure that illumination (or excitation) light from the laser is not mixed with the fluorescent emitted light when detecting it in the imaging system. The techniques that exploit fluorescence in microscopy includes (but are not limited to) epifluorescence, confocal (including Fluorescence recovery after photobleaching (FRAP), Fluorescence life time imaging (FLIM), etc), total internal reflection fluorescence (TIRF), light-sheet based techniques [such as

Selective plane illumination microscopy (SPIM), digitally scanned laser light (and all its variants using Gaussian, Bessel, Airy beams, etc)] and super resolution imaging techniques [such as stochastic optical reconstruction microscopy (STORM),

Photoactivation light microscopy (PALM), Stimulated emission depletion (STED), ground state depletion (GSD), Structured Illumination Microscopy (SIM) and all their respective variants].

For fluorescence microscopy, three main types of light sources are used: i) fluorescent lamps (such as xenon arc lamp or mercury-vapor lamp), ii) high-power LEDs and iii) lasers. Fluorescent lamps or LEDs are commonly used for widefield epifluorescence microscopy. Lasers, due to their intrinsic high intensity generated light, can also be used for confocal, light-sheet based and super resolution imaging techniques.

l In the case of lasers, given their limited tunability, any practical implementation of these imaging techniques normally requires a number of them to cover the whole visible excitation spectrum. As an example, a commercial confocal microscope system normally includes two diode lasers (at 408 nm and 561 nm, respectively) an Argon ion laser (at 457nm, 477nm, 488nm and 514nm) and a He-Ne laser (at 633nm). Therefore, these imaging systems are still bulky, requiring lots of space to hold the several laser systems (not only due to the size of the laser heads, but also for their respective power supplies), are clumsy to operate and maintenance intensive. In terms of diode lasers, although compact, cost effective and reliable, they are not tunable and cannot deliver all the wavelengths across the whole visible spectral range. Imaging systems based on Optical parametric oscillators (OPOs) can give a large tenability with the required output power. However, OPOs are large, difficult to operate and require expensive pump sources. An interesting alternative to the use of a OPOs or a collection of laser sources in microscopy is given by the super continuum generation. Such laser sources can cover the whole visible spectral range. However, they are bulky (shoe-box size) still require a powerful pump laser and their output power is dependent, through spectral slicing, on the selected spectral bandwidth which is typically of a few milli-watts per nanometer. Summary of the Invention

In accordance with a first aspect of the invention there is provided a compact broadly tunable visible spectrum laser source which can provide a continuous wave CW output or a pulsed output with a picosecond pulse length for use in fluorescence microscopy, the system comprising a quantum-dot external-cavity diode laser optically coupled with a periodically poled nonlinear crystal waveguide in which, two or more tunable wavelengths may be generated substantially simultaneously in the visible spectral range by using an one or more additional diffraction grating and a beam splitter in the external cavity setup to provide the coverage of a broad visible spectral range.

Preferably, wavelengths in the blue to red part of the visible spectrum are generated. Preferably, the laser source is used as a single source, to excite or photoactivate the fluorescent markers used for a fluorescence-based microscopy technique in the linear regime and across the whole visible range. Preferably, the quantum-dot external-cavity diode laser and the periodically poled nonlinear crystal waveguide use significant difference in the effective refractive indices of the high-order and low-order modes in multimode waveguides such that the difference between the effective refractive indices of the fundamental and SHG waves are shifted to match the period of poling in a very broad wavelength range limited mainly by the waveguide refractive index step.

Preferably, the tunability of the system is extended to cover the blue-to-red visible spectral range by adding another broadly-tunable laser diode with an emitting spectrum shifted to the short wavelength side and using the same crystal waveguide.

Advantageously, the nonlinear crystal waveguides provide an order-of-magnitude increase in the IR-to-visible conversion efficiency and enables a very different approach to second-harmonic generation (SHG) tunability in periodically-poled crystals, promising order-of-magnitude increase of wavelength range for SHG conversion.

Preferably, the laser source is widely tunability.

Preferably, the laser source is a quantum-dot external-cavity diode laser.

Preferably, the laser source has broad gain bandwidth.

Preferably, the laser source is a temperature insensitive. Advantageously the present invention replaces the plurality of lasers used in conventional confocal or LSFM to excite fluorescence with a single, robust, easy to use, cost effective, broadly tunable frequency doubled semiconductor laser.

Preferably, the laser source measures less than (10x10x10) cm in volume More preferably the laser will have a volume of around (5x5x5) cm. Optionally, the laser source is used in confocal microscopy.

Optionally, the laser source is used in light sheet fluorescence microscopy (LSFM).

Optionally, the laser source is used in selective plane illumination microscopy (SPIM).

Optionally, the laser source is used in stochastic optical reconstruction microscopy (STORM).

Furthermore, given the possibility of having two different output wavelengths the present invention may be used for other microscopy techniques for example super resolution imaging through the STORM technique. For this, the fluorescent markers should be activated using one wavelength and deactivated at a different one.

