CN120253628A - Cell spectral imaging device and method - Google Patents

Abstract

Translated fromChinese

本发明涉及光谱成像技术，具体提供了细胞光谱成像装置和方法，成像装置包括光源和探测器；还包括：调节单元反射所述光源的出射光，使得反射光在两个维度扫过通道内流动的细胞，所述细胞染有荧光素；分光单元将被激发细胞发出的荧光分光，所述探测器输出与位置和波长相关的三维光谱数据；处理单元用于对所述三维光谱数据进行解谱，获得多通道图像；控制器用于驱动所述调节单元和探测器采样触发。本发明具有分辨率高等优点，应用在单细胞分析中。

The present invention relates to spectral imaging technology, and specifically provides a cell spectral imaging device and method, wherein the imaging device includes a light source and a detector; further includes: an adjustment unit reflects the emitted light of the light source, so that the reflected light scans the cells flowing in the channel in two dimensions, and the cells are stained with fluorescein; a spectroscopic unit spectroscopically separates the fluorescence emitted by the excited cells, and the detector outputs three-dimensional spectral data related to the position and wavelength; a processing unit is used to despectrify the three-dimensional spectral data to obtain a multi-channel image; and a controller is used to drive the adjustment unit and the detector to sample and trigger. The present invention has the advantages of high resolution and is applied in single cell analysis.

Description

Cell spectrum imaging device and method

Technical Field

The invention relates to a spectrum imaging technology, in particular to a cell spectrum imaging device and a cell spectrum imaging method.

Background

Currently, fluorescence spectrum imaging of cells is difficult, specifically, after fluorescent staining of cells, cells are placed on a glass slide, and imaging of fluorescent dyes in cells is obtained by confocal laser microscopy scanning. This allows higher resolution quality of the cell fluorescence image, but suffers from the disadvantage:

1. the collection efficiency is lower, the imaging speed is low, meanwhile, the fluorescent channels which can be collected are limited and are subject to wavelength interference of fluorescent dyes, the use of the fluorescent channels is very limited, and the fluorescent channels are very unfavorable for observing a plurality of fluorescent channels.

As shown in FIG. 1, a plurality of tissues exist in the cell, different proteins are dyed with different luciferins through fluorescent staining, and from the perspective of space, a plurality of tissues are overlapped, and the traditional method can only carry out space imaging on single or a plurality of fluorescent dye marked proteins and cannot solve the problem of simultaneous imaging of multiple fluorescence.

2. The traditional laser confocal microscope can only scan a fixed sample point by point under a static condition, the spatial resolution is limited by Abbe diffraction limit, and meanwhile, polychromatic fluorescence depends on the center wavelength lambdac and half-width delta lambda of the optical filter, so that wave band overlapping and signal crosstalk are caused.

3. The existing flow cytometer realizes the throughput rate of 10⁴cells·s^-1 grades, but the sub-pixel level sub-band information cannot be analyzed by adopting a broadband integrated detector, so that the multi-fluorescence distribution in the cell sub-compartment is difficult to reveal.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a cell spectrum imaging device.

The invention aims at realizing the following technical scheme:

a cell spectrum imaging device comprises a light source and a detector, and further comprises:

a regulating unit that reflects the outgoing light of the light source such that the reflected light sweeps a cell flowing in a channel in two dimensions, the cell being stained with fluorescein;

A spectroscopic unit that splits fluorescence emitted from the excited cells, the detector outputting three-dimensional spectral data relating to position and wavelength;

The processing unit is used for carrying out spectrum decomposition on the three-dimensional spectrum data to obtain a multi-channel image;

And the controller is used for driving the adjusting unit and the detector to sample and trigger. .

The invention also aims to provide a cell spectrum imaging method, and the aim of the invention is achieved through the following technical scheme.

A method of cell spectroscopic imaging comprising the steps of:

the outgoing light of the light source is reflected by the adjusting unit, so that the reflected light sweeps cells flowing in the channel in two dimensions, and the cells are stained with fluorescein;

the excited cells emit fluorescence, and the fluorescence is received by the detector after being split, so that three-dimensional spectrum data related to the position and the wavelength are output;

The processing unit performs spectrum decomposition on the three-dimensional spectrum data to obtain a multi-channel image;

In the above process, the controller controls the movement of the adjusting unit and the sampling trigger of the detector.

