JPWO2020007761A5

JPWO2020007761A5 -

Info

Publication number: JPWO2020007761A5
Application number: JP2020572880A
Authority: JP
Publication date: 2022-06-23
Anticipated expiration: 2039-07-01

Claims

It comprises a light source (1, 2) and a spatial light modulator that modulates the light beam from the light source, the modulated light beam traversing a sample placed under the objective lens (21) of the microscope. Intended to be scanned, the sample is a programmable multipoint illuminator for an optical microscope with a fluorophore.
The spatial light modulator comprises a first acoustic optical deflector (8) and a second acoustic optical deflector (9), and the first acoustic optical deflector has a first modulation plane (81). The second acoustic-optical deflector has a second modulation plane (91), wherein the two acoustic-optical deflectors are arranged in a cascade to provide their respective deflections in different directions. The spatial light modulator can be scanned in two dimensions across the sample, and the spatial light modulator is a telescope for coupling the first modulation plane to the second modulation plane. Further comprising a relay (10), the illuminator is configured to synthesize optics, and the spatial light modulator simultaneously modulates the light beam in response to the hologram. Arbitrary waveform generator (13) arranged to inject a hologram of 1 into the first acoustic-optical deflector and such a second hologram into the second acoustic-optical deflector. Further equipped with optics.

The arbitrary waveform generator (13) is a multi-channel generator, at least one channel injects the hologram into the first acoustic optical deflector (8), and at least another channel injects the hologram into the second acoustic. Arranged to inject into the optical deflector (9), the arbitrary waveform generator (13) compensates for differences in the acoustic path of the acoustic optical cell and the hologram for the first acoustic optical deflector is the first. The illuminator according to claim 1, further comprising means for advancing or delaying the hologram sequence of one channel with respect to the hologram sequence of the other channel in order to align with the hologram for the acoustic-optical deflector of 2.

The illuminator according to claim 2 , wherein the arbitrary waveform generator (13) is configured to synthesize a hologram of arbitrary complexity.

The illuminator according to claim 1 , further comprising a tip-tilted optomechanical mount for each acoustic-optical deflector (8, 9).

The light source comprises a pulsed laser (1, 2), and the illuminator further comprises a dispersion compensator (3) for avoiding or reducing large velocity dispersion within the acoustic-optical deflector (8, 9). , The illuminator according to claim 1 .

Further equipped with a scanning lens (17) placed after the spatial optical modulator to project the reconstruction of the desired illumination pattern onto the intermediate image plane (18), the scanning lens is the tube lens (19) of the microscope. 1 ), wherein the 4f optical system in which the modulation plane (81, 91) of the acoustic-optical deflector (8, 9) is coupled to the input pupil (20) of the microscope objective lens (21) is formed. Illuminator.

The image sensor (26) comprising an electronic multipixel detector configured to allow real-time implementation of one digital pinhole around an image of any excited fluorescence position in the sample. The cofocal filter for an optical microscope having the illuminator according to claim 1 , further comprising a relay system (25) for focusing the fluorescence emitted by the sample on the image sensor.

A means (16) for synchronizing the arbitrary waveform generator (13) with the image sensor (26) in order to correctly configure a confocal emission image with the illuminator according to any one of claims 1 to 6. A confocal microscope comprising the confocal filter according to claim 7.

The method for operating the confocal microscope according to claim 8.
A step of causing the light source (1, 2) to emit a first light beam having a specific diameter (D ₁ ).
A step of extending the first light beam to a second light beam having a predetermined diameter (D ₂ ) and defining an illumination window on the first acoustic-optical deflector (8).
A step of injecting a first hologram into the first acoustic-optical deflector to modulate the second light beam and convert it into a third light beam.
The step of imaging the third light beam on the second modulation plane (91),
The diameter (D ₂ ) of the third light beam is the diameter of the second light beam, and the second light beam is zero-modulated so as to define an illumination window on the second acoustic-optical deflector (9). Steps to collimate the light beam of 3 and
A step of injecting a second hologram into the second acoustic-optical deflector to modulate the third light beam and convert it into a fourth light beam.
A step of imaging the fourth light beam in the input pupil (20) of the microscope objective lens (21).
A step of focusing the fourth light beam on the reconstruction plane (22) intersecting the sample.
A step of collecting the fluorescence emitted from the sample and focusing the light on the image sensor (26).
How to include.

A step of calculating the first synthetic radio frequency signal by the first digital holography algorithm and calculating the second synthetic radio frequency signal by the second digital holography algorithm.
Using the arbitrary waveform generator (13), the first hologram injected into the first acoustic optical deflector (8) from the first calculated signal and the second calculation. 9. The method of claim 9, further comprising synthesizing the second hologram injected into the second acoustic-optical deflector (9) from the resulting signal.

With respect to the hologram sequence in the channel of the arbitrary waveform generator (13) that synthesizes the first or second hologram, and the hologram sequence in another channel of the arbitrary waveform generator that synthesizes the other hologram. 10. The method of claim 10, further comprising a step of advancing or delaying.

The method of claim 10, further comprising a step of repeating each hologram for several cycles in succession and a step of introducing a blank period before switching to another hologram.

10. The method of claim 10, further comprising decomposing the hologram into separable components.

10. The method of claim 10, further comprising synchronizing the arbitrary waveform generator (13) with the image sensor (26) in order to correctly configure a confocal image.

13. The method of claim 13 or 14, further comprising constructing a confocal image as the sum of several processed emission images excited by a mathematically separable illumination pattern.