EP4479788A1 - Patterned light generating device for microscopy and apparatus thereof - Google Patents

Patterned light generating device for microscopy and apparatus thereof

Info

Publication number
EP4479788A1
EP4479788A1 EP23706109.8A EP23706109A EP4479788A1 EP 4479788 A1 EP4479788 A1 EP 4479788A1 EP 23706109 A EP23706109 A EP 23706109A EP 4479788 A1 EP4479788 A1 EP 4479788A1
Authority
EP
European Patent Office
Prior art keywords
waveguide
waveguides
angle
substrate
polarization maintaining
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23706109.8A
Other languages
German (de)
French (fr)
Inventor
Francesca BRAGHERI
Petra PAIÈ
Roberto Osellame
Matteo CALVARESE
Andrea Bassi
Federico SALA
Mark Neil
Hai GONG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ip2ipo Innovations Ltd
Consiglio Nazionale delle Richerche CNR
Politecnico di Milano
Original Assignee
Imperial College Innovations Ltd
Consiglio Nazionale delle Richerche CNR
Politecnico di Milano
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial College Innovations Ltd, Consiglio Nazionale delle Richerche CNR, Politecnico di Milano filed Critical Imperial College Innovations Ltd
Publication of EP4479788A1 publication Critical patent/EP4479788A1/en
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens

Definitions

  • the phase, and optionally the amplitude, of the waveguides are modulated to spatially translate and/or rotate the interference pattern.
  • An image of the sample is acquired for each translation and/ or rotation.
  • the acquired images of the sample are processed to reconstruct a super-resolved image of the sample.
  • the patterned light is used to illuminate a sample either on a glass slide or flowing in a microchannel.
  • a coherent SIM pattern is commonly achieved by interference of two or more laser beams on the sample.
  • a sinusoidal pattern can be generated by focusing two coherent laser beams into the pupil of the microscope. By changing the relative phase of beams, the pattern moves (spatially translates) through the field of view.
  • a diffraction grating created the two coherent beams, and a mechanical translator (and rotator) was used to change the pattern spatial phase and angle.
  • the beam is shaped with a spatial light modulator. This approach has the advantage to allow the creation of complex modulation patterns, with high phase precision; moreover the spatial light modulator can be further used to control the microscope aberrations.
  • the object of the present invention is, therefore, to provide a patterned light generating device for structured illumination microscopy and apparatus thereof that would be able to generate the translations and/ or rotations of the illumination pattern without employing any mechanical movements of optical elements of the device itself.
  • At least a first optical waveguide and a second optical waveguide the waveguides being configured for transmitting a polarized light beam to their ends; at least one coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide;
  • a substrate having one input port and at least a first output port and a second output port, said substrate being configured for housing the waveguides and the at least one coupler; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; and at least a first output polarization maintaining fibre fixed at the first output port and configured for being coupled with the first waveguide in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre and a second output polarization maintaining fibre fixed at the second output port and configured for being coupled with the second waveguide in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre; at least one phase shifter configured for creating a phase difference between the first waveguide and the second waveguide, the phase shifter being housed in the substrate; and a glass ferrule configured to being coupled with the output polarization fibres, the output polarization fibres ending into the ferrule in such a way to be
  • the device can be then coupled with at least one lens positioned in front of glass ferrule and configured to generate by interference a structured illumination pattern translatable and/ or rotatable according the phase difference between the waveguides, being the outputs radiation coherent, within the coherence length of the source of the laser beam.
  • the pattern is the Fourier transform of the light beams transmitted by the output polarization maintaining fibres to their ends.
  • the shape of the generated pattern can vary. For example, if two waveguides are employed, a sinusoidal pattern is generated, while if the device comprises three waveguides an hexagonal pattern is produced.
  • the movements of the illumination pattern are generated by means of mechanical translators and/ or rotators, that change the pattern spatial phase and angle, or by means of a spatial light modulator that shapes the light beam.
  • the relative phases and the amplitude of the waveguides are controlled by a voltage applied to the shifters and, optionally, to the amplitude modulator.
  • Thermal shifters are preferably used for controlling the phase and no mechanical components for generate movements of optical components are present.
  • SIM can be used also in combination with light sheet fluorescence microscopy (LSF), in order to obtain three-dimensional super-resolution images.
  • LSF light sheet fluorescence microscopy
  • the device of the present invention can be used also in a LSF microscope.
  • the latter will comprise: a laser beam source;
  • a device as described above, coupled with at least one lens positioned directly in front to the waveguides and configured to generate by interference the structured illumination pattern; a microfluidic chip comprising a channel configured for the housing of the sample, the sample being able to flow in a flowing direction; a camera;
  • the microfluidic chip is shaped as an equilateral triangular prism, each one of the output polarization fibres being configured to be coupled with one of the side face of said prism, the output polarization fibres ending into the channel in such a way to be oriented perpendicularly to the side faces of the prism.
  • microscopes listed and detailed above show how the present invention provides a single device that can be used in different microscopes and that, therefore, is highly versatile.
  • a further object of the present invention is to provide a patterned light generating device that guarantees a self-alignment of the light source.
  • the prior art devices are characterized by a bulk setup and, often, comprise mirrors that need of a fine regulation requiring an adjustment along the time.
  • the absence of a bulk setup, as the case of the device of the present invention, allows, instead a self-alignment without the necessity of regulation along the time.
  • Figure la is a lateral view of a first embodiment of the device of the present invention.
  • Figure lb is a front view of the first embodiment of the device of the present invention.
  • Figure 2a is a lateral view of a second embodiment of the device of the present invention.
  • Figure 2b is a front view of the second embodiment of the device of the present invention.
  • Figure 4a is a lateral view of a fourth embodiment of the device of the present invention.
  • Figure 5a is a lateral view of a second embodiment of the apparatus of the present invention.
  • Figure 12 shows two line plots of the pattern translated in the sample plane
  • a glass ferrule (30) configured to being coupled with the output polarization fibres (21, 22), the output polarization fibres (21, 22) ending into the ferrule (30) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (1, 2) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle
  • a glass substrate (207) having one input port, a first output port and a second output port, said substrate (207) being configured for the housing of the waveguides (201, 202) and the coupler (212); an input polarization maintaining fibre (210) fixed at the input port and configured for coupling the laser beam into the first waveguide (201); one phase shifter (211) housed on the substrate (207) and configured for creating a phase difference between the first waveguide (201) and the second waveguide (202).
  • a substrate (7) having one input port, a first output port, a second output port, and third output port, said substrate (7) being configured for housing the waveguides (1, 2, 3) and the couplers (12, 12); an input polarization maintaining fibre (10) fixed at the input port and configured for coupling the laser beam into the first waveguide (i); a first output polarization maintaining fibre (21) fixed at the first output port and configured for being coupled with the first waveguide (1) in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre (21); a second output polarization maintaining fibre (22) fixed at the second output port and configured for being coupled with the second waveguide (2) in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre (22); a third output polarization maintaining fibre (32) fixed at the third output port and configured for being coupled with the third waveguide (3) in such a way to transmit a light beam from the third waveguide to an end of the third output
  • a glass ferrule (30) configured to being coupled with the output polarization fibres (21, 22, 32), the output polarization fibres (21, 22, 32) ending into the ferrule (30) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (1, 2, 3) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
  • first optical waveguide 201
  • second optical waveguide 202
  • third optical waveguide 203
  • the waveguides 201, 202, 203 being configured for transmitting apolarized light beam to their ends
  • first coupler 212
  • second coupler 213
  • a glass substrate (207) having one input port, a first output port, a second output port, and a third output port, said substrate (207) being configured for the housing of the waveguides (201, 202, 202) and the couplers (212, 213); an input polarization maintaining fibre (210) fixed at the input port and configured for coupling the laser beam into the first waveguide (201);
  • a second phase shifter (213) housed on the substrate (207) and configured for creating a phase difference between the second waveguide (202) and the third waveguide (203).
  • the waveguides (201, 202, 203) are positioned on the substrate (207) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (201, 202, 203) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
  • the separation angle between the first and the second waveguide is equal to the separation angle between the second and the third waveguide and both are equal to 120°.
  • the device (300) generates a hexagonal pattern of structured illumination light, by the interference of the radiation emitted by the waveguides (201, 202, 23), being the outputs radiation coherent, within the coherence length of the source of the laser beam.
  • the generated hexagonal pattern can be translated according to the phase difference between the waveguides (201, 202, 203).
  • the hexagonal pattern is translated by varying the phase difference at least seven times.
