Patent Description:
Documents <CIT>, <CIT>, <NPL> and <NPL>) disclose devices for phase microscopy. The phase microscopy comprises many types of microscopy in which a signal wave is interfered with a reference wave in order to reveal information about the wavefront of the signal wave. Two prominent types of phase microscopy are: phase-contrast microscopy and spatial light interference microscopy, also known under the SLIM acronym.

Phase-contrast microscopy, also called Zernicke phase microscopy, is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image.

A phase microscope comprises a lighting device for illuminating a sample area. The lighting device comprises typically a light source and an optical element for concentrating the light onto the sample. The microscope further comprises an imaging device.

The imaging device comprises a camera, typically a 2D digital camera able to output a 2D matrix of pixels, some optical elements for directing the sample image onto the camera, i.e. the camera is at the conjugated plane of the sample. Other optical elements include lenses and a phase mask, typically a <NUM>° phase shift ring installed on the light path between the sample area and the camera.

Consequently, in the phase microscope, the interference is generated between light rays passing through the sample and scattered by the sample, the scattered light, and light rays unaffected by the sample and forming the reference light.

SLIM is a highly sensitive quantitative phase imaging method, which is used to study structure in biology and beyond. SLIM combines phase contrast microscopy with a phase-stepping algorithm. Quantitative information about the phase shifts induced by the sample is retrieved by taking and evaluating multiple images with different phase-shifts applied to the reference wave.

Phase microscopy is an invaluable tool for the study of biological specimens such as cells, and has found both scientific and diagnostic applications. For flat samples the high sensitivity of phase microscopy allows for the detection of atomic steps on surfaces, and for the unlabeled detection of single proteins.

However, biological specimens have typically variable thicknesses which generates variable phase shifts. These spatial variations often cover the full range (-ππ] and may even induce some phase wrapping. It is thus impossible to optimize the phase of a plane reference wave to achieve optimal sensitivity across the whole field of view.

Consequently, il will be very helpful to restore optimal sensitivity across the whole field of view. A phase microscope according to the invention is defined in claim <NUM>.

In a first embodiment, a device for phase microscopy comprises:.

is characterized in that it further comprises:.

Advantageously, the device allows to cancel the image artifacts due to thickness variations of the sample.

Advantageously, once properly initialized, the device allows acquiring quantitative phase imaging in a single frame.

This embodiment comprises other features, alone or in combination, such as:.

In a second embodiment, a phase microscope comprises a light path comprising at least a sample area, a lighting device for lighting said sample area and an imaging device for capturing a phase image of said sample, said phase image being a 2D matrix of pixels, and is characterized in that it further comprises a device as disclosed here above.

In a third embodiment, a method for obtaining a phase image of a sample by using a phase microscope as here above is defined in claim <NUM>.

In a fourth embodiment, a digital data storage medium is encoding a machine-executable program of instructions to perform the method disclosed here above.

Some embodiments of apparatus and/or methods in accordance with embodiments of the present invention are now described, by way of example only, and with reference to the accompagnying drawings, in which :.

In all figures, references to same or similar elements are identical.

In reference to <FIG>, standard phase microscopes are schematically drawn.

In <FIG>, a phase-contrast microscope <NUM> comprises a lighting device <NUM> for illuminating a sample area <NUM>.

The lighting device <NUM> comprises typically a light source <NUM>, an amplitude mask <NUM>, which can be a condenser annulus, and an optical element <NUM>, or condenser, for concentrating the light onto the sample.

The microscope <NUM> comprises also an imaging device <NUM>.

The imaging device <NUM> comprises a camera <NUM>, typically a 2D digital camera able to output a 2D matrix of pixels, for capturing the resulting image of the sample.

The imaging device <NUM> comprises also optical elements <NUM>, typically an objective and a tube lens, for directing the sample image onto the camera, i.e. the camera <NUM> is at the conjugated plane of the sample <NUM>.

Finally, the imaging device <NUM> comprises a phase mask <NUM>, typically a <NUM>° phase shift ring installed on the light path between the sample area <NUM> and the camera <NUM>.

<FIG> shows schematically a SLIM. The main difference with the phase-contrast microscope of <FIG> is that the phase mask <NUM> is replaced by a spatial light modulator <NUM>. This spatial light modulator <NUM> allows stepping the phase of the reference wave, which in turn enables a quantitative retrieval of the phase shifts imprinted on the signal wave.

It is worthwile to note that the phase mask <NUM> or the the spatial light modulator <NUM> are positioned in the Fourier plane of the sample plane <NUM>, the camera <NUM>, and the spatial light modulator <NUM>. It therefore allows shifting the reference wave with respect to the signal wave, but it does not allow subtracting phaseshifts from the signal wave in a spatially resolved way.

