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Title:
AN APPARATUS AND A METHOD FOR FLUORESCENCE IMAGING
Document Type and Number:
WIPO Patent Application WO/2023/135367
Kind Code:
A1
Abstract:
According to an example embodiment, a hyperspectral detection approach is used in combination with narrow linewidth illumination for fluorescence excitation. A more efficient fluorophores excitation and image capturing may be provided, and thus high-quality data for subsequent hyperspectral analysis may be obtained. An apparatus, a method, a system and a computer program are disclosed.

Inventors:
UUSIMAA PETTERI (FI)
FILIPCHUK ANNA (FI)
NECHAY KOSTIANTYN (FI)
PERTTILĂ„ ROBERT (FI)
Application Number:
PCT/FI2023/050027
Publication Date:
July 20, 2023
Filing Date:
January 11, 2023
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
MODULIGHT CORP (FI)
International Classes:
G01N15/14; G01N15/10; G01N21/64
Foreign References:
US9797836B12017-10-24
US20160299060A12016-10-13
CN111141713A2020-05-12
US20170176338A12017-06-22
Attorney, Agent or Firm:
PAPULA OY (FI)
Download PDF:
Claims:
27

CLAIMS

1 . An apparatus for fluorescence imaging, comprising : at least one narrow linewidth excitation light source configured to provide a beam for fluorescence excitation at an excitation region located at an optical path of the beam; at least one optical guiding component configured to guide fluorescence light generated by the beam at the excitation region to an image sensor for hyper- spectral imaging; and at least one image sensor comprising a spectral filtering component , the at least one image sensor configured to perform hyperspectral imaging based on the fluorescence light received via the at least one optical guiding component .

2 . The apparatus of claim 1 , wherein the at least one narrow linewidth excitation light source , the at least one optical guiding component and the at least one image sensor are arranged on a same side with respect to a sample holder comprising the excitation region such that the optical path of beam differs from an optical path of the fluorescence light received by the image sensor .

3 . The apparatus of claim 1 , further comprising : at least one optical focusing component positioned between the at least one narrow linewidth excitation light source and the excitation region and configured to focus the beam on the excitation region .

4 . The apparatus of claim 1 , further comprising : at least one bandpass filter positioned between the at least one narrow linewidth excitation light source and the excitation region, and configured to filter the beam into monochromatic light .

5 . The apparatus of claim 2 , wherein the bandpass filter is configured to limit spectral bandwidth of the beam to or below 1 nm .

6 . The apparatus of claim 1 , further comprising : at least one beam-shaping component positioned between the narrow linewidth excitation light source and the optical focusing component , and configured to adj ust at least one of a shape or a si ze of the beam .

7 . The apparatus of claim 1 , further comprising : at least one beam-shaping component positioned between the optical focusing component and the excitation region, and configured to adj ust at least one of a shape or a si ze of the beam .

8 . The apparatus of claim 1 , wherein the optical focusing component is configured based on at least one of a material at the excitation region, geometrical design of the material or a distance between the optical focusing element and the excitation region .

9 . The apparatus of claim 1 , wherein a position of the optical guiding component is determined based on at least one of material at the excitation region, geometrical design of the material , a distance between the second optical focusing component and the excitation region, magnification properties of the optical guiding component or a numerical aperture of the optical guiding component .

10 . The apparatus of claim 1 , wherein the apparatus comprises a plurality of narrow linewidth excitation light sources configured to provide beams for fluorescence excitation at two or more excitation regions ; and the optical guiding component is configured for guiding fluorescence light generated by the beams at the two or more excitation regions to different image sensors .

11 . The apparatus of claim 1 , wherein the apparatus comprises at least one narrow linewidth excitation light source configured to provide one or more beams for f luorescence excitation at one or more excitation regions ; and wherein the at least one narrow linewidth excitation light source comprises at least one of a single-frequency laser or a laser diode that have less than 1 nm spectral bandwidth .

12 . The apparatus of claim 11 , wherein the at least one narrow linewidth excitation light source is configured to be wavelength tuneable .

13 . The apparatus of claim 1 , comprising : at least one optical beam splitting component configured to split the fluorescence light according to a wavelength of the fluorescence to a first image sensor configured to receive the fluorescence light of a first wavelength and a second image sensor configured to receive the fluorescence light of a second wavelength .

14 . The apparatus of claim 11 , further comprising a mirroring component configured to guide the fluorescence light of the second wavelength to the second image sensor .

15 . The apparatus of claim 1 , wherein the apparatus comprises a plurality of narrow linewidth excitation light sources configured to operate simultaneously or with temporal separation .

16 . A method, the method comprising : providing, by at least one narrow linewidth excitation light source , a beam for fluorescence excitation at an excitation region located at an optical path of the beam; guiding, by at least one optical guiding component , fluorescence light generated by the beam at the excitation region to an image sensor for hyperspectral imaging; and performing hyperspectral imaging, by at least one image sensor comprising a spectral filtering component , based on the f luorescence light received via the at least one optical guiding component .

17 . The method of claim 14 , comprising : arranging the at least one narrow linewidth excitation light source , the at least one optical guiding component and the at least one image sensor on a same side with respect to the a sample holder comprising the excitation region such that the optical path of beam di ffers from an optical path of the fluorescence light received by the image sensor .

