The present application is a U.S. National Stage application of International PCT Application No. PCT/EP2013/001350 filed on May 7, 2013 which claims priority benefit of German Application No. DE 10 2012 010 207.0 filed on May 15, 2012, the contents of each are incorporated by reference in their entirety.
[1] Chen et al., Opt. Express 16, 18764 (2008)
[2] Wong et al., Appl. Opt. 48, 3237 (2009)
[3] WO2009/008838
[4] A. Leray and J. Mertz, Opt. Express 14, 10565 (2006)
[5] DE102011013613, DE102010013829
[6] EP500717 B2 Two-Photon Microscopy
[7] Sueda et al., Opt. Express 12, 3548 (2004)
[8] Chong et al., Biomedical Optics Express Vol. 1 No. 3
[9] Israel Rocha-Mendoza, Wolfgang Langbein, Peter Watson, and Paola Borri, “Differential coherent anti-Stokes Raman scattering microscopy with linearly chirped femtosecond laser pulses,” Opt. Lett. 34, 2258-2260 (2009)
The temporal focal modulation technique (FMM) is a method that is used in fluorescence microscopy, in order to be able to switch rapidly between different focusing fields. This feature is especially important when one would like to influence only the light in the focus of one objective lens. To this end, a method, with which a three dimensional imaging of optically thick samples with simultaneous discrimination of background light is possible, has already been demonstrated in the literature [1 to 4].
This method is based on the concept that a property (for example, the fluorescence), which is generated more or less in focus, is influenced in real time in such a way that this property (the fluorescence) is not modulated out of the focus.
To date this method has been based on the rapid switching of the optical phase in the pupil of an objective lens. To date it has been demonstrated, above all, that the phase is shifted in two half pupils.
Similar to the half pupil switching, described above, it may also be advantageous to use the switching between the optical phases of other partial beams in the pupil. Moreover, it is advantageously possible to use not only the phases, but also, in general, the field mode switching, in order to generate a temporal modulation of the emission from the focal volume. In this case the radiation is not modulated in real time outside the focal volume.
The figure shows one example for two focusing states in the cross section. In this case the switching can take place between these two focusing states. It is easily discernible that one of the states has a zero value on the optical axis. Out of focus, the zero value disappears.
In addition to switching phases solely in the pupil, switching of the polarization in combination with a phase plate has also been proposed [5] as an additional option, in order to modulate the radiation coming from the focal volume.
All of the possible focal modulation techniques benefit from modulation frequencies of several MHz. Therefore, in principle, they lend themselves well to an advantageous use in laser scanning microscopes (LSM), in order to increase the penetration depth without sacrificing the scan rate. The scan rate can be further increased by parallelization using multi-spot microscopy. However, a slower modulation is still possible and may be adjusted accordingly.
Owing to the advantageous high modulation frequencies it is possible to consider essentially rapid switching opto-electronic elements, such as acousto-optical modulators [AOMs] and electro-optical modulators [EOMs]. These components are capable of switching the polarization very rapidly in real time, as a result of which polarization-dependent phase deviations can be introduced in different spatial areas, preferably in an objective lens pupil (
Therefore, it is possible to achieve a switch-over of the phases by switching the polarization state. These solutions make it possible to switch a property that ultimately more or less affects the field in focus and, as a result, the focusing field is modulated, while the essential portions that are out of focus are not significantly modulated.
The phase plates, depicted in
Glass is listed here only as an example. Amorphous quartz (Suprasil) or other non-double refractive materials can also be used.
In
It should be added that in order to achieve the desired change in the polarization, it is also possible to use, for example, a nematic crystal, which, however, responds more slowly, or to use structures, which generate a different polarization by way of a path splitting; and this path changes rapidly, for example, by means of an AOM/AOTF (acousto-optic tunable filter).
In general, the polarization state can be varied in real time, for example, in either sinusoidal or rectangular waveform or some other advantageous waveform. This measure makes it possible to have a varied effect over time on the transition between the spatial field distributions in the focus of the microscope.
The advantages of the focal modulation microscopy (FMM) can be summarized as follows:
an increase in the depth of penetration by reducing the scattered light that is out of focus
optical cutting—reducing the fluorescence volume
an increase in the optical resolution
At this point it is clearly evident, in particular with reference to
According to the invention, it has now been recognized that surprisingly the intrinsic laser modulation frequency of the pulsed light source can be used to generate the focal modulation and, in particular, in an inexpensive way and without a great deal of technical complexity.
If a pulsed light source is used, then the light source itself already contributes advantageously to the modulation frequency so that the advantage to be gained is that it is possible to dispense with the fast switches described above.
Therefore, the technical complexity can be significantly reduced, if only passive elements are used, according to the invention, for the phase modulation.
Similar methods have already been described for other optical purposes. For example, the prior article [9] (see above) discloses a passive arrangement for the modulation in the coherent anti-Stokes Raman scattering [CARS] microspectroscopy. In this case, however, the optical phase is not modulated, but rather a time interval, which varies over time, is introduced between two laser pulses each in such a way that said time interval provides by changing to different contributions in the resulting CARS signal.
It has now been recognized in accordance with the invention that a modulation of the optical phase is possible with just the passive elements alone, wherein the modulation can be in the MHz range, if the laser has a suitably high repetition rate.
The invention is described in detail below with reference to the schematic drawings, in which
In
In the beam path, which is coming from the sample and contains the sample light, means (for example, main beam splitter), which are not shown in this embodiment, are provided in the direction of detection downstream of the objective lens, in order to deflect the sample light in the direction of a detection, which is not shown herein.