<FIG> shows schematically a first embodiment based on the phase-contrast microscope of <FIG>.

The light path of the microscope <NUM> is modified by inserting between the sample area <NUM> and the imaging device <NUM> a spatial light modulator <NUM>.

Connecting means <NUM> fix the spatial light modulator onto the microscope <NUM>, so that the spatial light modulator <NUM> is positioned in the light path in a conjugated plane of the sample area <NUM>. Connecting means comprises mechanical elements and optical elements, such as lenses, to direct correctly light through the light path.

In the embodiment of <FIG>, compared to the microscope of <FIG>, the spatial light modulator <NUM> "replaces" the camera <NUM> at the conjugated plane of the sample area <NUM> and a new conjugated plane of the spatial light modulator <NUM> is created on which the camera <NUM> is positioned.

The spatial light modulator <NUM> is controlled by a command device <NUM> which is, for instance, a computer.

The command device <NUM> is connected to the imaging device <NUM>, more particularly to the camera <NUM> in order to measure the phase shift of the pixels of the phase image.

The command device <NUM> commands then the spatial light modulator <NUM> in order to substract the measured phase shifts.

"Substracting the measured phase shifts" means that, for at least some regions, for instance for the light passing through the points Sx,y of the sample <NUM>, SLMx,y of the spatial light modulator <NUM> and captured as pixel Px,y by the camera <NUM>, the phase shift measured at pixel Px,y is substracted from the wave-front at point SLMx,y.

This operation may be applied for each pixel of the 2D matrix, for some pixels, for instance limited to the area of interest, and/or by grouping pixels in set of contiguous pixels defining basic areas for which an averaged phase shift is used.

In this last case, a compromise is done between the computer power and/or the speed of calculus on one side and the precision of the correction on the other side.

To determine the phase of a signal wave Es = |ES|eiφ, it has to be interfered with a reference wave ER = |ER|eiα. The resulting interference pattern reads I = |ES|<NUM> + |ER|<NUM> + <NUM>|ES||ER| cos(φ - α), and its sensitivity to small changes of φ is proportional to sin(φ - α). For measurements at optimal sensitivity α needs to be adjusted such that φ - α = (<NUM>n - <NUM>)π/<NUM>, with |n| = <NUM>,<NUM>,<NUM>,. At φ - α = nπ the sensitivity drops to zero.

Phase contrast microscopy is based on the insight that the optical field after sample interaction can be written as the sum of a plane reference wave and a scattered wave E = |E|eiψ(x,y) = |ER| + |ES(x, y) |eiφ(x,y). In the Fourier plane (after propagation through a 2f setup), the reference wave is focused onto a point, while the scattered wave is spread out across the plane, which allows adding a phase α to the reference wave, without significantly affecting the scattered wave. Propagation through another 2f setup yields an image of intensity :
<MAT>.

For α = ±π/<NUM>, the conventional schemes of positive and negative phase contrast are retrieved, which represent optimal choices only if φ(x, y) ≈ <NUM>.

The aim of the embodiment of <FIG> is to ensure this condition everywhere in the area of interest across the field of view of the image.

Consequently, the method, <FIG>, for using it is:.

If the error is non-negligible, step <NUM>, the procedure can be applied iteratively until ψ(x, y) - ψmeas(x,y) « <NUM> across the field of view. The choice of α = ± π-/<NUM> will now yield optimal sensitivity across the image. For dynamic studies this means that initialization of the spatial light modulator <NUM> at time t<NUM>, will allow for optimal sensitivity at all following times t<NUM> > t<NUM>, if the phase mask displayed on the spatial light modulator <NUM> can be updated fast enough.

The step <NUM> of firstly capturing a sample image may be replaced by a step of acquiring a plurality of images that allow for a coarse estimate of the phase-shifts induced by the sample.

In a second embodiment shown on <FIG>, the classical phase-contrast microscope <NUM> is replaced by the SLIM of <FIG>, i.e. the phase mask <NUM> is replaced by the spatial light modulator <NUM>. The advantage of this embodiment is that SLIM can be used to get a first coarse estimate of the phase-shifts induced by the sample.

In a third embodiment, <FIG>, the spatial light modulator <NUM> is still positioned in the conjugated plane of the sample but on the light path between the lighting device <NUM> and the sample area <NUM>. This embodiment allows using commercial phase objectives, in which the phase mask is realized as integral part of the microscope objective.