18 . The method of claim 14 , comprising : configuring a plurality of narrow linewidth excitation light sources to provide beams for fluorescence excitation at two or more excitation regions ; and configuring the optical guiding component to guide the f luorescence light generated by the beams at the two or more excitation regions to different image sensors .

Description:
AN APPARATUS AND A METHOD FOR FLUORESCENCE IMAGING

TECHNICAL FIELD

Various example embodiments generally relate to the field of fluorescence imaging . In particular, some example embodiments relate to an apparatus configured for fluorescence excitation and detection and a method thereof .

BACKGROUND

Hyperspectral flow cytometry imaging enables acquisition of hyperspectral images when performing flow cytometrical analysis . The hyperspectral images may be used, for example , for optical analysis and characteri zation of single-cells and other particles . Acquired spectral information, signali zing of the cell ' s material composition, can be collected along with spatial information in the form of spectral images . However, optimal excitation for the imaging may be difficult to achieve , and data analysis can be complicated due to multiple overlapping absorption and fluorescence emission bands of the target . It would be beneficial to improve imaging of biomedical samples to alleviate at least some of these drawbacks .

SUMMARY

This summary is provided to introduce a selection of concepts in a s implif ied form that are further described below in the detailed description . This summary is not intended to identify key features or essential features of the claimed subj ect matter, nor is it intended to be used to limit the scope of the claimed subj ect matter .

According to a first aspect , an apparatus for fluorescence imaging is provided . The apparatus may comprise at least one narrow linewidth excitation light source configured to provide a beam for fluorescence excitation at an excitation region located at an optical path of the beam; at least one optical guiding component configured to guide fluorescence light generated by the beam at the excitation region to an image sensor for hyperspectral imaging; and at least one image sensor comprising a spectral filtering component , the at least one image sensor configured to perform hyperspectral imaging based on the fluorescence light received via the at least one optical guiding component .

In an embodiment , the at least one narrow linewidth excitation light source , the at least one optical guiding component and the at least one image sensor are arranged on a same side with respect to a sample holder comprising the excitation region such that the optical path of beam dif fers from an optical path of the f luorescence light received by the image sensor .

In an embodiment , in addition or alternatively, the apparatus may further comprise at least one optical focusing component positioned between the at least one narrow linewidth excitation light source and the excitation region and configured to focus the beam on the excitation region .

In an embodiment , in addition or alternatively, the apparatus may further comprise at least one bandpass filter positioned between the at least one narrow linewidth excitation light source and the excitation region, and configured to filter the beam into monochromatic light .

In an embodiment , in addition or alternatively, the bandpass filter is configured to limit spectral bandwidth of the beam to or below 1 nm .

In an embodiment , in addition or alternatively, the apparatus may further comprise at least one beamshaping component positioned between the narrow linewidth excitation light source and the optical focusing component , and configured to adj ust at least one of a shape or a si ze of the beam .

In an embodiment , in addition or alternatively, the apparatus may further comprise at least one beamshaping component positioned between the optical focusing component and the excitation region, and configured to adj ust at least one of a shape or a si ze of the beam .

In an embodiment , in addition or alternatively, the optical focusing component is configured based on at least one of a material at the excitation region, geometrical design of the material or a distance between the optical focusing element and the excitation region .

In an embodiment , in addition or alternatively, a position of the optical guiding component is determined based on at least one of material at the excitation region, geometrical design of the material , a distance between the second optical focusing component and the excitation region, magnification properties of the optical guiding component or a numerical aperture of the optical guiding component .

In an embodiment , in addition or alternatively, the apparatus may comprise a plurality of narrow linewidth excitation light sources configured to provide beams for fluorescence excitation at two or more exci tation regions ; and the optical guiding component is configured for guiding fluorescence light generated by the beams at the two or more excitation regions to different image sensors .

In an embodiment , in addition or alternatively, the apparatus may comprise at least one narrow linewidth excitation light source configured to provide one or more beams for fluorescence excitation at one or more excitation regions ; and wherein the at least one narrow linewidth excitation light source comprises at least one of a s ingle-frequency laser or a laser diode that have less than 1 nm spectral bandwidth . In an embodiment , in addition or alternatively, the at least one narrow linewidth excitation light source is configured to be wavelength tuneable .

In an embodiment , in addition or alternatively, the apparatus may comprise at least one optical beam splitting component configured to split the fluorescence light according to a wavelength of the fluorescence to a first image sensor configured to receive the fluorescence light of a first wavelength and a second image sensor configured to receive the f luorescence light of a second wavelength .

In an embodiment , in addition or alternatively, the apparatus may further comprise a mirroring component configured to guide the fluorescence light of the second wavelength to the second image sensor .

In an embodiment , in addition or alternatively, the apparatus may comprise a plurality of narrow linewidth excitation light sources configured to operate simultaneously or with temporal separation .

According to a second aspect, a method is provided . The method may comprise providing, by at least one narrow linewidth excitation light source , a beam for fluorescence excitation at an excitation region located at an optical path of the beam; guiding, by at least one optical guiding component , fluorescence light generated by the beam at the excitation region to an image sensor for hyperspectral imaging; and performing hyperspectral imaging, by at least one image sensor comprising a spectral filtering component , based on the fluorescence light received via the at least one optical guiding component .