  • a fifth embodiment of the device of present invention consists in a patterned light generating device for microscopy comprising:
  • first optical waveguide, a second optical waveguide, a third optical waveguide and a fourth optical waveguide the waveguides being configured for transmitting, polarized light beam to their ends; a first coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide; a second coupler for splitting a laser beam entering the device between the second waveguide and the third waveguide; a third coupler for splitting a laser beam entering the device between the third waveguide and the fourth waveguide;
  • a substrate having one input port, a first output port, a second output port, a third output port and a fourth output port, said substrate being configured for housing the waveguides and the couplers; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; a first output polarization maintaining fibre fixed at the first output port and configured for being coupled with the first waveguide in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre; a second output polarization maintaining fibre fixed at the second output port and configured for being coupled with the second waveguide in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre; a third output polarization maintaining fibre fixed at the third output port and configured for being coupled with the third waveguide in such a way to transmit a light beam from the third waveguide to an end of the third output polarization maintaining fibre; a fourth output polarization maintaining fibre fixed at the fourth output port and configured
  • a glass ferrule configured to being coupled with the output polarization fibres, the output polarization fibres ending into the ferrule in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
  • the separation angle between the first and the second output polarization fibre is 90 ° and is equal to the separation angle between the second and the third output polarization fibre and to the separation angle between the third and the fourth output polarization fibre.
  • the device generates a two dimensional pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres, being the outputs radiation coherent, within the coherence length of the source of the laser beam.
  • the generated pattern can be translated according to the phase difference between the waveguides.
  • the pattern is translated by varying the phase difference at least seven times.
  • a sixth embodiment of the device of the present invention consists in a patterned light generating device for microscopy comprising:
  • first optical waveguide, a second optical waveguide, a third optical waveguide and a fourth optical waveguide the waveguides, being configured for transmitting apolarized light beam to their ends; a first coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide; a second coupler for splitting a laser beam entering the device between the second waveguide and the third waveguide; a third coupler for splitting a laser beam entering the device between the third waveguide and the fourth waveguide;
  • a glass substrate having one input port, a first output port, a second output port, a third output port, and a fourth output port, said substrate being configured for the housing of the waveguides and the couplers; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; a first phase shifter housed on the substrate and configured for creating a phase difference between the first waveguide and the second waveguide; a second phase shifter housed on the substrate and configured for creating a phase difference between the second waveguide and the third waveguide; and a third phase shifter housed on the substrate and configured for creating a phase difference between the third waveguide and the fourth waveguide.
  • the waveguides are positioned on the substrate in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
  • the separation angle between the first and the second waveguide fibre is 90 ° and is equal to the separation angle between the second and the third waveguide and to the separation angle between the third and the fourth output waveguide.
  • the device generates a two-dimensional pattern of structured illumination light, by the interference of the radiation emitted by the waveguides, being the outputs radiation coherent, within the coherence length of the source of the laser beam.
  • the generated pattern can be translated according to the phase difference between the waveguides. In a preferred use of the sixth embodiment of the device, the pattern is translated by varying the phase difference at least seven times.
  • the sixth embodiment of the present invention can comprise also four output polarization fibres, each of the output polarization fibres being fixed at one output port and configured for being coupled with one waveguide in such a way to transmit a light beam from the one waveguide to an end of the output polarization fibre.
  • a seventh embodiment of the device of present invention consists in a patterned light generating device for microscopy comprising:
  • a sixth output polarization maintaining fibre fixed at the sixth output port and configured for being coupled with the sixth waveguide in such a way to transmit a light beam from the sixth waveguide to an end of the sixth output polarization maintaining fibre; a first phase shifter configured for creating a phase difference between the first waveguide and the second waveguide, the phase shifter being housed in the substrate; a second phase shifter configured for creating a phase difference between the second waveguide and the third waveguide, the second phase shifter being housed in the substrate; a third phase shifter configured for creating a phase difference between the third waveguide and the fourth waveguide, the third phase shifter being housed in the substrate; and a fourth phase shifter configured for creating a phase difference between the fourth waveguide and the fifth waveguide, the fourth phase shifter being housed in the substrate; and a fifth phase shifter configured for creating a phase difference between the fifth waveguide and the sixth waveguide, the fifth phase shifter being housed in the substrate;
  • the separation angle between the first and the second output polarization fibre is 60 ° and is equal to the separation angle between the second and the third output polarization fibre, to the separation angle between the third and the fourth output polarization fibre, to the separation angle between the fourth and the fifth output polarization fibre and to the separation angle between the fifth and the sixth output polarization fibre.
  • the amplitude modulator is configured for turning on two waveguides at a time.
  • the device When a couple of waveguides is turned on, the device generates a sinusoidal pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres coupled to the waveguides that turned on, being the outputs radiation coherent, within the coherence length of the source of the laser beam.
  • the sinusoidal pattern can be rotated. More particularly, three different sinusoidal patterns are generated, a first pattern coming from the first and second waveguide, a second pattern coming from the third and fourth waveguide, a third pattern coming from the fifth and sixth waveguide, each of said patterns being rotated one respect to the other.
  • the pattern generated by a given couple of waveguides can be also translated according to the phase difference between the waveguides.
  • the pattern generated for each couple of the waveguides is translated by varying the phase difference at least three times.
  • An eight embodiment of the device of the present invention consists in a patterned light generating device for microscopy comprising:
  • the device when a couple of waveguides is turned on, the device generates a sinusoidal pattern of structured illumination light, by the interference of the radiation emitted by the waveguides that are turned on, being the outputs radiation coherent, within the coherence length of the source of the laser beam.
  • the sinusoidal pattern can be rotated generating three different sinusoidal patterns.
  • the pattern generated by a given couple of waveguides can be also translated according to the phase difference between the waveguides. Since, the pattern generated for each couple of the waveguides, can be translated by varying the phase difference three times, nine different sinusoidal pattern can be obtained.
  • a first embodiment of the apparatus of the present invention consists in an apparatus (500) for microscopy comprising: a patterned light generating device (300) for microscopy according its third embodiment as described above; one lens (501) positioned in front of glass ferrule (30) of the device (300).
  • the lens (501) is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres (21, 22) to their ends.
  • a second embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures, comprising: a patterned light generating device (100) for microscopy according its first embodiment as described above and as shown in Figure la and lb; one lens positioned in front of glass ferrule (30) of the device (100).
  • the lens is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres to their ends.
  • the lens is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the waveguides to their ends.
  • An eight embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures, comprising: a patterned light generating device for microscopy according its eight embodiment as described above; and one lens positioned in front of the ends of the waveguides.
  • the lens is configured to generate by interference a structured illumination pattern translatable and rotatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the waveguides to their ends.
  • a first embodiment of the microscope (1000) of the present invention consists in an optical microscope comprising:
  • a third embodiment (3000) of the microscope of the present invention comprises: a laser beam source; - the eight or the ninth embodiment of the apparatus described above; a microfluidic chip (3010) comprising a channel (2002, 3005) configured for the housing of the sample, the sample being able to flow in a flowing direction (3011);
  • the channel (2002, 3005) is placed in front to the cylindrical lenses (601, 601') of the apparatus, the channel and the lenses being oriented in such a way that one primary light sheet for each lens is generated in the channel, each of said primary light sheets being perpendicular to the flowing direction (3011) and in such a way that a resulting patterned light sheet is generated by interference of the primary light sheets
  • the microfluidic chip (3010) is shaped as an equilateral triangular prism, each one of the output polarization fibres (3001, 3002, 3003) being configured to be coupled with one of the side face (3004, 3007, 3008) of said prism, the output polarization fibres (3001, 3002, 3003) ending into the channel (3005) in such a way to be oriented perpendicularly to the side faces (3004, 3007, 3008) of the prism.
  • the example described here relates to an implementation of the third embodiment of the device of the present invention and to its use in a fluorescence microscopy (second embodiment of the microscope).
  • the device is composed of two integrated couplers, which act as beam splitters and distribute laser light among the three waveguides, according to their coupling ratio.
  • the fact that the splitting is obtained with a cascade of integrated couplers guarantees a rapid phase shift dynamic.
  • Laser light is coupled into one waveguide through an input polarization maintaining fibre.
  • the first splitter is designed with a coupling ratio of 33%, one third of the initial power continues travelling in the first arm, while the remaining is guided to the second splitter.
  • the latter is designed as a 3dB coupler, with a coupling ratio of 50% .
  • the initial power is ideally equally distributed among the three output ports.
  • the power ratio at the three outputs was 33%, 36% and 30% .
  • the optical circuit is integrated with two phase shifters to control the relative phase of the guided beams.
  • the shifters create a phase difference pij between each couple of guided beams i, j.
  • thermal phase shifters are used. They are constituted of a thin metal resistor which is placed on the top of one arm of each beam splitter. An applied voltage across them, causes a power dissipation into the device substrate, and a consequent refractive index difference between the beam paths by thermo-optic effect. This gives a phase shift between two adjacent beams. Using two thermal phase shifters is enough to control the relative phases of the three beams.
  • the three outputs of the device are coupled and glued to a polarization maintaining fibre array, which ends into a specifically designed glass ferrule.
  • the three fibres tips are arranged in a triangular configuration and their optical axis is aligned before gluing, so to place them in azimuthal polarization configuration.
  • the fabrication of both the device and ferrule is based on femtosecond laser micromachining.
  • the three-points at the ferrule plane act as the source for the pattern. Thanks to the Fourier transforming properties of lenses, imaging the three coherent points in the back focal plane of a lens, produces a hexagonal intensity pattern in its front focal plane.
