Bessel beam plane illumination microscope
A microscope has a light source for generating a light beam having a wavelength, λ, and beam-forming optics configured for receiving the light beam and generating a Bessel-like beam that is directed into a sample. The beam-forming optics include an excitation objective having an axis oriented in a first direction. Imaging optics are configured for receiving light from a position within the sample that is illuminated by the Bessel-like beam and for imaging the received light on a detector. The imaging optics include a detection objective having an axis oriented in a second direction that is non-parallel to the first direction. A detector is configured for detecting signal light received by the imaging optics, and an aperture mask is positioned.
1. A microscope comprising:
a light source for generating a light beam having a wavelength, λ;
beam-forming optics configured for receiving the light beam and generating a Bessel-like beam that is directed into a sample, the beam-forming optics including an excitation objective having a numerical aperture (NA) and an axis oriented in a first direction;
imaging optics configured for receiving signal light from a position within the sample that is illuminated by the Bessel-like beam and for imaging the received light on a detector, the imaging optics including a detection objective having an axis oriented in a second direction that is non-parallel to the first direction;
beam-translation optics configured for translating the position of the Bessel-like beam within the sample in discrete steps of more than or equal to λ/2NA to create a first excitation pattern of multiple Bessel-like beams having a spatial period, Λ, equal to the distance between beam positions of neighboring steps and configured to create N−1 additional excitation patterns that are spatially phase shifted from the first excitation pattern by (N−1)Λ/N;
a detector configured for detecting signal light received by the imaging optics, the detector having individual detection units; and
a processor configured to generate N images from the received signal light, each n image, for n=1 to N, being based on detected light due to excitation of the sample by the n th excitation pattern and further configured generate a final image of the sample through a linear combination of the N individual images.
2. The microscope of claim 1 , wherein generating the final image of the sample through a linear combination of the N individual images, includes combining the individual images according to
I
final
=
∑
n
=
1
N
I
n
exp
(
2
πⅈ
n
/
N
)
.
3. The microscope of claim 1 , wherein the signal light has a wavelength of λ/2.
4. The microscope of claim 1 , wherein the signal light is generated though a non-linear signal generation process.
5. The microscope of claim 1 , wherein N=3.
6. The microscope of claim 1 , wherein the step size is less than or equal to λ/NA.
7. The microscope of claim 1 , wherein N≧5.
8. The microscope of claim 7 , wherein the step size is greater than or equal to λ/NA.
9. The microscope of claim 1 , wherein the Bessel-like beam has a ratio of a Rayleigh length, z R to a minimum beam waist, w o , of more than 2πw o /λ and less than 100πw o /λ.
10. The microscope of claim 1 , wherein the Bessel-like beam has a non-zero ratio of a minimum numerical aperture to a maximum numerical aperture of less than 0.95.
11. The microscope of claim 1 , wherein the Bessel-like beam has a non-zero ratio of a minimum numerical aperture to a maximum numerical aperture of less than 0.90.
12. The microscope of claim 1 , wherein the Bessel-like beam has a minimum numerical aperture greater than zero and a ratio of energy in a first side of the beam to energy in the central lobe of the beam of less than 0.5.
13. The microscope of claim 1 , further comprising a coverslip that supports the sample, wherein a normal direction to a plane of the sample that supports the sample forms and angle with the first direction of more than 10 degrees and less than 80 degrees.
14. The microscope of claim 13 , wherein the sample is less than ten micrometers thick.
15. The microscope of claim 1 , further comprising an annular mask in a path of the light beam configured to generate an annular ring of light from which the Bessel-like beam is formed.
16. A method comprising:
generating a Bessel-like beam having a wavelength, λ;
directing the Bessel-like beam through an excitation objective having a numerical aperture (NA) and an axis oriented in a first direction and into a sample;
receiving signal light through a detection objective having an axis oriented in a second direction that is non-parallel to the first direction from a position within the sample that is illuminated by the Bessel-like beam and imaging the received light onto a detector, wherein the detector includes individual detection units;
translating the position of the Bessel-like beam within the sample in discrete steps of more than or equal to λ/2NA to create a first excitation pattern of multiple Bessel-like beams having a spatial period, Λ, equal to the distance between beam positions of neighboring steps and configured to create N−1 additional excitation patterns that are spatially phase shifted from the first excitation pattern by (N−1)Λ/N;
generating N images from the received signal light, each n image, for n=1 to N, being based on detected light due to excitation of the sample by the n th excitation pattern; and
generating a final image of the sample through a linear combination of the N individual images.
17. The method of claim 16 , wherein generating the final image of the sample through a linear combination of the N individual images, includes combining the individual images according to
I
final
=
∑
n
=
1
N
I
n
exp
(
2
πⅈ
n
/
N
)
.
18. The method of claim 16 , wherein the signal light has a wavelength of λ/2.
19. The method of claim 16 , wherein the signal light is generated though a non-linear signal generation process.
20. The method of claim 16 , wherein N=3.
21. The method of claim 20 , wherein the step size is less than or equal to λ/NA.
22. The method of claim 16 , wherein N≧5.
23. The method of claim 22 , wherein the step size is greater than or equal to λ/NA.
24. The method of claim 16 , wherein the Bessel-like beam has a ratio of a Rayleigh length, z R to a minimum beam waist, w o , of more than 2πw o /λ and less than 100πw o /λ.
25. The method of claim 16 , wherein the Bessel-like beam has a non-zero ratio of a minimum numerical aperture to a maximum numerical aperture of less than 0.95.
26. The method of claim 16 , wherein the Bessel-like beam has a non-zero ratio of a minimum numerical aperture to a maximum numerical aperture of less than 0.90.
27. The method of claim 16 , wherein the Bessel-like beam has a minimum numerical aperture greater than zero and a ratio of energy in a first side of the beam to energy in the central lobe of the beam of less than 0.5.