In an embodiment , the method may comprise arranging the at least one narrow linewidth excitation light source , the at least one optical guiding component and the at least one image sensor on a same side with respect to a sample holder compri sing the excitation region such that the optical path of beam differs from an optical path of the fluorescence light received by the image sensor .

In an embodiment , in addition or alternatively, the method may comprise configuring a plurality of narrow linewidth excitation light sources to provide beams for fluorescence excitation at two or more excitation regions ; and configuring the optical guiding component to guide the f luorescence light generated by the beams at the two or more excitation regions to different image sensors . Further features of the method may result from the features of the apparatus according to the first aspect .

Many of the attendant features wil l be more readily appreciated as they become better understood by reference to the following detailed description considered in connection with the accompanying drawings .

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings , which are included to provide a further understanding of the example embodiments and constitute a part of this specification, illustrate example embodiments and together with the description help to explain the example embodiments . In the drawings :

FIG . 1 illustrates an example of an apparatus for fluorescence imaging according to an example embodiment ;

FIG . 2 illustrates an example of an apparatus for fluorescence imaging comprising a plurality of image sensors for hyperspectral imaging, according to an example embodiment ;

FIG . 3 illustrates an example of a method for fluorescence imaging according to example embodiment .

Like references are used to designate equivalent or at least functionally equivalent parts in the accompanying drawings . DETAILED DESCRIPTION

Reference will now be made in detail to example embodiments , examples of which are illustrated in the accompanying drawings . The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utili zed . The description sets forth the functions of the example and the sequence of operations for constructing and operating the example . However , the same or equivalent functions and sequences may be accompl ished by different examples .

An embodiment may be configured to provide improved snapshot hyperspectral imaging for biological samples comprising fluorophores or producing auto-flu- orescence or self-f luorescence under illumination without a fluorophore . An apparatus and a method are provided accordingly, wherein a combination of narrow linewidth excitation and spectrally-resolving detection approaches may allow to achieve high-quality and high- information-content hyperspectral data from an examined specimen . For example , the apparatus may be configured to implement snapshot hyperspectral imaging for flow cytometry . Hence , the information content obtained in flow cytometry analysis may be increased . The apparatus may be also configured to resolve spatial spectral information in analysing other fluorescence labelled or auto-fluorescing biological samples , like RNA or DNA analysis or sequencing, or cell or tissue samples , or biopsies , that are stained for pathological diagnosis of cancer or other diseases . The apparatus can further be used to substitute a plurality of biological tests performed to obtain the same information .

Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles . In flow cytometry, a sample constituting of cells or particles may be suspended in a fluid and inj ected into a flow cytometer instrument , such as a microcapillary channel .

Fluorescence labelling may be used for DNA and RNA sequencing to enable easier and faster sequencing . For example , the order of nucleotides of the DNA may be determined based on a unique fluorescence emission profile of four nucleotides . The sequencing of the DNA may be carried out on a glass plate or flow cell excited with laser light , for example .

Optical biopsy may refer to a technique to assess different pathological conditions , particularly of cancer, with tissue or blood as sample . For example , in the case of cancer the biochemistry and biophysics of the cells of tissue may undergo significant changes , which may be highly fluorescent . The difference in the concentration and distribution of these fluorophores may be monitored, for example , by spectral features and/or by excited state decay times by fluorescence imaging .

A fluorophore is a fluorescent chemical compound that can re-emit l ight upon l ight excitation . In other words , a fluorophore may absorb light energy of a specific wavelength and re-emit light at a longer wavelength . Fluorescence may refer to the light emitted by the fluorophore . A fluorophore marker may refer to a fluorophore serving as a marker for affine or bioactive reagents , for example, to stain cells or materials in a variety of analytical methods including fluorescence imaging . Biological samples may also produce fluorescence signal without a fluorophore , called auto-fluo- rescence , when irradiated with light having high enough energy to excite some molecules of the cel ls or tis sue themselves . In fluorescence imaging, fluorescence is used to study properties of organic or inorganic substances . Hyperspectral imaging may be used to collect and process data from across an electromagnetic spectrum . In hyperspectral imaging, the spectrum may be obtained for each pixel in an image in order to identify materials or ongoing processes , for example . In snapshot hyperspectral imaging, hyperspectral images may be captured during a single integration time of a detector array . For example , a focal-plane array may be used to generate an image in an instant .