Iteration of this process allows numerous fluorophores to be localized and a super- resolution image to be constructed from the image data.

Brief Description of the Drawings

The present invention will now be described by way of example only, with reference to the accompanying drawings in which:

Figure 1 is a schematic diagram of an embodiment of a laser source in accordance with the present invention;

Figure 2 is a schematic diagram of a confocal microscope with a lasers source in accordance with the present invention which provides light at different wavelengths for exciting fluorescent markers; and Figure 3 is a schematic diagram of a SPIM/DSLM microscope with a lasers source in accordance with the present invention which provides light at different wavelengths for exciting fluorescent markers. Detailed Description of the Drawings

The present invention provides a compact broadly tunable visible laser source in CW and picosecond regimes for its use in fluorescence microscopy. Figure 1 shows a laser source for a fluorescence microscope 1 comprising gratings 3, 5, a beam splitter 6, lens 7, a gain chip 9, lens 1 1 , an optical isolator 13, a half wave plate 15, lens 17, a PPKTP waveguide, a lens 21 and a filter 23. In this embodiment of the invention the laser source is created by the frequency-doubling of the infrared light in a nonlinear crystal containing a waveguide. Nonlinear crystal waveguides that offer an order-of-magnitude increase in the IR-to-visible conversion efficiency also enable a very different approach to second-harmonic generation (SHG) tunability in periodically-poled crystals, promising order-of-magnitude increase of wavelength range for SHG conversion. In this respect, the wide tunability offered by the quantum-dot external-cavity diode lasers, due to the temperature insensitivity and the broad gain bandwidth, is very useful when creating a tunable visible source. A single laser device and a single crystal waveguide are used to provide a significant difference in the effective refractive indices of the high-order and low-order modes in multimode waveguides. To enable the difference between the effective refractive indices of the fundamental and SHG waves to be shifted to match the period of poling in a very broad wavelength range limited mainly by the waveguide refractive index step, Δη.

An all-room-temperature CW SHG source can be created with over 60 nm tunability from green to red (in the spectral region between 567.7 and 629.1 nm. in a periodically-poled potassium titanyl phosphate (PPKTP) waveguide pumped by a single broadly-tunable quantum dot laser diode.

In the present invention, the tunability of the system has been extended to cover the blue-to-red visible spectral range by adding another broadly-tunable laser diode with emitting spectrum shifted to the short wavelength side and using the same crystal waveguide.

Furthermore, by using a tunable mode-locked quantum dot external cavity laser as a pump source and a similar PPKTP waveguide, a compact all-room-temperature laser source broadly tunable in the visible spectral region in the picosecond regime was demonstrated. Two separately tunable wavelengths may be generated in the visible spectral range simultaneously by using an additional diffraction grating and a beam splitter in the external cavity setup as shown in figure 1.

Such compact laser system offering the coverage of a broad visible spectral range have may extend experimental options and replace a number of individual lasers used for microscopy. The present invention may be used as a single source, to excite (or photoactivate) in the linear regime and across the whole visible range, the different fluorescent markers used for any fluorescence-based (including

photoactivation, photobleaching, photoswitching, etc.) microscopy techniques while, at the same time, greatly reducing costs maintenance and size of the whole system, helping to significantly improve flexibility on the use for the most demanding biomedical applications.

Figure 2 shows an example of the use of a laser source in accordance with the present invention in a confocal light microscope.

In this embodiment, laser excitation light is generated throughout the visible spectrum using a laser source in accordance with the present invention. The excitation light is sent to a microscope through an excitation filter, a dichroic mirror (DM) a couple of xy scanning mirrors (SM) and the microscope objective (MO) that focuses this light inside the sample. The emitted fluorescent light is captured again and collimated by a microscope objective and de-scanned with the SM. Then, the fluorescent light is sent to the DM, focused through a pin hole and sent for detection (typically with a photomultiplier (PMT) or an avalanche detector). The pinhole rejects all the out-of-focused fluorescent light generated from the sample and a high resolution 2D image is formed by xy raster scanning the laser light. A 3D image is then formed by axially scanning either the sample or the MO lens. Figure 3 is a schematic diagram which shows a microscope 42, dichroic mirrors 43, collimating optics 45, an excitation filter 47 a SPIM 49. An illustration of the sample 50 is provided in the inset 51 which shows the excitation beam 53 and fluorescence light 55 emerging from the sample.

In the embodiment of the present invention shown with respect to figure 3, laser excitation light is generated throughout the visible spectrum using a laser source in accordance with the present invention in a light sheet fluorescence microscope which in this example is a selective plane illumination microscopy (SPIM).

