# US 2006/0132799 A1 · Digital holographic microscope

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- Number: US 2006/0132799 A1
- Document ID: US20060132799A1
- Status: Published application
- Kind: A1
- Application number: 11/284,768
- Filed: 2005-11-22
- Published: 2006-06-22
- Earliest claimed: 2001-06-29
- Assignees: Universite Libre de Bruxelles
- Inventors: Franck Dubois; Catherine Yourassowsky
- Web page: https://patentgrep.com/patent/US20060132799A1

## CPC

- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)}
- G03H 1/0005: {Adaptation of holography to specific applications (holographic optical element G02B5/32; holographic scanner G02B26/106; recognition using holographic mask G06V10/88; holographic memories G11B7/0065, G11C13/042)}
- G03H 1/0443: {Digital holography, i.e. recording holograms with digital recording means (holobject computation G03H1/0866)}
- G03H 1/041: {Optical element in the object space affecting the object beam, not otherwise provided for}
- G03H 1/0465: {Particular recording light; Beam shape or geometry (G03H1/06 takes precedence)}
- G03H 1/0866: {Digital holographic imaging, i.e. synthesizing holobjects from holograms}
- G03H 2001/005: {in microscopy, e.g. digital holographic microscope [DHM]}
- G03H 2001/0452: {arranged to record an image of the object}
- G03H 2001/0456: {Spatial heterodyne, i.e. filtering a Fourier transform of the off-axis record}
- G03H 2001/0458: {Temporal or spatial phase shifting, e.g. parallel phase shifting method}
- G03H 2222/24: Low coherence light normally not allowing valuable record or reconstruction (G03H1/06 takes precedence)
- G03H 2223/14: Diffuser, e.g. lens array, random phase mask
- G03H 2223/26: Means providing optical delay, e.g. for path length matching
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G03H: HOLOGRAPHIC PROCESSES OR APPARATUS (holograms, e.g. point holograms, used as ordinary optical elements G02B5/32; producing stereoscopic or other three-dimensional effects G02B30/00; diffraction-grating systems G02B27/44; systems using moiré fringes G02B27/60; optical logic elements G02F3/00; stereo-photography G03B35/00; photosensitive materials or processes for photographic purposes G03C; {stereo-photographic or similar processes G03C9/00}; apparatus for processing exposed photographic materials G03D; analogue computers performing mathematical operations with the aid of optical elements G06E3/00; authentication by radiation, of concealed information carried by holograms or diffraction gratings G06K19/16; holographic storage G11B7/0065, G11C13/04; {stereoscopic or other three dimensional effects in television systems H04N13/00})

## Abstract

A method and device for obtaining a sample with three-dimensional microscopy, in particular a thick biological sample and the fluorescence field emitted by the sample. One embodiment includes obtaining interferometric signals of a specimen, obtaining fluorescence signals emanating from the specimen, recording these signals, and processing these signals so as to reconstruct three-dimensional images of the specimen and of the field of fluorescence emitted by the specimen at a given time. Another embodiment includes a digital holography microscope, a fluorescence excitation source illuminating a specimen, where the microscope and the fluorescence excitation source cooperate to obtain interferometric signals of the specimen and obtain fluorescence signals emanating from the specimen, means for recording the interferometric signals and fluorescence signals, and means for processing the interferometric signals and the fluorescence signals so as to reconstruct three-dimensional images of the specimen and of the field of fluorescence emitted by the specimen at a given time.

## Claims

### Claim 1

A digital holographic microscope for obtaining without scanning three-dimensional images of a specimen to be studied, comprising a light source, an interferometer, a recorder defined by a sensitive plane and a processor,
said microscope being conceived in such a manner that in operating conditions said light source provides a partially spatially coherent light beam, said interferometer separates said partially spatially coherent light beam into a reference light beam and an object light beam, said object beam illuminates a specimen preliminary placed therein, said object and reference beams are recombined to produce interference patterns in the sensitive plane of said recorder, the recorder records said interference patterns and said processor processes said recorded interference patterns and reconstructs three-dimensional images of the specimen,
wherein the interferometer comprises:
a movable first subassembly that comprises a beam splitter and a mirror, able to form two parallel beams—an object beam and a reference beam, from the source light beam;
a fixed or movable second subassembly comprising a beam splitter and a mirror so as to combine said object beam and said reference beam into a recombined light beam;
an object cell, able to receive the specimen to be studied, said object cell being placed in the optical path of the object beam between the first subassembly and the second subassembly;
a first microscope objective located in the optical path of the object beam between the first subassembly and the second subassembly, downstream of the object cell;
a compensator able to optimize the equalization of the optical path lengths between the object beam and the reference beam, said compensator being located in the optical path of the reference beam between the first subassembly and the second subassembly;
a second microscope objective located in the optical path of the reference beam between the first subassembly and the second subassembly, downstream said compensator;
wherein the microscope further comprises a focuser defined by a back focal plane, said focuser being placed between the interferometer and the recorder in such a way that the back focal plane of said focuser is in the sensitive plane of the recorder.