In a system for flow cytometry, a flow cell may be positioned between an excitation and detection parts of the flow cytometry system . Flow cells may refer to samples cells , wherein liquid samples may be continuously flowed through a beam path for fluorescence excitation . Liquid samples may be focused along the flow path via various focusing means to provide precise interference points between the sample and the excitation source . A beam path may refer to an optical path or optical axis of a beam . For example , f low cell s may be used for samples that can be damaged by a light source . In flow cel ls , new samples may be continuous ly replenished so that the damage does not interfere with the analysis . Flow cells are also useful in situations where samples need to vary continuously, such as gradually changing concentration amounts . When the flow cell is positioned between the excitation and detection parts , a used optical system may depend on a flow cell geometry . The detection part of such system may be designed to detect a presence or absence of a studied fluorophore (quantitative data) . However, the detection part may not provide data on fluorophores distribution (qualitative data) within the studied cell . Thus , while the analysis may be fast , enabling higher throughput of the system, the information content of the data may be limited . Further, when a biological sample is illuminated from one side and fluorescence is detected directly from the other s ide , a lot of excitation l ight is allowed to be either transmitted or reflected onto the detector . In such configuration, most of the light hitting the detector or fluorescence collection optics can be , in fact , excitation light , producing noise in the detected signal . Filtering this unwanted light may not allow differentiating extremely weak fluorescence signals from the background noise . Similar quality and processing speed challenges may occur also with imaging static biological samples , like RNA or DNA in a flow cell or cell s or tis sue on a glass s lide or a microscope s lide prepared for fluorescence microscopy . Glass /microscope sl ides may be used to hold a specimen for examination, wherein the specimen may be placed between two thin sheets of glass .

Broad bandwidth excitation light sources (e . g . , LEDs or halogen lamps ) in combination with mul- tispectral detector approaches may be used for multi- spectral imaging flow cytometry . Broad bandwidth excitation light sources may be light sources that produce light across a wide range of wavelengths , as opposed to monochromatic light sources , which may produce only a single wavelength or color of light . However, the broad bandwidth excitation light sources may not provide optimal excitation conditions . The broad bandwidth excitation light sources may introduce additional noise to the multispectral detector, as most of the wavelengths present in broad bandwidth light are not used for fluor- ophore excitation . For example , the broadband excitation light sources may cause a lot of excessive reflected light , which may blur the images and lead to information loss . Having a single broadband excitation source in an imaging system may lead to a simpler geometrical arrangement , but it may also lead to the trade-off in image quality . Thus , data analysis may be complicated because subtraction of unwanted signal may be needed . Further, the multispectral system using broad bandwidth excitation light source may require additional filtering . Moreover, light intensity in LEDs is distributed unevenly through the different wavelengths . Hence , LEDs may not be manual ly adj usted for each separate wavelength of interest . Consequently, a high signal level required by some fluorophores for optimal excitation may not be achieved or may be completely missed due to dominating one fluorescence process over any other when using broadband illumination .

An obj ective is to improve efficiency and compactness of an apparatus for fluorescence imaging . An example embodiment enables improving functionality of a detection part for hyperspectral imaging to enable simultaneous collection of spatial and spectral information of a specimen . The hyperspectral detection approach may be used in combination with individually adj ustable narrow linewidth illumination which aims to provide more precise excitation of spectrally narrow transitions in fluorophores . This may lead to more efficient fluorophores excitation and image capturing . Hence , high-quality data may be provided for subsequent hyperspectral analysis . Optical modules of the apparatus may be positioned such that optical axes of excitation light and imaging are located on a same side with respect to a location of the specimen . Hence , an improved formfactor may be achieved with the geometrical design of the apparatus while improving quality of fluorescence imaging . Possible applications include clinical diagnostic tests and samples screening for RNA or DNA sequencing, or direct RNA and DNA sequencing or cell and tissue biopsy pathology .

FIG . 1 illustrates an example of an apparatus 100 for fluorescence imaging according to an example embodiment . The apparatus 100 may be configured for fluorescence excitation and detection . The apparatus 100 may be configured, for example, for flow cytometry, DNA sequencing, RNA sequencing or optical biopsy . The apparatus 100 may comprise at least one narrow linewidth excitation light source 102 . A narrow linewidth excitation light source may refer to a singlefrequency light source with a narrow optical emission spectrum . For example , the narrow linewidth excitation light source 102 may be configured to emit laser light . The narrow linewidth excitation light source 102 may be configured to emit , for example , a single-mode laser beam with a circular Gaussian profile . The at least one narrow linewidth excitation light source 102 may be configured to provide one or more beams for fluorescence excitation at an excitation region 104 . The beam for fluorescence excitation may also be referred to as an excitation beam . The apparatus 100 may be configured to provide the one or more beams , for example , as pulsed excitation light and/or in certain sequencies over time . The at least one narrow linewidth excitation light source 102 may also be configured to provide one or more beams for fluorescence excitation at more than one excitation regions 104 . The at least one narrow linewidth excitation light source 102 may comprise , for example , a single-frequency laser . In addition, or alternatively, the at least one narrow linewidth excitation light source 102 may comprise , for example , a laser diode . The single-frequency laser and the laser diode may be configured to have less than 1 nm spectral bandwidth . Further, the at least one narrow linewidth excitation light source 102 , such as the single-frequency laser and the laser diode , may be configured to be wavelength tuneable .

