US-12625356-B2 - Programmable multiple-point illuminator, confocal filter, confocal microscope and method to operate said confocal microscope

US12625356B2US 12625356 B2US12625356 B2US 12625356B2US-12625356-B2

Abstract

A programmable multiple-point illuminator for an optical microscope includes a light source and a spatial light modulator (SLM). The modulated light beam can scan across a sample placed under the microscope objective, the sample being provided with fluorophores. The SLM includes a first acousto-optic deflector and a second acousto-optic deflector, the first acousto-optic deflector having a first modulation plane and the second acousto-optic deflector having a second modulation plane, said two acousto-optic deflectors being arranged in cascade. The SLM includes a telescope relay to conjugate the first modulation plane with the second modulation plane. The illuminator includes an arbitrary waveform generator that can synthesize holograms, and is arranged to simultaneously inject a first such hologram into the first acousto-optic deflector and a second such hologram into the second acousto-optic deflector, in order for the SLM to modulate the light beam in response to said holograms.

Inventors

Mario Montes Usategui
Raul Bola Sampol
Estela Martin Badosa
Dorian TREPTOW

Assignees

UNIVERSITAT DE BARCELONA

Dates

Publication Date: 20260512
Application Date: 20231115
Priority Date: 20180702

Claims (8)

1 . A programmable multiple-point illuminator for an optical microscope, comprising: one continuous laser or a plurality of continuous wave lasers coupled into a single light beam, the lasers being configured to emit uninterrupted light beams; and a spatial light modulator to modulate a single or combined light beam from the continuous wave lasers to produce a modulated light beam configured to scan across a sample placed under an objective of the microscope, the sample being provided with fluorophores, the spatial light modulator comprising: a first acousto-optic deflector; and a second acousto-optic deflector, the first acousto-optic deflector having a first modulation plane, and the second acousto-optic deflector having a second modulation plane, said first and second acousto-optic deflectors being arranged in cascade to provide respective deflection in different directions, the spatial light modulator being configured to scan in two dimensions across the sample, the first and second modulation planes being conjugated with an input pupil of the objective of the microscope, the illuminator further comprising: a waveform generator configured to synthesize holograms and arranged to simultaneously inject a first such hologram into the first acousto-optic deflector and a second such hologram into the second acousto-optic deflector, for the spatial light modulator to modulate the single or combined light beam in response to said holograms, the objective of the microscope acting as a Fourier transform lens with respect to the conjugated modulation planes and focusing the single or combined light beam on a Fourier reconstruction plane that intersects the sample.
2 . The illuminator according to claim 1 , wherein the first and second acousto-optic deflectors are high-resolution, high deflection angle devices.
3 . A confocal filter for an optical microscope having the illuminator of claim 1 , comprising an imaging sensor provided with an electronic multi-pixel detector configured to enable real-time implementation of one digital pinhole around an image of any excited fluorescence location in the sample, further comprising a relay system to focus a fluorescent light emitted by the sample on the imaging sensor.
4 . A confocal microscope comprising the confocal filter of claim 3 , further comprising means to synchronize the waveform generator with the imaging sensor in order to correctly compose a confocal emission image.
5 . A method of operating the confocal microscope of claim 4 , comprising: making the single or combined continuous wave lasers to emit a first light beam of a certain diameter (D 1 ); expanding the first light beam into a second light beam having a prescribed diameter (D 2 ) to define an illumination window on the first acousto-optic deflector; injecting a first hologram into the first acousto-optic deflector to modulate the second light beam and transform it into a third light beam; imaging the third light beam on the second modulation plane; collimating the third light beam at zero modulation, so that the diameter (D 2 ) of the third light beam is that of the second light beam and defines an illumination window on the second acousto-optic deflector; injecting a second hologram into the second acousto-optic deflector to modulate the third light beam and transform it into a fourth light beam; imaging the fourth light beam on the input pupil of the microscope objective; focusing the fourth light beam on the Fourier reconstruction plane that intersects the sample; repeating the first and second holograms a number of cycles in a row before switching to another hologram, to prevent the first and second holograms to be only partially displayed when traveling through their respective illumination windows; and collecting the fluorescent light emitted by the sample and focusing said light on the imaging sensor.
6 . The method according to claim 5 , further comprising introducing a blank period before switching to another hologram.
7 . The illuminator according to claim 2 , wherein the high-resolution, high deflection angle devices provide higher than 500×500 resolvable spots.
8 . The method according to claim 5 , wherein no action is taken to center the first and second holograms.