  • the pattern was initially characterized by placing the ferrule in front of a planoconvex 50 mm lens. This configuration creates an interference pattern which can be collected by a camera placed in the focal plane of the lens (Fig. 10a). As expected, the pattern shows hexagonal symmetry with clear zeros.
  • an image of the three point-sources has to be created in the microscope's pupil.
  • this is possible by adding a lens in front of the chip, that in combination with the tube lens (TL) of the microscope forms a magnified image of the point sources.
  • This lens was selected to be a 5X microscope objective (Mitutoyo, 0.14 NA).
  • a telescope was added at the output port of the microscope, composed of two 150 mm lenses.
  • the three point-sources were on a circle with radius 0.25 mm at the ferrule plane and the circle was magnified to a 1.25 mm radius at the back focal plane of the objective.
  • the pattern period at the sample plane depends on the illumination objective used to form the pattern, its value is affected both by the numerical aperture and the physical size of the pupil.
  • a filling factor FF can be defined, as the ratio between the radius of the three points in the back focal plane and the pupil radius. The higher this value is, the closer the pattern period gets to the theoretical maximum resolution of the objective.
  • FIG. 10b shows a fluorescent cell (MitoTracker from Invitrogen #1 slides), illuminated with the pattern. The hexagonal lattice is superimposed to the fluorescence signal. A controlled shift of the pattern on the cell was proved to be possible, by applying different consecutive voltages to the thermal shifters, and recording the corresponding images.
  • Figures Ila and 11b show a subsection of the cell, acquired with two different values of phase shifts. In the line plot of Fig. 12, the pattern translation in the sample plane is also visible (the pattern translation relative to Fig.
  • Ila is plotted in dashed line whereas the pattern translation relative to Fig. 11b is plotted in solid line) .
  • This measurement indicates that the chip allows the generation of a SIM pattern on the object plane of the microscope that can be spatially translated in the sample plane by changing the relative phase of the output beams.
  • the reconstruction algorithm reduces the background in the image, significantly improving its contrast, relative to the widefield case.

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Abstract

A patterned light generating device (100, 300) for microscopy comprising: at least a first optical waveguide (1) and a second optical waveguide (2), the waveguides (1, 2) being configured for transmitting a polarized light beam to their ends; at least one coupler (12) for splitting a laser beam entering the device (100, 300) between the first waveguide (1) and the second waveguide (2); a substrate (7), preferably of glass, having one input port and at least a first output port and a second output port, said substrate (7) being configured for housing the waveguides (1, 2) and the at least one coupler (12); an input polarization maintaining fibre (10) fixed at the input port and configured for coupling the laser beam into the first waveguide (1); and at least a first output polarization maintaining fibre (21) fixed at the first output port and configured for being coupled with the first waveguide (1) in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre (21) and a second output polarization maintaining fibre (22) fixed at the second output port and configured for being coupled with the second waveguide (2) in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre (22); at least one phase shifter (11) configured for creating a phase difference between the first waveguide (1) and the second waveguide (2), the phase shifter (11) being housed in the substrate (7); and a glass ferrule (30) configured to being coupled with the output polarization fibres (21, 22), the output polarization fibres (21, 22) ending into the ferrule (30) in such a way to be oriented one respect to the other by an angle equal to the round angle divided by the number of waveguides (1, 2).

Description

PATTERNED LIGHT GENERATING DEVICE FOR MICROSCOPY AND APPARATUS THEREOF
DESCRIPTION
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a patterned light generating device for structured illumination microscopy and apparatus thereof. The device consists in an optical circuit incorporating optical waveguides, couplers and phase shifters controlling the relative phases of the waveguides. Such a circuit can be placed in the illumination path of widefield microscope, optically conjugated with the microscope objective's pupil. Therefore, the present invention also relates to a structured illumination microscope comprising such optical circuit. The microscope objective creates a structured illumination pattern on the sample, by interference of the outputs of the device's waveguides. The pattern illuminates a sample in the object plane of the microscope and an image of the sample is captured by a widefield camera placed in primary or secondary image plane of the microscope. The phase, and optionally the amplitude, of the waveguides are modulated to spatially translate and/or rotate the interference pattern. An image of the sample is acquired for each translation and/ or rotation. The acquired images of the sample are processed to reconstruct a super-resolved image of the sample. The patterned light is used to illuminate a sample either on a glass slide or flowing in a microchannel.
STATE OF THE ART
Structured illumination microscopy (SIM) is an optical technique that is continuously growing in terms of rate of use for imaging at high resolution and beyond the classical diffraction limit. Initially used for obtaining optical sectioning, SIM is now commonly and commercially used for super-resolution microscopy. Structured illumination microscopy can double the resolution of a diffraction limited microscope, if it is implemented with linear excitation, where the fluorescence emission is proportional to the excitation intensity. It can further increase the resolution down to the tens of nanometer scale, if operated in a nonlinear regime. Recently, structured illumination has opened new possibilities in three-dimensional localization microscopy, light sheet microscopy and far field optical nanoscopy, among others.
The generation of a coherent SIM pattern is commonly achieved by interference of two or more laser beams on the sample. In an epifluorescence microscope, a sinusoidal pattern can be generated by focusing two coherent laser beams into the pupil of the microscope. By changing the relative phase of beams, the pattern moves (spatially translates) through the field of view. In its original implementation, a diffraction grating created the two coherent beams, and a mechanical translator (and rotator) was used to change the pattern spatial phase and angle. In recent SIM applications the beam is shaped with a spatial light modulator. This approach has the advantage to allow the creation of complex modulation patterns, with high phase precision; moreover the spatial light modulator can be further used to control the microscope aberrations. However, even in modular implementation, an optical setup for SIM pattern generation that requires mechanical movements of the optical components has the disadvantage to be cumbersome and to require day-by-day adjustments. Recently, a method to generate a SIM pattern, based on optical fibres and temporal phase modulation and without mechanical movements of the optical elements, has been proposed [Hinsdale, T. A., Stallinga, S., & Rieger, B. (2021). High-speed multicolor structured illumination microscopy using a hexagonal single mode fiber array. Biomedical Optics Express, 12(2), 1181-1194], However, even in this latter case, the optical layout consists in a bulk setup that still requires free space elements and is not fully integrated.
OBTECTS AND SUMMARY OF THE INVENTION
The object of the present invention is, therefore, to provide a patterned light generating device for structured illumination microscopy and apparatus thereof that would be able to generate the translations and/ or rotations of the illumination pattern without employing any mechanical movements of optical elements of the device itself.
This object is achieved by the present invention consisting in patterned light generating device for structured illumination microscopy comprising:
- at least a first optical waveguide and a second optical waveguide, the waveguides being configured for transmitting a polarized light beam to their ends; at least one coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide;
- a substrate having one input port and at least a first output port and a second output port, said substrate being configured for housing the waveguides and the at least one coupler; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; and at least a first output polarization maintaining fibre fixed at the first output port and configured for being coupled with the first waveguide in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre and a second output polarization maintaining fibre fixed at the second output port and configured for being coupled with the second waveguide in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre; at least one phase shifter configured for creating a phase difference between the first waveguide and the second waveguide, the phase shifter being housed in the substrate; and a glass ferrule configured to being coupled with the output polarization fibres, the output polarization fibres ending into the ferrule in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle. Optionally, the aforementioned device can also comprise at least one amplitude modulator configured for creating an amplitude difference between the at least two waveguides.
The device can be then coupled with at least one lens positioned in front of glass ferrule and configured to generate by interference a structured illumination pattern translatable and/ or rotatable according the phase difference between the waveguides, being the outputs radiation coherent, within the coherence length of the source of the laser beam. The pattern is the Fourier transform of the light beams transmitted by the output polarization maintaining fibres to their ends.
According to the number of the waveguides and, thus, of the output polarization fibres, the shape of the generated pattern can vary. For example, if two waveguides are employed, a sinusoidal pattern is generated, while if the device comprises three waveguides an hexagonal pattern is produced.
As described above, in the devices of the state of art, the movements of the illumination pattern are generated by means of mechanical translators and/ or rotators, that change the pattern spatial phase and angle, or by means of a spatial light modulator that shapes the light beam. Differently from these prior art solutions, in the device of the present invention, the relative phases and the amplitude of the waveguides are controlled by a voltage applied to the shifters and, optionally, to the amplitude modulator. Thermal shifters are preferably used for controlling the phase and no mechanical components for generate movements of optical components are present.
A second object of the present invention is to provide a patterned light generating device for structured illumination microscopy and apparatus thereof that would be extremely compact that it can be miniaturized.
This object is achieved by the present invention consisting in a patterned light generating device for structured illumination microscopy comprising: - at least a first optical waveguide and a second optical waveguide, the waveguides being configured for transmitting a polarized light beam to their ends; at least one coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide;
- a glass substrate having one input port and at least a first output port and a second output port, said substrate being configured for the housing of the waveguides and the at least one coupler; and an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; at least one phase shifter housed on the substrate and configured for creating a phase difference between the first waveguide and the second waveguide.