SPIM provides an incident static sheet of excitation light on the sample plane using a cylindrical lens. Then, the fluorescence light emerging from this plane is collected through a microscope objective (MO) placed along the axis orthogonal to the excitation sheet. This uncoupling between the excitation and collection branches provides SPIM with: i) 2D optical sectioning capability in large fields of view that does not require point-scanning, and ii) decoupled resolution in the transversal and axial directions, determined by the collection numerical aperture (NA) and light-sheet thickness, respectively. Perhaps the most valuable benefit of this technique is the reduction of the photodamage to the sample, due to the restriction of the irradiation to the plane under observation. Since it also can provide rapid acquisition speed, SPIM is a powerful tool for in vivo time lapse studies, from single cells to whole organisms and tissues. An alternative implementation of SPIM relies on the generation of the light sheet by scanning in one direction a focused Gaussian beam. The scanning direction is perpendicular to the optical axis in a such way that a plane, equivalent to the SPIM, is formed. This technique is termed digital scanned (laser) light sheet microscopy (DSLM). There are several advantages to this implementation over SPIM: i) The full power of the excitation light is concentrated into the single scanned line providing better illumination efficiency and lower exposure times, ii) each line in the specimen is illuminated with the same intensity generating a homogenous light-sheet, where the height can be easily controlled with the amplitude of the scanning. Furthermore, DSLM can be easily implemented using Bessel beams (BB) instead of Gaussian beams. The self-healing properties of these beams allow alleviating the deleterious effect of scattering on scanned sheet microscopy. For example, this technique allows imaging 50% deeper inside human skin when compared with Gaussian beams. Finally, the DSLM technique can be implemented using two-photon excited fluorescence (TPEF) (2p-DSLM). for live imaging of fruit fly and zebra fish embryos. They show the advantages of using 2p-DSLM for imaging large highly scattering samples over the conventional 2p-LSM and 1 p-DSLM. Basically, the use of TPEF increases the penetration depth, improves background rejection and reduces phototoxic effects. In addition, the line scanning configuration improves the excitation efficiency and increase the tolerance to aberrations. These advantages allow deep, fast, non-phototoxic imaging of living organisms.

The present invention replaces the different lasers used in conventional confocal or LSFM to excite fluorescence with a single, robust, easy to use, cost effective, match box size, broadly tunable frequency doubled semiconductor laser.

Furthermore, given the possibility of having two different output wavelengths as shown in figure 1 , other microscopy techniques such as super resolution imaging through the STORM technique can use laser sources in accordance with the present invention. In that case, the fluorescent markers are activated using one wavelength and deactivated at a different one. Iteration of this process allows numerous fluorophores to be localized and a super-resolution image to be constructed from the image data. Improvements and modifications may be incorporated herein without deviating from the scope of the invention.

Claims

1. A compact broadly tunable visible spectrum laser source for providing a continuous wave (CW) output or a pulsed output with a picosecond pulse length for use in fluorescence microscopy, the laser source comprising:

a quantum-dot external-cavity diode laser optically coupled with a periodically poled nonlinear crystal waveguide in which, two or more tunable wavelengths are generated substantially simultaneously in the visible spectral range by using an one or more additional diffraction grating and a beam splitter in the external cavity setup to provide the coverage of a broad visible spectral range.

2. A laser source as claimed in claim 1 wherein a single source is used to excite or photoactivate fluorescent markers used for a fluorescence-based microscopy technique in the linear regime and across the whole visible range.

3. A laser source as claimed in claim 1 or claim 2 wherein, the quantum-dot external-cavity diode laser and the periodically poled nonlinear crystal waveguide use significant difference in the effective refractive indices of the high-order and low- order modes in multimode waveguides such that the difference between the effective refractive indices of the fundamental and SHG waves are shifted to match the period of poling in a very broad wavelength range limited mainly by the waveguide refractive index step.

4. A laser source as claimed in any preceding claim wherein the tunability of the system is extended to cover the blue-to-red visible spectral range by adding another broadly-tunable laser diode with an emitting spectrum shifted to the short wavelength side and using the same crystal waveguide.

5. A laser source as claimed in any preceding claim wherein, the laser source has broad gain bandwidth.

6. A laser source as claimed in any preceding claim wherein, the laser source is a temperature insensitive.

7. A laser source as claimed in any preceding claim wherein, the laser source measures less than (10x10x10)cm in volume

8. A laser source as claimed in any preceding claim wherein the laser source measures less than (5x5x5) cm in volume.

9. A laser source as claimed in any preceding claim wherein, the laser source is used in confocal microscopy.

10. A laser source as claimed in any of claims 1 to 8 wherein, the laser source is used in light sheet fluorescence microscopy (LSFM).

11. A laser source as claimed in any of claims 1 to 8 wherein, the laser source is used in selective plane illumination microscopy (SPIM).

12. A laser source as claimed in any of claims 1 to 8 wherein, the laser source is used in stochastic optical reconstruction microscopy (STORM).

13. A confocal microscope with a laser source as claimed in claims 1 to 8.

14. A light sheet fluorescence microscope (LSFM) with a laser source as claimed in claims 1 to 8.

15. A selective plane illumination microscope (SPIM) with a laser source as claimed in claims 1 to 8.

16. A stochastic optical reconstruction microscope (STORM)) with a laser source as claimed in claims 1 to 8.