The apparatus 100 may comprise a plurality of narrow linewidth excitation sources 102 configured to provide one or more beams for fluorescence excitation at one or more excitation regions 104 . Hence , multiple narrow linewidth excitation sources 102 may be configured to provide excitation light at the same excitation region 104 . Alternatively, multiple narrow linewidth excitation sources 102 may be configured to provide excitation light at different excitation regions 104 . The one or more narrow linewidth excitation sources 102 may comprise , for example , a fixed-wavelength or a wavelength-tunable laser source , or a combination thereof . The one or more narrow excitation sources 102 may be configured for producing the excitation light with different pulse length and/or timing at one or more different excitation regions 104 . With one or more pul sed excitation light sources , the apparatus 100 may be configured for time-separated fluorescence excitation and detection . The apparatus 100 may comprise , for example , two or more excitation light sources , as illustrated in FIG . 1 . The two or more narrow linewidth excitation light sources 102 may be configured for having a different optical axis for excitation with respect to each other, each directed at least one of at a different excitation region 104 or at a same excitation region 104 . The two or more excitation sources 102 may be configured to generate the excitation beam at the same time for imaging . The optical axis of an excitation light source 102 may be fixed or changeable (by translation and/or rotation) for any or al l of the one or more narrow linewidth excitation light sources 102 . The two or more narrow linewidth excitation light sources 102 may be positioned symmetrically around a detection part of the apparatus 100 . The detection part of the apparatus 100 may comprise , for example , at least one image sensor and optics for guiding fluorescence light to the image sensor .

The apparatus 100 may be configured to perform fluorescence excitation and detection for a sample flowing through or sitting on the one or more excitation regions 104 . The excitation region 104 may comprise one or more specimen of the sample . The specimen may comprise , for example , cells to be studied . The cells may be located in a buf fer solution flowing through a flow channel 114 , such as a microcapillary channel , which serves as a propagation medium for the studied cells containing f luorophores . One or more excitation regions 104 may be located at the flow channel 114 . An excitation region may also be referred to as an observation region . The flow channel 114 may be part of the apparatus 100 , or separate from the apparatus 100 . However , the apparatus 100 may also be configured for measurement of stationary samples . Hence , instead of the flow channel 114 , the one or more excitation regions may be located, for example , at a sample chamber, glass slides or microscope slides for the purpose of fluorescence excitation of the sample . A flow channel , a sample chamber, glass slides or microscope slides may be referred to as a sample container or a sample holder . The apparatus 100 may be configured to provide excitation light at the sample containing f luorophores , for example at a given pulsed wavelength . In response to the caused excitation at the sample , fluorescence light emission may take place . This fluorescence may be emitted in all directions from the sample . The emitted fluorescence may represent a point light source at the excitation region 104 .

The detection part of the apparatus 100 and the at least one narrow linewidth excitation light source 102 may be positioned at a same side from the excitation region 104 . Further, the position of the at least one narrow linewidth excitation light source 102 may be inclined with respect to the detection part such that optical path of the beam is non-parallel to an optical path of light guided for imaging by the image sensor . When the components are located at the same side , it may refer to a same side with respect to an axis determined based on a flowing direction of the samples , such as below the microcapillary channel .

The excitation beam may be collimated and/or focused, for example by using lenses . In general , the apparatus 100 may comprise an optical system for exci tation between any or all of the excitation regions 104 and the corresponding narrow linewidth excitation light sources 102 for providing the excitation light to the excitation region 104 . The optical system for excitation may also be part of the narrow linewidth excitation light source 102 , for example integrated thereto , or separate therefrom . The optical systems for any or all different excitation regions 104 may be separate from each other . The optical system may comprise any of collimation, filtering and focusing optics for the excitation light .

For example , the apparatus 100 may comprise at least one bandpass filter 110 configured to f ilter the light emitted from the narrow linewidth excitation light source 102 . The at least one bandpass filter 110 may be positioned between the one or more narrow linewidth excitation light sources 102 and the one or more excitation regions 104 along a respective optical axis of the excitation light . The optical axis may also be referred to as an optical path . The bandpass filter 110 may be also part of the narrow linewidth excitation source 102 , for example integrated thereto . The bandpass filter 110 may be configured to provide improved monochromaticity . The monochromaticity may be beneficial in flow cytometry and other applications of fluorescence imaging, since energy required for an electron transition in fluoro- phores may have to match precise energy excitation values . The bandpass filter 110 may be configured to limit a spectral bandwidth of the excitation beam to 1 nm, preferably below 1 nm . Thus , the bandpass filter 110 may enable achieving improved fluorophore excitation by the excitation beam, wherein the spectral bandwidth may not exceed the 1 nm limit . When the spectral bandwidth is limited to 1 nm or below, it may make it easier for the excitation beam to hit certain molecular absorption lines . The bandpass filter 110 may enable the highest transmission of light at the desired excitation region 104 and filtering of the rest of the spectrum .

The apparatus 100 may comprise an optical focusing component 112 , such as a focus ing lens . The optical focusing component may be positioned between the one or more narrow linewidth excitation sources 102 and the one or more excitation regions 104 along a respective optical axis of the beam for f luorescence excitation . The optical focusing component 112 may be configured to focus the fluorescence excitation light provided by the beam on the excitation region 104 . The optical focusing component 112 may be positioned, for example , after the bandpass filter 110 such that the filtered fluorescence excitation light is focused on the excitation region 104 . Precise focusing on an imaged area, i . e . , the excitation region 104 , may enable to avoid power losses and excessive excitation of the fluoro- phore , which might damage the sample , such as the studied cell . The optical focusing component 112 may be selected based on parameters comprising at least one of a material of the sample , a geometrical design of the sample , and/or a distance between the optical focusing component 112 and the excitation region 104 .