The waveguides are positioned on the substrate in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle
In this case, the device can be coupled with at least one lens positioned directly in front of the waveguides and configured to generate by interference a structured illumination pattern translatable and/or rotatable according the phase difference between the waveguides, being the outputs radiation coherent, within the coherence length of the source of the laser beam. The pattern is the Fourier transform of the light beams transmitted by the waveguides themselves to their ends.
The absence of the glass ferrule together with the other features mentioned above allows for a design that is completely integrated, extremely compact and potentially miniaturizable.
A third object of the present invention is to provide a patterned light generating device that would be highly versatile and suitable for all the typologies of microscope. To this aim, the device of the present invention can be mounted in the illumination path of a widefield microscope, optically conjugated with the microscope objective's pupil. Such a microscope can be, for example, an optical microscope comprising: a camera;
- a tube lens; a plane configured for housing a sample; a laser beam source;
- a beam splitter;
- a device, as described above, positioned together with the at least one lens, between the laser beam source and the beam splitter, the beam splitter being configured to reflect the pattern generated by the lens, generating a reflected pattern; a relay lens system configured for receiving the reflected pattern of the apparatus and for relaying the pattern to the tube lens, generating a relayed pattern; and an objective lens configured for receiving the relayed pattern and to transmit the relayed pattern to the plane.
The optical microscope can be also a fluorescence microscope. In this case, a fluorescence filter will be positioned between the camera and the beam splitter will be a dichroic mirror configured to transmit to the camera a fluorescence radiation emitted by the sample.
As known, SIM can be used also in combination with light sheet fluorescence microscopy (LSF), in order to obtain three-dimensional super-resolution images. The device of the present invention can be used also in a LSF microscope. The latter will comprise: a laser beam source;
- a device, as described above, coupled with at least one lens positioned directly in front to the waveguides and configured to generate by interference the structured illumination pattern; a microfluidic chip comprising a channel configured for the housing of the sample, the sample being able to flow in a flowing direction; a camera;
- a tube lens; an objective lens having an optical axis parallel to the flowing direction; and
- a bandpass filter between the objective lens and the tube lens.
The channel is placed in front to the lenses (601, 601') the channel and the lenses being oriented in such a way that one primary light sheet for each lens is generated in the channel, each of said primary light sheets being perpendicular to the flowing direction and in such a way that a resulting patterned light sheet is generated by interference of the primary light sheets.
The microfluidic chip is shaped as an equilateral triangular prism, each one of the output polarization fibres being configured to be coupled with one of the side face of said prism, the output polarization fibres ending into the channel in such a way to be oriented perpendicularly to the side faces of the prism.
The examples of microscopes listed and detailed above, show how the present invention provides a single device that can be used in different microscopes and that, therefore, is highly versatile.
Finally, a further object of the present invention is to provide a patterned light generating device that guarantees a self-alignment of the light source. The prior art devices are characterized by a bulk setup and, often, comprise mirrors that need of a fine regulation requiring an adjustment along the time. The absence of a bulk setup, as the case of the device of the present invention, allows, instead a self-alignment without the necessity of regulation along the time.
These and further objects will be made clearer by the following detailed description of some preferred embodiments of the present invention, to be understood by way of a non-limiting example of the more general concepts claimed.
BRIEF DESCRIPTION OF THE DRAWINGS The following description refers to the accompanying drawings, in which:
Figure la is a lateral view of a first embodiment of the device of the present invention;
Figure lb is a front view of the first embodiment of the device of the present invention;
Figure 2a is a lateral view of a second embodiment of the device of the present invention;
Figure 2b is a front view of the second embodiment of the device of the present invention;
Figure 3a is a lateral view of a third embodiment of the device of the present invention;
Figure 3b is a front view of the third embodiment of the device of the present invention;
Figure 4a is a lateral view of a fourth embodiment of the device of the present invention;
Figure 4b is a front view of the fourth embodiment of the device of the present invention;
Figure 5a is a lateral view of a second embodiment of the apparatus of the present invention;
Figure 5b is a lateral view of a fourth embodiment of the apparatus of the present invention;
Figure 6 is a schematic representation of a ninth embodiment of the apparatus of the present invention;
Figure 7 is a schematic representation of a tenth embodiment of the apparatus of the present invention;
Figure 8 is a three-dimensional view of a second embodiment of the microscope of the present invention;
Figure 9 is a schematic representation of a third embodiment of the microscope; Figure 10a shows the structured illumination pattern generated by the second embodiment of the apparatus of the present invention;
Figure 10b shows a fluorescent cell (MitoTracker from Invitrogen #1 slides), acquired by the second embodiment of the microscope of the present invention and illuminated with the structured illumination pattern generated by the second embodiment of the apparatus of the present invention;
Figure Ila shows a subsection of the cell shown in Fig, 10b, acquired with a first values of phase shifts;
Figure 11b shows a subsection of the cell shown in Fig. 10b, acquired with a second values of phase shifts;
Figure 12 shows two line plots of the pattern translated in the sample plane;
Figure 13a shows a widefield image of a mitochondrial membrane with a zoom relative to a filament of said membrane; and
Figure 13b shows a SIM reconstructed image of the same membrane showed in Figure 13a with a zoom relative to the same particular zoomed in Figure 13a.
DETAILED DESCRIPTION OF THE INVENTION
Referring to Figure la and lb, a first embodiment of the device (100) of the present invention consists in a patterned light generating device (100) for microscopy comprising:
- a first optical waveguide (1) and a second optical waveguide (2), the waveguides (1, 2) being configured for transmitting a polarized light beam to their ends; one coupler (12) for splitting a laser beam entering the device (100) between the first waveguide (1) and the second waveguide (2); - a substrate (7) having one input port, a first output port and a second output port, said substrate (7) being configured for housing the waveguides (1, 2) and the coupler (12); an input polarization maintaining fibre (10) fixed at the input port and configured for coupling the laser beam into the first waveguide (i); a first output polarization maintaining fibre (21) fixed at the first output port and configured for being coupled with the first waveguide (1) in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre (21); a second output polarization maintaining fibre (22) fixed at the second output port and configured for being coupled with the second waveguide (2) in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre (22); one phase shifter (11) configured for creating a phase difference between the first waveguide (1) and the second waveguide (2), the phase shifter (11) being housed in the substrate (7); and
- a glass ferrule (30) configured to being coupled with the output polarization fibres (21, 22), the output polarization fibres (21, 22) ending into the ferrule (30) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (1, 2) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle
Preferably, the separation angle is equal to the first angle. The device (100) generates a sinusoidal pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres (21, 22), being the outputs radiation coherent, within the coherence length of the source of the laser beam. The generated sinusoidal pattern can be translated according to the phase difference between the waveguides (1, 2).
Referring to Figure 2a, 2b and 6, a second embodiment of the device (200) of the present invention consists in a patterned light generating device (200) for optical microscopy comprising:
- a first optical waveguide (201) and a second optical waveguide (202), the waveguides (201, 202) being configured for transmitting a polarized light beam to their ends; one coupler (212) for splitting a laser beam entering the device between the first waveguide (201) and the second waveguide (202);
- a glass substrate (207) having one input port, a first output port and a second output port, said substrate (207) being configured for the housing of the waveguides (201, 202) and the coupler (212); an input polarization maintaining fibre (210) fixed at the input port and configured for coupling the laser beam into the first waveguide (201); one phase shifter (211) housed on the substrate (207) and configured for creating a phase difference between the first waveguide (201) and the second waveguide (202).
The waveguides (201, 202) are positioned on the substrate (207) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (201, 202) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle. Preferably, the separation angle is equal to the first angle. The device (200) generates a sinusoidal pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres (201, 202), being the outputs radiation coherent, within the coherence length of the source of the laser beam. The generated sinusoidal pattern can be translate according to the phase difference between the waveguides (201, 202).
Additionally, the second embodiment (200) of the present invention can comprise also two output polarization fibres (2003, 2004), each of the output polarization fibres (2003, 2004) being fixed at one output port and configured for being coupled with one waveguide (201, 202) in such a way to transmit a light beam from the one waveguide (201, 202) to an end of the output polarization fibre (2003, 2004).