Compared with the prior art, the invention has the following beneficial effects:

According to the invention, the rapid two-dimensional scanning of the single cell dyed with the fluorescein is realized by utilizing the adjusting unit, the fluorescence full spectrum is obtained, and then the fluorescent component is obtained through the light splitting and the spectrum splitting, so that the fluorescent signal on each pixel point of the cell is obtained, and the imaging of the flow type rapid single cell multi-fluorescent component is realized.

1. Achieving synchronization of a spatial resolution of less than 2 μm with a spectral resolution of less than 0.15 nm;

2. The acquisition time of each cell is less than 2 mu s, and can reach 5 multiplied by 10⁴cells·s^-1 real-time multi-fluorescence mapping;

3. by using regularized non-negative matrix factorization, the upper limit of the fluorescence channel is raised to 40+, and the crosstalk suppression ratio is raised by 8dB.

Drawings

The present disclosure will become more readily understood with reference to the accompanying drawings. It is to be understood by those skilled in the art that these drawings are for illustrative purposes only and are not intended to limit the scope of the present invention. In the figure:

FIG. 1 is a schematic diagram of a cell;

FIG. 2 is a schematic diagram of a cell spectrum imaging device according to the present invention;

FIG. 3 is a schematic representation of a two-dimensional scan of cells according to the present invention;

fig. 4 is a schematic illustration of a solution according to the present invention.

Detailed Description

Fig. 2-4 and the following description depict alternative embodiments of the invention to teach those skilled in the art how to make and reproduce the invention. In order to teach the technical solution of the present invention, some conventional aspects have been simplified or omitted. Those skilled in the art will appreciate variations or alternatives derived from these specific embodiments that fall within the scope of the invention. Those skilled in the art will appreciate that the features described below can be combined in various ways to form multiple variations of the invention. Thus, the invention is not limited to the following alternative embodiments, but only by the claims and their equivalents.

Example 1.

As shown in fig. 2, the cellular spectrum imaging apparatus of the present embodiment includes:

the light source and the detector, the light source usually adopts a laser, and the detector adopts a linear array photoelectric detector.

An adjustment unit, such as a galvanometer or a rotating prism, is used to reflect the outgoing light of the light source, as shown in fig. 3, so that the reflected light sweeps cells flowing in the channel in two dimensions, the cells being stained with fluorescein.

And the light splitting unit is used for splitting the fluorescence emitted by the excited cells, and the detector outputs three-dimensional spectrum data related to the position and the wavelength.

And the processing unit is used for carrying out spectrum decomposition on the three-dimensional spectrum data to obtain a multi-channel image, such as a spectrum decomposition software, as shown in fig. 4.

And the controller is used for driving the adjusting unit and the detector to sample and trigger.

In order to improve the speed and accuracy of the solution spectrum, further, the processing unit solves through a non-negative matrix factorization algorithm with regularization term, and the objective function is that,S=MC+ε。

S is a spectrum matrix output by the detector, M is a reference spectrum base matrix, C is an abundance matrix to be calculated, alpha is a space smooth regular weight (a dynamic range with a value of 0.01-0.1,10 times covers a prototype test interval of 2dB-20dB of common SNR),Is the gradient of C in the x, y dimension of the pixel grid,Is the Frobenius norm and epsilon is the measurement noise.

In order to control the operation of the adjusting unit, further, the adjusting unit adopts a galvanometer, and the scanning frequency f_scan and the center flow velocity v_flow in the channel satisfy the following conditions:

f_scan=v_flow/Δx, Δx is the pixel step size, referring to the sampling pitch on the lateral pixel grid. The vibrating mirror is driven by triangular waves, and the angular frequency omega=2pi f_scan.

For spectral imaging, further, the collimating lens, the variable diffraction diaphragm, the adjusting unit, the objective lens and the flow pipeline are sequentially arranged on the emergent light path, the flow pipeline, the light splitting unit and the detector are sequentially arranged on the fluorescent light path, and emergent light forms an Airy spot incident on cells after the objective lens.