Each of the narrow linewidth excitation light sources 102 may be associated with a dedicated optical system for excitation, such as comprising the bandpass filter 110 and optical focusing element 112 . Alternatively, two narrow linewidth excitation light sources 102 may be coupled into a same waveguide or to an optical fiber on the same optical axi s . The two or more narrow linewidth excitation sources 102 may be configured to provide excitation beams having different wavelengths . A waveguide may be configured to support the propagation of both wavelengths entering it . An example of a waveguide is an optical f ibre designed to support propagation of different wavelengths or a photonic integrated circuit delivering or combining multiple wavelength light in waveguides produced for example by silicon dioxide or silicon nitride on a silicon . In this case , the bandpass filter 110 and the optical focusing element 112 may be configured to enable transmission of two or more excitation wavelengths .

The apparatus 100 may further comprise a beamshaping component . The beam shaping component may be conf igured to correct a profile of the excitation beam when the profile does not meet beam shape requirements . The beam shape requirements may depend on, for example , properties of the sample . The beam-shaping component may be pos itioned to a location before the excitation beam is collimated by the optical focusing component 112 . The beam-shaping component may be configured, for example , between the bandpass filter 110 and the focusing component 112 . In addition, or alternatively, the beam-shaping component may be configured between the focusing component 112 and the excitation region 104 . The beamshaping component may comprise one or more cylindrical lenses . The beam-shaping component may comprise , for example , a diffractive optical element (DOE ) . The beamshaping component may be configured to adj ust a geometrical shape and/or si ze of the beam emitted by the narrow linewidth excitation light source 102 . The geometrical shape may comprise any of a line , a rectangular, a square , or a circular shape , for example . The geometrical shape and/or si ze may be adj usted based on used flow cell parameters . The beam-shaping component may be also configured to correct the beam shape to obtain a multispot geometry if required by the application . A multispot geometry may refer to a pattern of multiple circular shapes . A single beam-shaping component may be configured to produce two or more beam shapes and/or beam si zes . Moreover, a combination of two or more beamshaping components may be configured to provide further shape and/or si ze adj ustment of the beam . Hence , performance of the apparatus 100 may be optimi zed . The beam-shaping component ( s ) and desired output may be defined by the flow cell arrangement , such as a material or a thickness of the sample . Thus , the beam-shaping components may be configured to be switchable to enable the apparatus 100 to be compatible with various flow cell designs .

The apparatus 100 may comprise an optical guiding component 106 for guiding the fluorescence light emitted from the excitation region 104 after receiving the excitation beam . The optical guiding component 106 may comprise one or more lenses . The optical guiding component 106 may comprise , for example , a microscope obj ective . For example , the microscope obj ective may be configured to collimate the emitted fluorescence light to guide the fluorescence light to an image sensor 108 . The optical guiding component 106 may be selected based on parameters comprising at least one of a material of the sample , a geometrical design of the sample , a distance between the optical focusing component 112 and the excitation region 104 , magnification properties and/or a numerical aperture (NA) . Position of the optical guiding component 106 may be selected such that a maj or fraction of cells passing through a flow cell may be configured in focus when recorded by the image sensor 108 .

The apparatus 100 may further comprise at least one image sensor 108 . The image sensor 108 may be configured for hyperspectral imaging . The image sensor 108 may comprise , for example , a spectrally-resolving sensor . An example of a spectrally-resolving sensor is a spectrally resolving detection array ( SRDA) . The image sensor 108 may further compri se a spectral fi lter . The spectral filter may comprise , for example , a static pixeli zed filter or an adj ustable dynamic filtering element . The spectral filter may be configured to have any mosaic-like geometry . The static pixeli zed filter may comprise , for example, a mosaic filter . An example of a mosaic filter may be a Fabry-Perot interference filter or a Bayer filter . The Fabry-Perot interference filter may be also known as an optical Fabry-Perot filter or a narrow band-pass filter . Geometry of the spectral filter can be that of Fabry-Perot ( FP) interference filters , consisting of two highly reflective plates constituting a resonating cavity . Wavelength selectivity inside the resonating cavity may be achieved by multiple beam interferences . The FP filters may also be reali zed with adj ustable resonance by electric field, for example , based on a liquid crystal layer (LCD) or a microelectromechanical system (MEMS ) . The FP filters may be directly appl ied on top of , for example , a CMOS ( complementary metal-oxide-semiconductor ) or a CCD ( chargecouple device ) . For example , the apparatus 100 may be configured to cause the emitted fluorescence light to be imaged onto the SRDA sensor after filtering by a monolithic mosaic filter . The spectral filter may comprise a plurality of filters on top of the image sensor arranged in a mosaic like manner, so that data recorded by the plurality of filters with one wavelength can afterwards be combined to form a spectral sub image . The image sensor 108 may comprise , for example , a hyper- spectral snapshot camera .

The apparatus 100 may be further configured to send the image data to an output device . The output device may comprise , for example , a display . The apparatus 100 may be further configured to send the image data to a computing device for analysis . The output device and the computing device may be external devices , or the apparatus 100 may comprise at least one of the devices .