Referring to Figure 3a and 3b, a third embodiment of the device (300) of the present invention consists in a patterned light generating device (300) for microscopy comprising:
- a first optical waveguide (1), a second optical waveguide (2) and a third waveguide (3), the waveguides (1, 2, 3) being configured for transmitting apolarized light beam to their ends; a first coupler (12) for splitting a laser beam entering the device (300) between the first waveguide (1) and the second waveguide (2); a second coupler (13) for splitting a laser beam entering the device (300) between the second waveguide (2) and the third waveguide (3);
- a substrate (7) having one input port, a first output port, a second output port, and third output port, said substrate (7) being configured for housing the waveguides (1, 2, 3) and the couplers (12, 12); an input polarization maintaining fibre (10) fixed at the input port and configured for coupling the laser beam into the first waveguide (i); a first output polarization maintaining fibre (21) fixed at the first output port and configured for being coupled with the first waveguide (1) in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre (21); a second output polarization maintaining fibre (22) fixed at the second output port and configured for being coupled with the second waveguide (2) in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre (22); a third output polarization maintaining fibre (32) fixed at the third output port and configured for being coupled with the third waveguide (3) in such a way to transmit a light beam from the third waveguide to an end of the third output polarization maintaining fibre (32); a first phase shifter (11) configured for creating a phase difference between the first waveguide (1) and the second waveguide (2), the phase shifter (11) being housed in the substrate (7); a second phase shifter (11') configured for creating a phase difference between the second waveguide (2) and the third waveguide (3), the second phase shifter (11') being housed in the substrate (7); and
- a glass ferrule (30) configured to being coupled with the output polarization fibres (21, 22, 32), the output polarization fibres (21, 22, 32) ending into the ferrule (30) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (1, 2, 3) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
Preferably, the separation angle between the first and the second output polarization fibre is equal to the separation angle between the second and the third output polarization fibre and both are equal to 120°. The device (300) generates a hexagonal pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres (21, 22, 32), being the outputs radiation coherent, within the coherence length of the source of the laser beam. The generated hexagonal pattern can be translated according to the phase difference between the waveguides (1, 2, 3). In a preferred use of the third embodiment of the device (300), the hexagonal pattern is translated by varying the phase difference at least seven times.
Referring to Figure 4a, 4b and 7, a fourth embodiment of the device (400) of the present invention consists in a patterned light generating device (400) for microscopy comprising:
- a first optical waveguide (201), a second optical waveguide (202), a third optical waveguide (203), the waveguides (201, 202, 203) being configured for transmitting apolarized light beam to their ends; a first coupler (212) for splitting a laser beam entering the device between the first waveguide (201) and the second waveguide (202); a second coupler (213) for splitting a laser beam entering the device between the second waveguide (202) and the third waveguide (203);
- a glass substrate (207) having one input port, a first output port, a second output port, and a third output port, said substrate (207) being configured for the housing of the waveguides (201, 202, 202) and the couplers (212, 213); an input polarization maintaining fibre (210) fixed at the input port and configured for coupling the laser beam into the first waveguide (201);
- a first phase shifter (211) housed on the substrate (207) and configured for creating a phase difference between the first waveguide (201) and the second waveguide (202); and
- a second phase shifter (213) housed on the substrate (207) and configured for creating a phase difference between the second waveguide (202) and the third waveguide (203). The waveguides (201, 202, 203) are positioned on the substrate (207) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (201, 202, 203) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
Preferably, the separation angle between the first and the second waveguide is equal to the separation angle between the second and the third waveguide and both are equal to 120°.
The device (300) generates a hexagonal pattern of structured illumination light, by the interference of the radiation emitted by the waveguides (201, 202, 23), being the outputs radiation coherent, within the coherence length of the source of the laser beam. The generated hexagonal pattern can be translated according to the phase difference between the waveguides (201, 202, 203). In a preferred use of the fourth embodiment of the device (400), the hexagonal pattern is translated by varying the phase difference at least seven times.
Additionally, the fourth embodiment (400) of the present invention can comprise also three output polarization fibres (3001, 3002, 3003), each of the output polarization fibres (3001, 3002, 3003) being fixed at one output port and configured for being coupled with one waveguide (201, 202, 203) in such a way to transmit a light beam from the one waveguide (201, 202, 203) to an end of the output polarization fibre (3001, 3002, 3003).
A fifth embodiment of the device of present invention, not shown in the figures consists in a patterned light generating device for microscopy comprising:
- a first optical waveguide, a second optical waveguide, a third optical waveguide and a fourth optical waveguide, the waveguides being configured for transmitting, polarized light beam to their ends; a first coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide; a second coupler for splitting a laser beam entering the device between the second waveguide and the third waveguide; a third coupler for splitting a laser beam entering the device between the third waveguide and the fourth waveguide;
- a substrate having one input port, a first output port, a second output port, a third output port and a fourth output port, said substrate being configured for housing the waveguides and the couplers; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; a first output polarization maintaining fibre fixed at the first output port and configured for being coupled with the first waveguide in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre; a second output polarization maintaining fibre fixed at the second output port and configured for being coupled with the second waveguide in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre; a third output polarization maintaining fibre fixed at the third output port and configured for being coupled with the third waveguide in such a way to transmit a light beam from the third waveguide to an end of the third output polarization maintaining fibre; a fourth output polarization maintaining fibre fixed at the fourth output port and configured for being coupled with the fourth waveguide in such a way to transmit a light beam from the fourth waveguide to an end of the fourth output polarization maintaining fibre; a first phase shifter configured for creating a phase difference between the first waveguide and the second waveguide, the phase shifter being housed in the substrate; a second phase shifter configured for creating a phase difference between the second waveguide and the third waveguide, the second phase shifter being housed in the substrate; a third phase shifter configured for creating a phase difference between the third waveguide and the fourth waveguide, the third phase shifter being housed in the substrate; and
- a glass ferrule configured to being coupled with the output polarization fibres, the output polarization fibres ending into the ferrule in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
Preferably, the separation angle between the first and the second output polarization fibre is 90 ° and is equal to the separation angle between the second and the third output polarization fibre and to the separation angle between the third and the fourth output polarization fibre.
The device generates a two dimensional pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres, being the outputs radiation coherent, within the coherence length of the source of the laser beam. The generated pattern can be translated according to the phase difference between the waveguides. In a preferred use of the fifth embodiment of the device, the pattern is translated by varying the phase difference at least seven times.
A sixth embodiment of the device of the present invention, not shown in the figures, consists in a patterned light generating device for microscopy comprising:
- a first optical waveguide, a second optical waveguide, a third optical waveguide and a fourth optical waveguide, the waveguides, being configured for transmitting apolarized light beam to their ends; a first coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide; a second coupler for splitting a laser beam entering the device between the second waveguide and the third waveguide; a third coupler for splitting a laser beam entering the device between the third waveguide and the fourth waveguide;
- a glass substrate having one input port, a first output port, a second output port, a third output port, and a fourth output port, said substrate being configured for the housing of the waveguides and the couplers; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; a first phase shifter housed on the substrate and configured for creating a phase difference between the first waveguide and the second waveguide; a second phase shifter housed on the substrate and configured for creating a phase difference between the second waveguide and the third waveguide; and a third phase shifter housed on the substrate and configured for creating a phase difference between the third waveguide and the fourth waveguide.
The waveguides are positioned on the substrate in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
Preferably, the separation angle between the first and the second waveguide fibre is 90 ° and is equal to the separation angle between the second and the third waveguide and to the separation angle between the third and the fourth output waveguide. The device generates a two-dimensional pattern of structured illumination light, by the interference of the radiation emitted by the waveguides, being the outputs radiation coherent, within the coherence length of the source of the laser beam. The generated pattern can be translated according to the phase difference between the waveguides. In a preferred use of the sixth embodiment of the device, the pattern is translated by varying the phase difference at least seven times.
Additionally, the sixth embodiment of the present invention can comprise also four output polarization fibres, each of the output polarization fibres being fixed at one output port and configured for being coupled with one waveguide in such a way to transmit a light beam from the one waveguide to an end of the output polarization fibre.
In all its embodiments described above, the device of the present invention can comprise also one or more amplitude modulators configured for creating an amplitude difference between two waveguides.
A seventh embodiment of the device of present invention, not shown in the figures consists in a patterned light generating device for microscopy comprising:
- a first optical waveguide, a second optical waveguide, a third optical waveguide, a fourth optical waveguide, a fifth optical waveguide and a sixth optical waveguide, the waveguides being configured for transmitting polarized light beam to their ends; a first coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide; a second coupler for splitting a laser beam entering the device between the second waveguide and the third waveguide; a third coupler for splitting a laser beam entering the device between the third waveguide and the fourth waveguide; a fourth coupler for splitting a laser beam entering the device between the fourth waveguide and the fifth waveguide; a fifth coupler for splitting a laser beam entering the device between the fifth waveguide and the sixth waveguide; - a substrate having one input port, a first output port, a second output port, a third output port, a fourth output port, a fifth output port and a sixth output port, said substrate being configured for housing the waveguides and the couplers; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; a first output polarization maintaining fibre fixed at the first output port and configured for being coupled with the first waveguide in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre; a second output polarization maintaining fibre fixed at the second output port and configured for being coupled with the second waveguide in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre; a third output polarization maintaining fibre fixed at the third output port and configured for being coupled with the third waveguide in such a way to transmit a light beam from the third waveguide to an end of the third output polarization maintaining fibre; a fourth output polarization maintaining fibre fixed at the fourth output port and configured for being coupled with the fourth waveguide in such a way to transmit a light beam from the fourth waveguide to an end of the fourth output polarization maintaining fibre; a fifth output polarization maintaining fibre fixed at the fifth output port and configured for being coupled with the fifth waveguide in such a way to transmit a light beam from the fifth waveguide to an end of the fifth output polarization maintaining fibre;
- a sixth output polarization maintaining fibre fixed at the sixth output port and configured for being coupled with the sixth waveguide in such a way to transmit a light beam from the sixth waveguide to an end of the sixth output polarization maintaining fibre; a first phase shifter configured for creating a phase difference between the first waveguide and the second waveguide, the phase shifter being housed in the substrate; a second phase shifter configured for creating a phase difference between the second waveguide and the third waveguide, the second phase shifter being housed in the substrate; a third phase shifter configured for creating a phase difference between the third waveguide and the fourth waveguide, the third phase shifter being housed in the substrate; and a fourth phase shifter configured for creating a phase difference between the fourth waveguide and the fifth waveguide, the fourth phase shifter being housed in the substrate; and a fifth phase shifter configured for creating a phase difference between the fifth waveguide and the sixth waveguide, the fifth phase shifter being housed in the substrate;
- a glass ferrule configured to being coupled with the output polarization fibres, the output polarization fibres ending into the ferrule in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle; and at least one amplitude modulator configured for creating an amplitude difference between the waveguides.
