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.