Excitation and detection of fluorescence can be implemented in two ways . In a first approach, the apparatus 100 may be configured to generate spatially separated excitation beams on the microcapillary channel , leading to fluorescence image spatial separation on the image sensor, which allows selecting regions of interest . The regions of interest may comprise, for example , regions of intersection of excitation beams with the microcapillary channel , where the emission of fluorescence occurs . The apparatus 100 may be configured to cause data read-out from the selected regions of interest , which may increase frame per second rate of snapshot hyperspectral imaging . In the second approach, the apparatus 100 may be configured to generate spatially overlapping but temporally separated excitation beams . Such approach may permit hyperspectral imaging of a light-emitting particle synchroni zed with pulsed illumination during its movement along the microcapillary channel . This may allow the apparatus to record the hyperspectral images of the particle from non-overlapping pixels at different time moments with subsequent image reconstruction . I llumination duration and sensor exposure time of the apparatus 100 may be configured in accordance with light-emitting particle velocity and its emiss ion intens ity to decrease motion blur of the particle image .

The combination of the narrow bandwidth illumination based on excitation components of the apparatus 100 and multispectral imaging approach based on detection components of the apparatus 100 may provide improved conditions for high-quality imaging of biological cells or other particles of interest . The excitation components of the apparatus may comprise the one or more narrow linewidth excitation sources , the focusing component , the band-pass filter and/or the beam-shaping component . The detection components of the apparatus 100 may comprise to the optical guiding component and the image sensor .

On the detection side , employing the spectrally-resolving sensor such as the SRDA as a detector in the snapshot imaging scheme may al low to reduce the si ze of the apparatus 100 . The si ze of the apparatus may be reduced since dispersing/scanning optical elements may not be needed . Instead, the image sensor may employ a mosaic filter which works as spectrally selective element for each detector pixel . Such configuration may allow obtaining spectral and spatial information at the same time , for example , to fill a spectral data cube . Further, design of the apparatus is simple as there may not be need for the dispersive or scanning elements . Another advantage is an amount of received detectable light emitted by the sample , which may be significantly decreased due to light loss in the dispersive optical elements .

The image sensor and the spectral filter of the apparatus may be selected based on the application . For example , a region of interest in flow cytometry may comprise a visible part of the spectrum ( 400 - 800 nm) , since most of the fluorophores emit light in the region . Hence , in this case the image sensor may be conf igured for cytometrical applications to detect light in the region of 400 - 800 nm specifically . An increment in a number of spectral channels recorded by the image sensor may enable more precise detection of larger number of fluorophores . However, the increment in the number of the spectral channels may decrease the resolution of the images after a de-mosaicing process , since unless a si ze of the image sensor is increased, the lesser number of pixels may be used for composing an image of single bandwidth . The trade-off between quantity and quality may be thus optimised depending on the application . Alternatively, more than one image sensors may be used simultaneously for increasing the number of spectral channels without affecting the quality of the images .

FIG . 2 illustrates an example of an apparatus for fluorescence imaging comprising a plurality of image sensors for hyperspectral imaging, according to an example embodiment . The apparatus 100 may comprise the at least one narrow linewidth excitation light source 102 , the optical system for excitation compri sing any or all of the bandpass filter 110 , the optical focusing element 112 or the beam-shaping element , and an optical system for detection comprising the optical guiding component 106 , as illustrated in FIG . 1 . Therefore , details of the components already discussed are not repeated herein .

The optical system for detection may further comprise an optical beam splitting component 120 configured to split the fluorescence light into a transmitted beam 126 and a reflected beam 124 . The optical beam splitting element 120 may be configured to split the fluorescence light according to its wavelength . The optical beam splitting component 120 may comprise , for example , two triangular glass prism which are attached together such that half of the fluorescence light incident through one side of the first prism is reflected through the first prism and the other half is transmitted through the second prism due to frustrated total internal reflection . Alternatively, the optical beam splitting component 120 may comprise for example a halfsilvered mirror or a dichroic mirrored prism assembly . The optical beam splitting component 120 may be positioned between the optical guiding element 106 and a first image sensor 108 .

The optical system for detection may further comprise at least one mirroring component 122 , such as a mirror . The mirroring component ( s ) 122 may be configured to guide the reflected beam 124 to a second image sensor 108 .

The apparatus 100 may comprise a plurality of image sensors 108 . Although depicted with the first and the second image sensor receiving the transmitted 126 and reflected 124 light , the apparatus 100 may comprise more than two image sensors 108 by arranging a plurality of optical beam splitting components 120 and/or mirroring component 122 for guiding the fluorescence light to the plurality of image sensors 108 . Each image sensor 108 may comprise a spectrally resolving sensor and a spectral filter . For example , any or all of the plurality of image sensors 108 may comprise a Fabry- Perot filter or a Bayer filter configured to convert complementary CMOS /CCD array into a respective SRDA . Alternatively, any existing or a future spectrally-re- solving sensor and/or spectral filtering component may be part of the image sensor 108 . In general , an operational principle of Fabry-Perot or Bayer filter may rely on Fabry-Perot resonance which is tuned to work as a spectral filter for a corresponding pixel in the CMOS /CCD array . Depending on the fluorophores of interest and optimal resolution, the image sensor 108 may enable resolving up to a few tens of spectral bands within a span of several hundreds of nm . The number of resolved spectral channels ( spectral resolution) may be reversely proportional to the spatial resolution of the detector . As mentioned, this may lead to a trade-off between the number of spectral channels and resolution of the images after a de-mosaicing process . Hence , the apparatus 100 may comprise several SRDAs with mosaic filters designed to cover different spectral bands and configured to be used simultaneously in order to increase detectable spectral range of the hyperspectral imaging .