Preferably, the separation angle between the first and the second output polarization fibre is 60 ° and is equal to the separation angle between the second and the third output polarization fibre, to the separation angle between the third and the fourth output polarization fibre, to the separation angle between the fourth and the fifth output polarization fibre and to the separation angle between the fifth and the sixth output polarization fibre.
More particularly, the amplitude modulator is configured for turning on two waveguides at a time.
When a couple of waveguides is turned on, the device generates a sinusoidal pattern of structured illumination light, by the interference of the radiation emitted by the output polarization maintaining fibres coupled to the waveguides that turned on, being the outputs radiation coherent, within the coherence length of the source of the laser beam. By turning on two waveguides at a time, the sinusoidal pattern can be rotated. More particularly, three different sinusoidal patterns are generated, a first pattern coming from the first and second waveguide, a second pattern coming from the third and fourth waveguide, a third pattern coming from the fifth and sixth waveguide, each of said patterns being rotated one respect to the other.
Moreover, the pattern generated by a given couple of waveguides, can be also translated according to the phase difference between the waveguides. In a preferred use of the seventh embodiment of the device, the pattern generated for each couple of the waveguides, is translated by varying the phase difference at least three times. In conclusion, in the seventh embodiment of the device, nine different sinusoidal pattern can be obtained.
An eight embodiment of the device of the present invention, not shown in the figures, consists in a patterned light generating device for microscopy comprising:
- a first optical waveguide, a second optical waveguide, a third optical waveguide, a fourth optical waveguide, a fifth optical waveguide, a sixth optical waveguide, the waveguides, being configured for transmitting a, polarized light beam to their ends; a first coupler for splitting a laser beam entering the device between the first waveguide and the second waveguide; a second coupler for splitting a laser beam entering the device between the second waveguide and the third waveguide; a third coupler for splitting a laser beam entering the device between the third waveguide and the fourth waveguide; a fourth coupler for splitting a laser beam entering the device between the fourth waveguide and the fifth waveguide; a fifth coupler for splitting a laser beam entering the device between the fourth waveguide and the fifth waveguide;
- a glass substrate having one input port, a first output port, a second output port, a third output port, a fourth output port, a fifth output port and a sixth output port, said substrate being configured for the housing of the waveguides and the couplers; an input polarization maintaining fibre fixed at the input port and configured for coupling the laser beam into the first waveguide; a first phase shifter housed on the substrate and configured for creating a phase difference between the first waveguide and the second waveguide; a second phase shifter housed on the substrate and configured for creating a phase difference between the second waveguide and the third waveguide; a third phase shifter housed on the substrate and configured for creating a phase difference between the third waveguide and the fourth waveguide; a fourth phase shifter housed on the substrate and configured for creating a phase difference between the fourth waveguide and the fifth waveguide; a third phase shifter housed on the substrate and configured for creating a phase difference between the fifth waveguide and the sixth waveguide; and at least one amplitude modulator configured for creating an amplitude difference between the waveguides.
The waveguides are positioned on the substrate in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
Preferably, the separation angle between the first and the second waveguide is 60 ° and is equal to the separation angle between the second and the third waveguide, to the separation angle between the third and the fourth waveguide, to the separation angle between the fourth and the fifth waveguide and to the separation angle between the fifth and the sixth waveguide.
The amplitude modulator is configured for turning on two waveguides at a time.
As described above referring to the seventh embodiment of the device, when a couple of waveguides is turned on, the device generates a sinusoidal pattern of structured illumination light, by the interference of the radiation emitted by the waveguides that are turned on, being the outputs radiation coherent, within the coherence length of the source of the laser beam. By turning on two waveguides at a time, the sinusoidal pattern can be rotated generating three different sinusoidal patterns. The pattern generated by a given couple of waveguides, can be also translated according to the phase difference between the waveguides. Since, the pattern generated for each couple of the waveguides, can be translated by varying the phase difference three times, nine different sinusoidal pattern can be obtained.
Referring to Figure 3a, 3b and 5a a first embodiment of the apparatus of the present invention consists in an apparatus (500) for microscopy comprising: a patterned light generating device (300) for microscopy according its third embodiment as described above; one lens (501) positioned in front of glass ferrule (30) of the device (300). The lens (501) is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres (21, 22) to their ends.
A second embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures, comprising: a patterned light generating device (100) for microscopy according its first embodiment as described above and as shown in Figure la and lb; one lens positioned in front of glass ferrule (30) of the device (100).
The lens (501) is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres (21, 22) to their ends.
A third embodiment of the apparatus of the present invention consists in an apparatus, for microscopy, not shown in the figures, comprising: a patterned light generating device for microscopy according its fifth embodiment as described above; one lens positioned in front of glass ferrule of the device.
The lens is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres to their ends.
A fourth embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures: a patterned light generating device for microscopy according its seventh embodiment as described above; one lens positioned in front of glass ferrule of the device.
The lens is configured to generate by interference a structured illumination pattern translatable and rotatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres to their ends.
Referring to Figure 4a, 4b and 5b a fifth embodiment of the apparatus of the present invention consists in an apparatus (500') for microscopy comprising: a patterned light generating device (400) for microscopy according its fourth embodiment as described above; and one lens (501') positioned in front of the ends of the waveguides (201, 202, 203);
The lens (501') is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides (201, 202, 203), the pattern being the Fourier transform of the light beams transmitted by the waveguides (201, 202, 203) to their ends.
A sixth embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures, comprising: a patterned light generating device (200) for microscopy according its second embodiment as described above and as shown in Figure 2a and 2b; one lens positioned in front of the ends of the waveguides.
The lens (501') is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the waveguides (201,202) to their ends.
A seventh embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures, comprising: a patterned light generating device for microscopy according its sixth embodiment as described above; and one lens positioned in front of the ends of the waveguides.
The lens is configured to generate by interference a structured illumination pattern translatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the waveguides to their ends. An eight embodiment of the apparatus of the present invention consists in an apparatus for microscopy, not shown in the figures, comprising: a patterned light generating device for microscopy according its eight embodiment as described above; and one lens positioned in front of the ends of the waveguides.
The lens is configured to generate by interference a structured illumination pattern translatable and rotatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the waveguides to their ends.
Referring to Figure 2a, 2b and 6, a ninth embodiment of the apparatus of the present invention consist in an apparatus for light sheet microscopy, comprising:
- a patterned light generating device (200) according its second embodiment, said device (200) comprising: two output polarization fibres (2003, 2004), said output polarization fibres (2003, 2004) being fixed at one output port and configured for being coupled with one waveguide (201, 202) in such a way to transmit a light beam from the one waveguide (201, 202) to an end of the output polarization fibre (2003, 2004);
- one lens (601, 601') for each output polarization maintaining fibres (2003, 2004) positioned in front of the end of the output polarization maintaining fibres (2003, 2004), said lens (601, 601') being a cylindrical lens (601, 601').
Referring to Figure 4a, 4b and 7, a tenth embodiment of the apparatus of the present invention consist in an apparatus for light sheet microscopy, comprising:
- a patterned light generating device (400) according its fourth embodiment, said device (400) comprising: three output polarization fibres (3001, 3002, 3003), said output polarization fibres (3001, 3002, 3003) being fixed at one output port and configured for being coupled with one waveguide (201, 202, 203) in such a way to transmit a light beam from the one waveguide (201, 202, 203) to an end of the output polarization fibre (3001, 3002, 3003); - one lens (602, 602', 602") for each output polarization maintaining fibres (3001, 3002, 3003) positioned in front of the end of the output polarization maintaining fibres (3001, 3002, 3003), said lens (602, 602', 602") being a cylindrical lens (602, 602', 602").