### Claim 2 (depends on 1)

The microscope according to claim 1, wherein the position of the focuser relatively to the interferometer and the recorder is such as to obtain in operating conditions an invariant positioning of the object beam and the reference beam on the sensitive plane of the recorder.

### Claim 3 (depends on 1)

The microscope according to claim 1, wherein the first subassembly and the second subassembly are movable in rotation.

### Claim 4 (depends on 3)

The microscope according to claim 3, wherein the movements in rotation of the second subassembly are such as to allow in operating conditions the implementation of a Fourier transform method by the processor in order to process the interference patterns.

### Claim 5 (depends on 1)

The microscope according to claim 1, wherein the interferometer further comprises a first focuser capable of focusing the object beam onto the object cell, and a second focuser capable of focusing the reference beam onto said compensator, the first subassembly being movable in rotation, the second subassembly being fixed in rotation and the mirror of the second subassembly being movable in translation.

### Claim 6 (depends on 5)

The microscope according to claim 5, wherein the movements in translation of the mirror of the second subassembly are such as to allow in operating conditions the implementation of a phase-stepping method by the processor in order to process the interference patterns.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the mirror of the second subassembly is mounted on a piezo-electric motor.

### Claim 8 (depends on 6)

The microscope according to claim 6, further comprising a director device able to direct the source light beam onto the first subassembly at the beam splitter.

### Claim 9 (depends on 8)

The microscope according to claim 8, wherein the director device comprises mirrors.

### Claim 10 (depends on 1)

The microscope according to claim 1, wherein the partially spatially coherent light source comprises the following elements: a source emitting light of small spectral width, a first lens, a first aperture, a second lens, a second aperture and a third lens, the relative arrangement of these various elements being such that, when the source is operating, the light emitted by the light-emitting source encounters, in succession, the first lens, the first aperture, the second lens, the second aperture and the third lens, and the light at the exit of the third lens is collimated light.

### Claim 11 (depends on 1)

The microscope according to claim 1, wherein the partially spatially coherent light source comprises: a source emitting light of small spectral width, an optical fiber, a first lens, a second lens, a third lens and an adjustable aperture, the relative arrangement of these various elements being such that, when the source is operating, the light emitted by the light-emitting source passes firstly through the optical fiber so as to have a homogeneous light field and then encounters the first lens, then the second lens and the third lens in order to obtain collimated light at the aperture.

### Claim 12 (depends on 1)

The microscope according to claim 1, wherein the partially spatially coherent light source of said microscope comprises: a laser source, a first lens, a rotating diffuser, a second lens and an aperture, the relative arrangement of these various elements being such that, when the source is operating, the laser beam emitted by the laser source firstly passes through the first lens, then the rotating diffuser, which is placed behind the focal point of said first lens, then the second lens, placed at its focal length with respect to the plane of the rotating diffuser, and then said aperture.

### Claim 13 (depends on 1)

The microscope according to claim 1, wherein the partially spatially coherent light source comprises: a laser source, a first lens, a rotating diffuser, and a second lens, the relative arrangement of these various elements being such that, when the source is operating, the laser beam emitted by the laser source firstly passes through the first lens, then the diffuser and then the second lens, the laser beam at the exit of the first lens being focused at a focal point located upstream of the diffuser at an adjustable distance from the diffuser.

### Claim 14 (depends on 13)

The microscope according to claim 13, wherein the rotating diffuser comprises a ground glass plate.

### Claim 15 (depends on 1)

The microscope according to claim 1, wherein said compensator comprises a reference cell similar to the object cell but not including the specimen to be studied, or a transparent material of suitable ness and suitable composition.

### Claim 16 (depends on 2)

The microscope according to claim 2, wherein the first subassembly and the second subassembly are movable in rotation.

### Claim 17 (depends on 16)

The microscope according to claim 16, wherein the movements in rotation of the second subassembly are such as to allow in operating conditions the implementation of a Fourier transform method by the processor in order to process the interference patterns.