Instead of using a separate sensor for each wavelength detection, a separate image sensor may be used to cover different spectral regions . Thus , even despite the apparatus 100 may comprise a dispersive element such as a glas s prism, the si ze of the apparatus 100 may be kept compact with a smaller amount of image sensors .

A geometrical arrangement of the apparatus 100 illustrated in FIGS 1 and 2 may employ a geometry, where all the components of the apparatus 100 for excitation and detection are located on the same side with respect to the one or more excitation regions 104 . In other words , the excitation region 104 may be located, for example , above or below each component of the apparatus 100 . For example , each of the narrow l inewidth excitation light source ( s ) , the imaging sensor arrangement , and associated optical components may be located on a same side with respect to the flow channel 114 , or in general a sample holder . Thus , the whole optical system provided by the apparatus 100 can be compatible with any flow cell design after focus calibration of the optical focusing component 112 and the optical guiding component 106 . Further, the narrow linewidth excitation light sources 102 may be in an angled position in relation to the detection components of the apparatus 100 . Hence , optical path of the excitation beam may dif fer from an optical path of the fluorescence light used for imaging . The narrow l inewidth excitation sources 102 may be inclined, for example , such that an angle between the optical path 116 of the generated excitation beam is less than 90 degrees or less than 45 degrees with respect to the optical path 118 , 124 , 126 of the fluorescence light received by the image sensor 108 . This enables , that the emitted or ref lected excitation l ight may not cause information loss on the fluorescence imaging .

FIG . 3 il lustrates an example of a method 300 for fluorescence imaging according to example embodiment . The method may be carried out, for example , by the apparatus 100 .

At an operation 302 , the method may comprise providing, by at least one narrow linewidth excitation light source , a beam for f luorescence excitation at an excitation region located at an optical path of the beam .

At an operation 304 , the method may comprise guiding, by at least one optical guiding component , fluorescence light generated by the beam at the excitation region to an image sensor for hyperspectral imaging .

At an operation 306 , the method may comprise performing hyperspectral imaging, by at least one image sensor comprising a spectral filtering component , based on the fluorescence light received via the at least one optical guiding component .

Further features of the method ( s ) directly result , for example , from functionalities of the apparatuses described throughout the specification and in the appended claims and are therefore not repeated here . Different variations of the method ( s ) may be also appl ied, as described in connection with the various example embodiments . It is obvious to a person skilled in the art that with the advancement of technology, the basic idea of the disclosure may be implemented in various ways . The di sclosure and the embodiments are thus not limited to the examples described above , instead they may vary within the scope of the claims .

An apparatus may be configured to perform or cause performance of any aspect of the method ( s ) described herein . Further, a computer program may comprise instructions for causing, when executed, an apparatus to perform any aspect of the method ( s ) described herein . Further, an apparatus may comprise means for performing any aspect of the method ( s ) described herein . According to an example embodiment , the means compri ses at least one processor, and memory including program code , the at least one processor, and program code configured to , when executed by the at least one processor, cause performance of any aspect of the method ( s ) .

Any range or device value given herein may be extended or altered without losing the effect sought . Also , any embodiment may be combined with another embodiment unless explicitly disallowed . Although the subj ect matter has been described in language specific to structural features and/or acts , it is to be understood that the subj ect matter defined in the appended claims is not necessarily limited to the specific features or acts described above . Rather, the specific features and acts described above are disclosed as examples of implementing the claims and other equivalent features and acts are intended to be within the scope of the claims .

It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments . The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benef its and advantages . It wi ll further be understood that reference to ' an ' item may refer to one or more of those items .

The operations of the methods described herein may be carried out in any suitable order, or simultaneously where appropriate . Additionally, individual blocks may be deleted from any of the methods without departing from the scope of the subj ect matter described herein . Aspects of any of the embodiments described above may be combined with aspects of any of the other embodiments described to form further embodiments without losing the effect sought .

The term ' comprising ' is used herein to mean including the method, blocks , or elements identified, but that such blocks or elements do not comprise an exclusive list and a method or apparatus may contain additional blocks or elements .

Although subj ects may be referred to as ' first ' or ' second' subj ects , this does not necessarily indicate any order or importance of the subj ects . Instead, such attributes may be used solely for the purpose of making a difference between subj ects . It wi ll be understood that the above description is given by way of example only and that various modif ications may be made by those ski lled in the art . The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments . Although various embodiments have been described above with a certain degree of particularity, or with reference to one or more individual embodiments , those skilled in the art could make numerous alterations to the disclosed embodiments without departing from scope of this specification .