The cell spectrum imaging method of the embodiment of the invention, namely the working method of the imaging device of the embodiment, comprises the following steps:

The outgoing light of the light source is reflected by the regulating unit such that the reflected light sweeps in two dimensions over cells flowing in the channel, said cells being stained with fluorescein.

The excited cells emit fluorescence, which is received by a detector after being split, thereby outputting three-dimensional spectral data related to position and wavelength.

And the processing unit performs spectrum decomposition on the three-dimensional spectrum data to obtain a multi-channel image.

In the above process, the controller controls the movement of the adjusting unit and the sampling trigger of the detector.

In order to improve the speed and accuracy of the spectrum decomposition, further, the spectrum decomposition method comprises the following steps:

A reference spectral base matrix M is constructed and solved using an alternate direction multiplier method.

,S=MC+ε。

S is a spectrum matrix output by the detector, C is an abundance matrix to be solved, alpha is a space smooth regular weight,Is the gradient of C in the x, y dimension of the pixel grid,Is the Frobenius norm and epsilon is the measurement noise.

Mapping each row in the abundance matrix C to be solved to a pixel grid (x, y) to obtain a multichannel image G (x, y).

In this embodiment, the weight α is obtained in the following manner:

The first term in the objective function ensures that the solution spectrum residual is minimal, and the second term is constrained by pixel-spectral gradientsThe suppression of streaks/noise, α, is the weight of both terms.

The dimension and the scale are unified.

Normalization, namely column vector l_2-norm normalization is carried out on the original spectrum S and the base matrix M, so that |S|_F|MC|_F is carried out.

Scaling the gradient term, if the pixel step length Deltax of each frame is different, the methodMultiplying by Deltax (or Deltat) is normalized so that the two norms|And F falls approximately between 0 and 1.

Through this step, |S-MC|_F2 and|on the same datasetThe magnitude of F2 is comparable, which is why the interval 0.01-0.1 can cover 10 times the dynamic range, but not too much or too little.

Example 2.

Application example of the cell spectrum imaging apparatus and method according to embodiment 1 of the present invention.

1. As shown in fig. 2, in the outgoing light path, the light source uses a laser, and the outgoing light has a center wavelength λ₀ =488 nm and a power P_exc =30 nW. The emergent light sequentially passes through the collimating lens L₁ and the variable diffraction diaphragm.

The adjusting unit adopts a two-dimensional scanning galvanometer G₁ to drive with triangular waves, and the angular frequency ω=2pi f_scan.

The cell diameter is 12 μm, the target lateral pixel step size Δx=0.4 μm, f_scan=v_flow/Δx≈5·10⁵ Hz, single pixel dwell time τ_pix=1/f_scan =2 μs.

On the outgoing light path, an objective lens (na=0.95, f=200 mm) forms an airy spot radius W₀≈1.22λ₀/(2 NA) =0.31 μm.

On the fluorescence path, the light-splitting unit adopts a transmission type prismatic grating light-splitting module (linear density is 1200 lp mm^-1), and the spectral resolution is estimated as follows:

δλ=d/(mNcos β) ≡0.12nm, diffraction order m=1, grating constant d=0.833 μm, illumination groove number n=2400, diffraction angle β=15 degrees.

2. In the flow path, cell suspension 10⁶cells mL^-1 was arranged, and the sheath: sample=100:1, and the liquid injected into the capillary was 50. Mu.L. In the flow line, the capillary inner diameter D_h = 120 μm, the flow velocity v_flow=0.2m·s^-1. The cells were centered after focusing by sheath flow with radial drift less than 1 μm.

The corresponding reynolds number re=ρ·v_flow·D_h/a≡24, the laminar structure is formed, ρ is the fluid density and a is the dynamic viscosity.

3. The controller takes 200MHzFPGA as a core and generates CMOS multichannel APD sampling trigger of the galvanometer driving and detecting device. The time alignment error δt of the same pixel is smaller than 5ns, and may be regarded as δy=v_flow ·δt not larger than 1nm, which is negligible.

4. The spectrum resolution mode of the processing unit is as follows:

the detector outputs a three-dimensional hyperspectral data cube I (x, y, λ), (x, y) being a grid of pixels.