Similarly, the optical elements <NUM> and those of the connecting means <NUM> may be reorganized in the best pratical way. For instance, the tube lens <NUM> may stay in position after the sample area <NUM> and the optical means <NUM> are used to project the plane of the spatial light modulator <NUM> onto the camera <NUM>.

The man skilled in the art notices that the <FIG> show a spatial light modulator <NUM> of refracting mode, but a spatial light modulator of reflecting mode can also be used.

A setup using reflecting spatial light modulator with a SLIM is shown at <FIG>.

It is built as an add-on to a commercial inverted microscope. The sample <NUM> is illuminated with light from an LED <NUM>, and imaged onto a spatial light modulator <NUM> using a 50x (NA = <NUM>) objective <NUM>. After reflection from the spatial light modulator <NUM>, a 4f lens configuration is used to image the wave onto a camera <NUM>. To perform phase microscopy, matched aperture and phase masks <NUM>, <NUM> have to be inserted in conjugated planes before the sample <NUM> and after the spatial light modulator <NUM>. These allow for selective application of α to the unscattered wave. While these masks are typically ring-shaped, a more random (but known) pattern is used here, which allows reducing imaging artifacts. The aperture mask <NUM> is laser printed, the phase mask <NUM> is realized using a spatial light modulator. A folded configuration can be used to allow for using two regions of a single spatial light modulator to realize the patterns required on spatial light modulator <NUM> and spatial light modulator <NUM>.

To test the setup of <FIG> under real imaging conditions, biological samples are introduced in the sample plane of the inverted phase microscope. <FIG> shows the results obtained for red blood cells. Negative, positive, and corrected images are shown in the top row. The bottom row shows the results of a differential measurement, in which a checkerboard phase distribution has been added to the wavefront. <FIG> shows that the sensitivity to these additional phase shifts is improved by being more homogeneous, and quantitative when corrected by the phase-shift subtraction.

A person of skill in the art would readily recognize that steps of various above-described methods can be performed by programmed computers. Herein, some embodiments are also intended to cover program storage devices, e.g., digital data storage media, which are machine or computer readable and encode machine- executable or computer-executable programs of instructions, wherein said instructions perform some or all of the steps of said above-described methods. The program storage devices may be, e.g., digital memories, magnetic storage media such as a magnetic disks and magnetic tapes, hard drives, or optically readable digital data storage media. The embodiments are also intended to cover computers programmed to perform said steps of the above-described methods.

Moreover, explicit use of the term "processor" or "controller" should not be construed to refer exclusively to hardware capable of executing software, and may implicitly include, without limitation, digital signal processor (DSP) hardware, network processor, application specific integrated circuit (ASIC), field programmable gate array (FPGA), read only memory (ROM) for storing software, random access memory (RAM), and non volatile storage. Similarly, any routers shown in the figures are conceptual only.

Claim 1:
Phase microscope (<NUM>) comprising a light path comprising at least a sample area (<NUM>), a lighting device (<NUM>) for lighting said sample area and an imaging device (<NUM>) for capturing a phase image of said sample, said phase image being a 2D matrix of pixels,
wherein it further comprises a device for phase microscopy comprising:
• a spatial light modulator (<NUM>), and:
• a connecting means (<NUM>) adapted to fix the spatial light modulator onto said phase microscope (<NUM>) so that the spatial light modulator (<NUM>) is positioned in the light path in a conjugated plane of the sample area (<NUM>) and
• a command (<NUM>) of the spatial light modulator (<NUM>) connected to the imaging device (<NUM>) and characterized in that said command is adapted to:
∘ command a step b) of measuring the phase shift of a plurality of pixels of the phase image, wherein the phase image is generated by an optical field (E) written as the sum of a plane reference wave (ER) and a scattered wave (ES): E = |E|eiψ(x,y) = |ER| + |ES(x,y)|eiφ(x,y) , where:
ψ(x,y) is the phase of the optical field E,
|ER| is the field amplitude of the plane reference wave ER,
φ(x,y) is the phase of the scattered wave ES,
|ES| is the field amplitude of the scattered wave ES,
which yields to an intensity of the phase image I(x,y) = |ES(x,y)|<NUM> + |ER|<NUM> + <NUM>|ES(x, y)||ER| cos(φ(x,y) - α),
where:
- α is chosen to be equal to ±π/<NUM>,
∘ command a step c) of controlling (<NUM>) the spatial light modulator (<NUM>) so that it subtracts the measured phase-shift from the light ray associated with the pixel,
∘ command a repetition of the steps a) to c), recursively, and until ψ(x,y) - ψmeas(x,y) « <NUM>