Referring to the Figure 8, a first embodiment of the microscope (1000) of the present invention consists in an optical microscope comprising:
- a camera (1008);
- a tube lens (1003); a plane (1002) configured for housing a sample; a laser beam source;
- a beam splitter (1006); any of the embodiments from the first to the eight of the apparatus (500, 500') described above, said apparatus (500, 500') being positioned between the laser beam source and the beam splitter (1006), the beam splitter (1006) being configured to reflect the pattern generated by the lens (501, 501') of the apparatus (500, 500'), generating a reflected pattern; a relay lens system (1005, 1005') configured for receiving the reflected pattern of the apparatus and for relaying the pattern to the tube lens (1003), generating a relayed pattern; and an objective lens (1001) configured for receiving the relayed pattern and to transmit the relayed pattern to the plane (1002).
Referring to Figure 8, a second embodiment of the microscope of the present invention comprises all the components of the first embodiment and a fluorescence filter (1007) positioned between the camera (1008) and the beam splitter (1006). The latter, is a dichroic mirror (1006) configured to transmit to the camera (1008) a fluorescence radiation emitted by the sample.
Referring to Figure 9, a third embodiment (3000) of the microscope of the present invention comprises: a laser beam source; - the eight or the ninth embodiment of the apparatus described above; a microfluidic chip (3010) comprising a channel (2002, 3005) configured for the housing of the sample, the sample being able to flow in a flowing direction (3011);
- a camera (3008);
- a tube lens; an objective lens (3001) having an optical axis parallel to the flowing direction (3011); and
- a bandpass filter (3007) between the objective lens (3001) and the tube lens;
The channel (2002, 3005) is placed in front to the cylindrical lenses (601, 601') of the apparatus, the channel and the lenses being oriented in such a way that one primary light sheet for each lens is generated in the channel, each of said primary light sheets being perpendicular to the flowing direction (3011) and in such a way that a resulting patterned light sheet is generated by interference of the primary light sheets
The microfluidic chip (3010) is shaped as an equilateral triangular prism, each one of the output polarization fibres (3001, 3002, 3003) being configured to be coupled with one of the side face (3004, 3007, 3008) of said prism, the output polarization fibres (3001, 3002, 3003) ending into the channel (3005) in such a way to be oriented perpendicularly to the side faces (3004, 3007, 3008) of the prism.
EXAMPLE
The example described here relates to an implementation of the third embodiment of the device of the present invention and to its use in a fluorescence microscopy (second embodiment of the microscope).
The device is composed of two integrated couplers, which act as beam splitters and distribute laser light among the three waveguides, according to their coupling ratio. The fact that the splitting is obtained with a cascade of integrated couplers guarantees a rapid phase shift dynamic. Laser light is coupled into one waveguide through an input polarization maintaining fibre. The first splitter is designed with a coupling ratio of 33%, one third of the initial power continues travelling in the first arm, while the remaining is guided to the second splitter. The latter is designed as a 3dB coupler, with a coupling ratio of 50% . With such power splitting, the initial power is ideally equally distributed among the three output ports. In the implementation of the device of this example, the power ratio at the three outputs was 33%, 36% and 30% .
The optical circuit is integrated with two phase shifters to control the relative phase of the guided beams. The shifters create a phase difference pij between each couple of guided beams i, j. To obtain a phase shift control for the output beams, thermal phase shifters are used. They are constituted of a thin metal resistor which is placed on the top of one arm of each beam splitter. An applied voltage across them, causes a power dissipation into the device substrate, and a consequent refractive index difference between the beam paths by thermo-optic effect. This gives a phase shift between two adjacent beams. Using two thermal phase shifters is enough to control the relative phases of the three beams.
The three outputs of the device are coupled and glued to a polarization maintaining fibre array, which ends into a specifically designed glass ferrule. The three fibres tips are arranged in a triangular configuration and their optical axis is aligned before gluing, so to place them in azimuthal polarization configuration.
The fabrication of both the device and ferrule is based on femtosecond laser micromachining.
The three-points at the ferrule plane act as the source for the pattern. Thanks to the Fourier transforming properties of lenses, imaging the three coherent points in the back focal plane of a lens, produces a hexagonal intensity pattern in its front focal plane. The pattern was initially characterized by placing the ferrule in front of a planoconvex 50 mm lens. This configuration creates an interference pattern which can be collected by a camera placed in the focal plane of the lens (Fig. 10a). As expected, the pattern shows hexagonal symmetry with clear zeros.
In order to generate the hexagonal pattern in the object plane, an image of the three point-sources has to be created in the microscope's pupil. In a commercial inverted microscope this is possible by adding a lens in front of the chip, that in combination with the tube lens (TL) of the microscope forms a magnified image of the point sources. This lens was selected to be a 5X microscope objective (Mitutoyo, 0.14 NA). To have more flexibility on the alignment of the illumination and detection path, a telescope was added at the output port of the microscope, composed of two 150 mm lenses.
The three point-sources were on a circle with radius 0.25 mm at the ferrule plane and the circle was magnified to a 1.25 mm radius at the back focal plane of the objective. The pattern period at the sample plane depends on the illumination objective used to form the pattern, its value is affected both by the numerical aperture and the physical size of the pupil. A filling factor FF can be defined, as the ratio between the radius of the three points in the back focal plane and the pupil radius. The higher this value is, the closer the pattern period gets to the theoretical maximum resolution of the objective.
An Olympus 60X oil-immersion objective (1.4 NA) was used. In this configuration, a pattern period (distance between two zeros of the hexagonal pattern) of ~450nm was obtained, which correspond to a filling factor of ~0.5. The filling factor determines the resolution improvement of the SIM reconstruction, as the final resolution can be roughly estimated as pSIM is the widefield resolution, limited by the microscope objective lens. In the best case the filling factor is 1 and the resolution improvement is two-fold. The tests were limited to a filling factor of 0.5, in order to have a clearly visible pattern at the image plane and to demonstrate that the technique is able to generate and move the pattern precisely. Considering that the waist of the beam at the fibres plane is 2.7 pm, at the pupil the waist of each of the sources is 13.5 |im, giving rise to a modulation area with a diameter of ~ 80 gm (FWHM), at the object. Figure 10b shows a fluorescent cell (MitoTracker from Invitrogen #1 slides), illuminated with the pattern. The hexagonal lattice is superimposed to the fluorescence signal. A controlled shift of the pattern on the cell was proved to be possible, by applying different consecutive voltages to the thermal shifters, and recording the corresponding images. Figures Ila and 11b show a subsection of the cell, acquired with two different values of phase shifts. In the line plot of Fig. 12, the pattern translation in the sample plane is also visible (the pattern translation relative to Fig. Ila is plotted in dashed line whereas the pattern translation relative to Fig. 11b is plotted in solid line) . This measurement indicates that the chip allows the generation of a SIM pattern on the object plane of the microscope that can be spatially translated in the sample plane by changing the relative phase of the output beams. The hexagonal pattern with active phase control allows one to perform demodulation and to reconstruct super-resolved images acquiring N=7 images with a well precise combination of relative phases between the three laser beams.
A cell slide (FluoCells Prepared Slides #1) was imaged using a 60x/1.4NA oil immersion objective. The 532 nm laser excites the MitoTracker Red CMXRos that was used to stain the mitochondria of the cells. After a self-calibration process, N=7 images with the correct phases were acquired and the reconstruction process was performed. Figure 13a and 13b compare a widefield image with the reconstruction obtained with SIM. As expected, the reconstructed SIM image shows a an improved resolution when imaging the mitochondrial membranes. By taking a line profile of the filament shown in the particulars surrounded by the white square in Figures 13a and 13b a resolution improvement of 50%, in line with the used pupil fill factor was estimated. Remarkably the reconstruction algorithm reduces the background in the image, significantly improving its contrast, relative to the widefield case. These measurements demonstrate that the device of the present invention can be used as an add-on system to a standard fluorescence microscope, to implement structured illumination microscopy. Even if in this example, the HexSIM configuration, with a pattern formed by a 3-beam interference, is showed, several waveguides could be exploited to be used in N-beams SIM.