Furthermore, the scanning means are not shown; these scanning means are disposed upstream of the objective lens on the illumination side, in order to guide an illuminating beam, which is focused by means of O, a beam distribution or an illuminating line in one or two directions over the sample, through which the sample light passes again backwards in the direction of detection before being blocked in the direction of detection to the descanned detection.
A lambda/2 plate Wp is disposed downstream of the laser L, which emits short pulses Pu. The illuminating light L reaches a first polarizing beam splitter Pst1 and is split into two pulsed components Pu1 and Pu2. The one transmitted component Pu1 passes through a phase plate PP of the pulse train Pu1, said phase plate being disposed preferably in the objective lens pupil or disposed in a plane conjugate to said objective lens pupil, and a second polarizing beam splitter Pst2 in the direction of the sample through the objective lens O.
The second pulse train Pu2 is reflected at S1 and travels by way of the deflecting mirrors S2 and S2 with a time delay with respect to Pu1 to the second polarizing beam splitter Pst2 and is also deflected at said second polarizing beam splitter in the direction of the sample through the objective lens O.
Thus,
(1) The linear polarization of the light pulses from the laser is polarized by means of a lambda/2 or lambda/4 plate in such a way that the polarizing beam splitter Pst1 divides the incoming pulse train into two partial beams or more specifically into two pulse trains that are identical in terms of energy. These two partial beams differ only in the polarization.
(2) However, one of these pulse trains also passes through the phase plate PP in the pupil (
(3) Moreover, the two pulse trains are displaced in time in relation to each other, for example, in order not to undershoot, half of the repetition time, and if possible, half of the repetition time should not drop below the fluorescence lifetime, i.e., repetition rates of less than 100 MHz are recommended.
The time delays are caused by the different light paths in the partial beam paths, which can also be adjusted preferably by moving the mirrors S1, S2 perpendicular to the optical axis.
After the polarizing beam splitter PST2 there is, in chronological order, as shown, a change between the pulses PU2 and the pulse trains, which are influenced by PP, with partially delayed phase position in accordance with the FMM principle.
These pulse trains meet in an alternating manner in the objective lens focus (in the sample).
The net result of the principle, depicted in
The embodiments, described in
Pulsed lasers that can also be used include a continuous wave (CW) laser, which is switched on and off by means of appropriate rapid switching means.
For the sake completeness it should be noted that the configuration, shown in
At variance with the literature [8], the AOMs are used in this case for the pulsed on and off switching of the laser beam in the two light paths.
In this way the pulse sequence, shown in
The amplitude modulation in the two light paths can be designed, for example, sinusoidal or rectangular, as a function of time.
It should be noted that the method, shown in the
This method can be combined advantageously with other methods: point scanning high resolution method: stimulated emission depletion (STED) microscopy, reversible saturable optical (fluorescence) transitions (RESOLFT) microscopy, saturated patterned excitation microscopy (SPEM), structured illumination microscopy (SIM), and FLIM—fluorescence lifetime imaging microscopy.
While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.
Number | Date | Country | Kind |
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10 2012 010 207 | May 2012 | DE | national |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2013/001350 | 5/7/2013 | WO | 00 |
Publishing Document | Publishing Date | Country | Kind |
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WO2013/170940 | 11/21/2013 | WO | A |
Number | Name | Date | Kind |
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20100014156 | Iketaki | Jan 2010 | A1 |
20100328674 | Berguiga | Dec 2010 | A1 |
20120268812 | Anhut | Oct 2012 | A1 |
Number | Date | Country |
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10 2004 034 959 | Feb 2006 | DE |
10 2004 034 998 | Feb 2006 | DE |
10 2010 013 829 | Sep 2011 | DE |
10 2010 013 830 | Sep 2011 | DE |
10 2011 013 613 | Apr 2012 | DE |
10 2011 013613 | Apr 2012 | DE |
0 500 717 | Nov 2003 | EP |
2003 270551 | Sep 2003 | JP |
2004 317646 | Nov 2004 | JP |
2006058477 | Mar 2006 | JP |
2011 028208 | Feb 2011 | JP |
2011028208 | Feb 2011 | JP |
2011 197609 | Oct 2011 | JP |
2011197609 | Oct 2011 | JP |
2012 078802 | Apr 2012 | JP |
WO 2009008 838 | Jan 2009 | WO |
WO 2012041502 | Apr 2012 | WO |
Entry |
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Notification of Transmittal of Translation of the International Preliminary report on Patentability (Chapter I or Chapter II). |
Wong, Chee Howe, et al.; “Simple spatial phase modulator for focal modulation microscopy”; Applied Optics 2009; 48(17):3237-3242. |
Rocha-Mendoza, I., et al.;“Differential coherent anti-Stokes Raman scattering microscopy with linearly chirped femtosecond laser pulses”; Optics Letters 2009; 34(15):2258-2260. |
Chen, Nanguang, et al.; “Focal modulation microscopy”; Optics Express 2008; 16(23):18764-18769. |
Chong, Shau Poh, et al.; “High-speed focal modulation microscopy using acousto-optical modulators”; Biomedical Optics Express 2010; 1(3):1026-1033. |
Leray, Aymeric, et al., “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging”, Optics Express 2006; 14(22):10565-10573. |
Sueda, K., et al., “Laguerre-Gaussian beam generated with a multilevel spiral phase plate for high intensity laser pulses”, Optics Express 2004; 12(15):3548-3553. |
English translation of the Notification of Reasons for Rejection from the Japanese Patent Office. |
Number | Date | Country | |
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20150116807 A1 | Apr 2015 | US |