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System and method for providing enhanced background rejection in thick tissue with differential-aberration two-photon microscopy

US20090084980 A1. Filed on Sept. 29, 2008, published on April 2, 2009. view in google patents

A system for providing enhanced background rejection in thick tissue contains an aberrating element for introducing controllable extraneous spatial aberrations in an excitation beam path; at least one mirror capable of directing received laser pulses to the aberrating element; an objective; a beam scanner imaged onto a back aperture of the objective so that the beam scanner steers beam focus within the thick tissue; and a detector for recording signals produced by the tissue. An associated method comprises the steps of acquiring two-photon excited fluorescence of thick tissue without extraneous aberrations; introducing an extraneous aberration pattern in an excitation beam path; acquiring two-photon excited fluorescence of the thick tissue having the introduced extraneous aberration pattern; and subtracting the two-photon excited fluorescence with extraneous aberrations from the acquired standard two-photon excited fluorescence of the thick tissue without extraneous aberrations.

Schematic illustrating the general principle of differential-aberration two-photon excited fluorescence (DA-TPEF).

FIG. 1A, FIG. 1B, and FIG. 1C are schematic diagrams illustrating the general principle of differential-aberration two-photon excited fluorescence (DA-TPEF), in accordance with the present system and method. Specifically, when focusing a laser beam 10 into thick tissue 12, such as by use of an objective 14, as described hereinafter with regard to FIG. 2, power of the laser beam 10 becomes largely depleted by scattering before the laser attains the beam focus. As shown by FIG. 1A, TPEF background can then arise from out-of-focus ballistic excitation 16, particularly near the sample (also referred to as a medium) 12 surface, or from “snakelike” scattered excitation 18 near the beam focus 22, both of which can produce background fluorescence that is non-negligible compared to in-focus TPEF signal 20 fluorescence.

As shown by FIG. 1B, in accordance with the present invention, the introduction of extraneous aberrations 24 in an illumination pupil, or objective 14, leads to a spreading of the ballistic excitation profile that is more pronounced near the beam focus 22 than away from the beam focus 22, thereby preferentially quenching the in-focus TPEF signal 20 and associated focal spot, while leaving the out-of-focus TPEF background relatively unchanged. As shown herein, in accordance with the present invention, the subtraction of a TPEF image with extraneous aberrations, as shown by FIG. 1B from an image without extraneous aberrations, as shown by FIG. 1A, results in an enhanced out-of-focus TPEF background rejection, as shown by FIG. 1C. This process is described in more detail below.

An example of a system that may be used in accordance with the present invention.

An example of a system 50 that may be used in accordance with the present invention, is illustrated by FIG. 2. In accordance with one exemplary embodiment of the invention, the system 50 contains a laser source 52, a computer 54, a two-photon excited fluorence microscope with the addition of a differential aberrating element (DA-TPEF) 60. The laser source 52 may be one of many categories of pulse laser sources used for providing non-linear interactions in matter. As an example, the laser source 52 may be a Titanium-sapphire (Ti:Sa) laser. Of course, the laser source 52 is not intended to be limited to a Ti:Sa laser.

A pulse laser of the laser source 52 is directed to the DA-TPEF microscope 60. As is shown by FIG. 2, the microscope 60 contains a first mirror 62 and a second mirror 64 for directing laser pulses from the laser source 52 to a deformable mirror 66. It should be noted that the microscope 60 may have more or fewer mirrors for directing laser pulses from the laser source 52 to the deformable mirror 66. It should also be noted that the deformable mirror 66 is only an example of a switchable aberrating element. Other types of switchable aberrating elements could also be implemented.

The deformable mirror 66 is located in an excitation beam path of the microscope 60. The deformable mirror 66 is, in turn, imaged onto a beam scanner 68. The beam scanner 68 is imaged onto a back aperture of the objective 14, so that, ultimately, the beam scanner 68 steers the beam focus within the sample of interest. The deformable mirror 66 is therefore located in a conjugate plane of the objective 14 back aperture, meaning that height deformations in the deformable mirror 66 effectively translate to phase deformations (aberrations) in the pupil function governing the excitation beam focus. The deformable mirror 66 may provide one or more of many aberration profiles, such as, but not limited to, quadrant or spiral phase aberration profiles. Such profiles would be caused by providing different voltage patterns to the deformable mirror 66.

An example of a deformable mirror that may be used in accordance with the present system and method includes, but is not limited to, a pDMS-Multi deformable mirror with a 3.5 maximum stroke, by Boston Micromachines Corporation, of Cambridge, Mass.

TPEF resulting from the laser source 52 is collected (typically through the microscope objective) and directed onto a detector, typically with the use a dichroic mirror 74. The detector records the signal produced by the sample and can be, but is not restricted to, a photomultiplier tube (PMT) 72. The dichroic mirror 74, if used, separates laser illumination from the signal produced by the sample. It should be noted that there is no communication between the deformable mirror 66 and the PMT 72, as a result, patterns are applied to the deformable mirror 66 that are independent of what is received by the PMT 72.

The microscope 60 also can contain a filter 74 that is capable of removing stray laser light prior to signal being received by the PMT 72.

The present system and method enables a separation of the excitation light from the laser source 52 into two components, namely, ballistic and scattered. These are respectively defined as the components of the excitation light that have not and have undergone scattering inside the sample 12. The power of the ballistic excitation in a scattering medium can be quite high near the medium surface, but decays exponentially as it progresses toward the beam focus 22. The power density of the ballistic excitation can therefore be locally peaked at both the sample surface and at the beam focus 22.

Defining FS to be the TPEF signal 20 generated by the ballistic excitation beam near its focus, FB to be the superficial background TPEF generated by the ballistic excitation far from focus (such as near the medium surface), and FNF to be the near-focus background TPEF generated by scattered excitation, which, for weakly scattering media, is largely confined to a blurred area around the beam focus 22, total TPEF in a sample can be expressed by the following equation 1.

\(F 0 =F S +F B +F NF\)  (Eq. 1)

As previously mentioned, when extraneous aberrations are introduced into the excitation beam path, these preferentially quench the signal TPEF (FS) while leaving the background TPEF (FB+FNF) relatively unaffected. That is, the total TPEF with extraneous aberrations is given by the following equation 2.

\(F \Phi \approx F B +F NF \)  (Eq. 2)

Subtracting equation 1 from equation 2 recovers the signal fluorescence, as illustrated by the following equation 3.

\( \Delta F=F 0 −F \Phi \approx F S \)  (Eq. 3)

The computer 54 of FIG. 2, performing functions in accordance with software stored therein, is capable of controlling types of aberrations introduced by the deformable mirror 66. Specifically, the computer 54 is capable of controlling voltage levels applied to the deformable mirror 66, thereby resulting in different types of aberrations, such as, but not limited to, quadrant and spiral phase aberrations. In addition, the computer 54 is capable of controlling timing of aberration introduction by the deformable mirror 66. Specifically, the computer 54 is capable of controlling when voltages are applied to the deformable mirror 66, thereby controlling when the deformable mirror 66 is activated.