Description

TECHNICAL FIELD The present disclosure is related to a programmable multiple-point illuminator for an optical microscope, and to a confocal filter to make said microscope a confocal one. The disclosure is also related to a method to operate the confocal microscope. The illuminator comprises a light source and a spatial light modulator for modulating a light beam from the light source, the modulated light beam being intended to scan across a sample placed under an objective of the microscope, the sample being normally provided with fluorophores. The expression ‘the sample is placed under the microscope objective’ means that the light beam is to be focused by the objective in the sample (that expression is not to be understood as meaning that the sample is always located below the objective). BACKGROUND ART Confocal microscopy is the reference technique for sample visualization in all fields of cellular biology, and it is widely acknowledged as one of the most important inventions ever made in optical microscopy. Confocal microscopes have enjoyed a tremendous explosion in popularity in recent years and most universities and scientific institutions worldwide, and increasingly many individual laboratories, own confocal microscopes. Confocal microscopes come essentially in two different modalities: single-point and multi-point scanning instruments. They are often used in combination with fluorescent tags (fluorescent molecules or fluorophores) that selectively label the structure of interest and that respond to an illumination laser by emitting light at a longer wavelength (Stokes shift). This wavelength shift permits an easy isolation of the excitation and emission optical trains by means of dichroic mirrors and filters. Single-point confocal microscopes are based on a single laser beam that progressively scans the sample on a point-by-point basis, which results in a high resolution, high contrast and optically-sectioned image after the light emitted by the sample is filtered out by a small pinhole aperture conjugated to the laser spot. Light emitted by excited fluorophores above and below the focused plane are intercepted by the pinhole and do not reach the detector, minimizing the light haze that plagues non-confocal microscopes when imaging thick samples. However, this point-by-point scanning method entails an obviously slow image acquisition, which is the main limitation of single-point confocals. Live samples such as cells have often to be fixed (i.e. killed) to obtain images without motion blur, as the instrument is unable to resolve the temporal dynamics of many cellular phenomena. Faster scanning has been therefore a crucial vector in the development of modern confocal microscopy. However, scanning using a single laser spot cannot be made arbitrarily fast: a high scan rate means that the laser spot can only illuminate any sample point during a very short exposure time, in the scale of microseconds. In order to compensate for this small excitation time, the laser power falling on the sample has to be increased, which very quickly saturates the fluorophores. Laser power increments above the saturation threshold do not result in an equivalent increment in the fluorescent emission rate, in such a way that the total amount of photons reaching the detector will decrease with decreasing exposure times, thus setting a limit to the scanning speed around a few frames per second. The only technical solution that enables a fast confocal operation is the use of several laser spots scanning the sample in parallel. Multi-point confocals have been developed in response to this need. They use thousands of laser beamlets to simultaneously scan the sample, thus being able to reach frames rates in the range of hundreds of frames per second. An added advantage of splitting the total power into many laser foci is that these instruments are considerably gentler with the biological samples ( 1/15 less damaging than a single point confocal in comparable conditions), minimizing photobleaching and phototoxicity. However, commercial implementations of the multi-point scanning principle are based on disks covered by arrays of tiny microlenses and pinholes (Nipkow disk) that spin at high speed, which make the system inflexible and optically inefficient. Indeed, spinning disk microscopes cannot scan arbitrary regions of interest in the sample and are matched to a single objective, usually with high magnification and high numerical aperture lenses. Also, a typical Nipkow disk has around 4% optical efficiency, requiring powerful excitation lasers, which are costly. A further difficulty is the reduced confocality arising from the crosstalk between pinholes, especially in thick samples (crosstalk is due to leakage on a detected signal from other optical signals). Spurious light excited by one laser spot can reach the detector (e.g. a camera) through neighbouring pinholes, resulting in a noticeably lower resolution image when compared wit