Claims

CLAIMS A patterned light generating device (100, 300) for microscopy comprising:
- at least a first optical waveguide (1) and a second optical waveguide (2), the waveguides (1, 2) being configured for transmitting a polarized light beam to their ends; at least one coupler (12) for splitting a laser beam entering the device (100, 300) between the first waveguide (1) and the second waveguide (2);
- a substrate (7) having one input port and at least a first output port and a second output port, said substrate (7) being configured for housing the waveguides (1, 2) and the at least one coupler (12); an input polarization maintaining fibre (10) fixed at the input port and configured for coupling the laser beam into the first waveguide
(1); and at least a first output polarization maintaining fibre (21) fixed at the first output port and configured for being coupled with the first waveguide (1) in such a way to transmit a light beam from the first waveguide to an end of the first output polarization maintaining fibre (21) and a second output polarization maintaining fibre (22) fixed at the second output port and configured for being coupled with the second waveguide (2) in such a way to transmit a light beam from the second waveguide to an end of the second output polarization maintaining fibre (22); said device (100, 300) being characterized in that it comprises: at least one phase shifter (11) configured for creating a phase difference between the first waveguide (1) and the second waveguide
(2), the phase shifter (11) being housed in the substrate (7); and - a glass ferrule (30) configured to being coupled with the output polarization fibres (21, 22), the output polarization fibres (21, 22) ending into the ferrule (30) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (1, 2) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle. A device (200) for microscopy comprising:
- at least a first optical waveguide (201) and a second optical waveguide (202), the waveguides (201, 202) being configured for transmitting a polarized light beam to their ends; at least one coupler (212) for splitting a laser beam entering the device between the first waveguide (201) and the second waveguide (202);
- a glass substrate (207) having one input port and at least a first output port and a second output port, said substrate (207) being configured for the housing of the waveguides (201, 202) and the at least one coupler (212); and an input polarization maintaining fibre (210) fixed at the input port and configured for coupling the laser beam into the first waveguide (201); said device (200) being characterized in that it comprises at least one phase shifter (211) housed on the substrate (207) and configured for creating a phase difference between the first waveguide (201) and the second waveguide (202) and in that the waveguides (201, 202) are positioned on the substrate (207) in such a way to be positioned one respect to the other by said separation angle being comprised within a range, the upper limit of the range being the sum of a first angle and a second angle, the first angle being equal to the round angle divided by the number of waveguides (201, 202) and the second angle being equal to the 10% of the first angle; and the lower limit of the range being the difference between the first angle and the second angle.
3. The device (400) according to claim 2, comprising:
- a third optical waveguide (203) housed in the substrate (207), the substrate (207) having a third output port, the third waveguide (3) being configured for transmitting a polarized light beam to its end and the waveguides (201, 202, 203) being configured to transmit polarized light; a coupler (213) for splitting a laser beam entering the second waveguide (202) between the second waveguide (202) and the third waveguide (203), the third waveguides (203) being configured to transmit a light beam from the coupler (213) for splitting a laser beam between the second waveguide (202) and the third waveguide (203), to the end of the third waveguide (203); and a phase shifter (211') housed on the substrate (207) and configured for creating a phase difference between the second waveguide (202) and the third waveguide (203); wherein the separation angle between the second and the third waveguides is equal to the separation angle between the first and the second waveguides or the separation angle between the second and the third waveguides is different from the separation angle between the first and the second waveguides.
4. The device (300) according to claim 1, comprising:
- a third optical waveguide (3) housed in the substrate (7), the substrate (7) having a third output port, the third waveguide (3) being configured for transmitting a polarized light beam to its end and the waveguides (1, 2, 3) being configured to transmit polarized light; a coupler (13) for splitting a laser beam entering the second waveguide (2) between the second waveguide (2) and the third waveguide (3); a third output polarization maintaining fibre (32) fixed at the third output port and configured for being coupled with the third waveguide (3) in such a way to transmit a light beam from the third waveguide (3) to an end of the third output polarization maintaining fibre (32); and a phase shifter (11') housed on the substrate (7) configured for creating a phase difference between the second waveguide (2) and the third waveguide (3); wherein the separation angle between the second and the third waveguides is equal to the separation angle between the first and the second waveguides or the separation angle between the second and the third waveguides is different from the separation angle between the first and the second waveguides. The device according to claim 4, comprising:
- a fourth optical waveguide housed in the substrate (7), the substrate (7) having a fourth output port, the fourth waveguide being configured for transmitting a polarized light beam to its end and the waveguides being configured to transmit polarized light; a coupler for splitting a laser beam entering the third waveguide (3) between the third waveguide (3) and the fourth waveguide; a fourth output polarization maintaining fibre fixed at the fourth output port and configured for being coupled with the fourth waveguide in such a way to transmit a light beam from the fourth waveguide to an end of the fourth output polarization maintaining fibre; and a phase shifter housed on the substrate (7) and configured for creating a phase difference between the third waveguide (3) and the fourth waveguide.
6. A device (100, 200, 300, 400) according to any of the preceding claims, comprising at least one amplitude modulator configured for creating an amplitude difference between two waveguides (1, 2, 3, 201, 202, 203).
7. The device according to claim 5 and 6, comprising:
- a fifth and a sixth optical waveguides housed in the substrate (7), the substrate (7) having a fifth and a sixth output ports, respectively and the fifth and the sixth waveguides being configured for transmitting a polarized light beam to their ends; a coupler for splitting a laser beam entering the fourth waveguide between the fourth waveguide and the fifth waveguide; a coupler for splitting a laser beam entering the fifth waveguide between the fifth waveguide and the sixth waveguide; a fifth output polarization maintaining fibres fixed at the fifth output port and configured for being coupled with the fifth waveguide in such a way to transmit a light beam from the fifth waveguide to an end of the fifth output polarization maintaining fibre;
- a sixth output polarization maintaining fibres fixed at the sixth output port and configured for being coupled with the sixth waveguide in such a way to transmit a light beam from the sixth waveguide to an end of the sixth output polarization maintaining fibre; a phase shifter housed on the substrate (7) and configured for creating a phase difference between the fourth waveguide and the fifth waveguide; a phase shifter housed on the substrate (7) and configured for creating a phase difference between the fifth waveguide and the sixth waveguide; wherein the at least one amplitude modulator is configured for turning on two waveguides at a time. The device (400) according to claim 2 or 3 comprising one output polarization maintaining fibres (3001, 3002, 3003) for each waveguide (201, 202, 203), said one output polarization maintaining fibres (3001, 3002, 3003) being fixed at one output port and configured for being coupled with the waveguide (201, 202, 203) in such a way to transmit a light beam from the one waveguide to an end of the output polarization maintaining fibre (3001, 3002, 3003). An apparatus (500) for optical microscopy comprising: a device (100, 300) according to any of the claims from 4 to 8 when dependent on claim 1;
- at least one lens (501) positioned in front of glass ferrule (30) of the device (100, 300); wherein the lens (501) is configured to generate by interference a structured illumination pattern translatable and/or rotatable according the phase difference between the waveguides, the pattern being the Fourier transform of the light beams transmitted by the output polarization maintaining fibres (21, 22) to their ends. . An apparatus (600) for optical microscopy comprising: a device (200, 400) according to claim 2 or 3; and
- at least one lens (501') positioned in front of the ends of the waveguides (201, 202, 203); wherein the at least lens (501') is configured to generate by interference a structured illumination pattern translatable and/ or rotatable according the phase difference between the waveguides (201, 202, 203), the pattern being the Fourier transform of the light beams transmitted by the waveguides (201, 202, 203) to their ends.
11. An apparatus (700) for light sheet microscopy comprising:
- a device according to claim 8;
- one lens (601, 601', 602, 602', 602") for each output polarization maintaining fibres (2003, 2004, 3001, 3002, 3003) positioned in front of the end of the output polarization maintaining fibres (2003, 2004, 3001, 3002, 3003), said lens (601, 601', 602', 602', 602") being a cylindrical lens (601, 601', 602, 602', 602").-
12. A optical microscope (1000) comprising
- a camera (1008);
- a tube lens (1003); a plane (1002) configured for housing a sample; a laser beam source;
- a beam splitter (1006);
- an apparatus (500, 500') according claim 9 or 10 positioned between the laser beam source and the beam splitter (1006), the beam splitter (1006) being configured to reflect the pattern generated by the lens (501, 501') of the apparatus (500, 500'), generating a reflected pattern; a relay lens system (1005, 1005') configured for receiving the reflected pattern of the apparatus and for relaying the pattern to the tube lens (1003), generating a relayed pattern; and an objective lens (1001) configured for receiving the relayed pattern and to transmit the relayed pattern to the plane (1002).
13. A microscope (1000) according to the previous claim, comprising a fluorescence filter (1007) positioned between the camera (1008) and the beam splitter (1006); wherein the beam splitter (1006) is a dichroic mirror configured to transmit to the camera (1008) a fluorescence radiation emitted by the sample.
14. A light sheet fluorescence microscope (3000) comprising: a laser beam source; an apparatus according to claim 11; a microfluidic chip (2001, 3010) comprising a channel (2002, 3005) configured for the housing of the sample, the sample being able to flow in a flowing direction (3011);
- a camera (3008);
- a tube lens; an objective lens (3001) having an optical axis parallel to the flowing direction (3011); and
- a bandpass filter (3007) between the objective lens (3001) and the tube lens; wherein the channel (2002, 3005) is placed in front to the cylindrical lenses (601, 601', 602, 602', 602") of the apparatus, the channel and the lenses being oriented in such a way that one primary light sheet for each lens is generated in the channel, each of said primary light sheets being perpendicular to the flowing direction (3011) and in such a way that a resulting patterned light sheet is generated by interference of the primary light sheets.
15. A light sheet fluorescence microscope (3000) according to the previous claim wherein the microfluidic chip (3010) is shaped as an equilateral triangular prism, each one of the output polarization fibres (3001, 3002, 3003) being configured to be coupled with one of the side face (3004, 3007, 3008) of said prism, the output polarization fibres (3001, 3002, 3003) ending into the channel (3005) in such a way to be oriented perpendicularly to the side faces (3004, 3007,
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