A reference spectral base matrix M (mxn, cells stained with n dyes) was constructed and solved using the alternate direction multiplier method.

,S=MC+ε。

S is the spectral matrix (m wavelength samples x p pixels) output by the detector, C is the abundance matrix to be solved (n x p), alpha is the spatially smooth canonical weight,Is the gradient of C in the x, y dimension of the pixel grid,Is the Frobenius norm and epsilon is the measurement noise.

Mapping each row in the abundance matrix C to be solved to a pixel grid (x, y) to obtain a multichannel image G (x, y).

Average photon number per pixelQuantum efficiency η=0.35, h is planck constant, v=c/λ, c is speed of light, corresponding to snr≡21dB.

Claims (10)

1. The cell spectrum imaging device comprises a light source and a detector, and is characterized by further comprising:

a regulating unit that reflects the outgoing light of the light source such that the reflected light sweeps a cell flowing in a channel in two dimensions, the cell being stained with fluorescein;

A spectroscopic unit that splits fluorescence emitted from the excited cells, the detector outputting three-dimensional spectral data relating to position and wavelength;

The processing unit is used for carrying out spectrum decomposition on the three-dimensional spectrum data to obtain a multi-channel image;

and the controller is used for driving the adjusting unit and the detector to sample and trigger.

2. The imaging apparatus of claim 1, wherein the processing unit solves by a non-negative matrix factorization algorithm with regularization term, the objective function being,S=MC+ε,

S is a spectrum matrix output by the detector, M is a reference spectrum base matrix, C is an abundance matrix to be solved, alpha is a space smooth regular weight,Is the gradient of C in the x, y dimension of the pixel grid,Is the Frobenius norm and epsilon is the measurement noise.

3. The imaging apparatus of claim 1, wherein the adjustment unit employs a galvanometer, a scanning frequency f_scan, and an in-channel center flow velocity v_flow satisfying:

f_scan=v_flow/Δx, Δx is the pixel step size.

4. An imaging device according to claim 3, wherein the galvanometer is driven with a triangular wave, with an angular frequency ω = 2ρf_scan.

5. The imaging apparatus of claim 1, wherein the light source is a laser and the light splitting unit is a transmissive grid light splitting module.

6. The imaging apparatus of claim 5, wherein a collimating lens and an iris diaphragm, an adjusting unit, an objective lens and a flow path are sequentially disposed on the outgoing light path, and a flow path, a spectroscopic unit and a detector are sequentially disposed on the fluorescent light path, and the outgoing light forms airy spots incident on cells after the objective lens.

7. A method of cell spectroscopic imaging comprising the steps of:

the outgoing light of the light source is reflected by the adjusting unit, so that the reflected light sweeps cells flowing in the channel in two dimensions, and the cells are stained with fluorescein;

the excited cells emit fluorescence, and the fluorescence is received by the detector after being split, so that three-dimensional spectrum data related to the position and the wavelength are output;

The processing unit performs spectrum decomposition on the three-dimensional spectrum data to obtain a multi-channel image;

In the above process, the controller controls the movement of the adjusting unit and the sampling trigger of the detector.

8. The imaging method of claim 7, wherein the means for resolving the spectrum is:

constructing a reference spectrum base matrix M, solving by using an alternate direction multiplier method,

,S=MC+ε,

S is a spectrum matrix output by the detector, C is an abundance matrix to be solved, alpha is a space smooth regular weight,Is the gradient of C in the x, y dimension of the pixel grid,Is the Frobenius norm and epsilon is the measurement noise;

Mapping each row in the abundance matrix C to be solved to a pixel grid (x, y) to obtain a multichannel image G (x, y).

9. The imaging method of claim 7, wherein the adjustment unit employs a galvanometer, a scanning frequency f_scan, and an in-channel center flow velocity v_flow that satisfy:

f_scan=v_flow/Δx, Δx is the pixel step size.

10. The imaging method of claim 7, wherein the outgoing light passes through the collimating lens and the variable diffraction diaphragm in order, is reflected by the adjusting unit, and passes through the objective lens to form an airy disk incident on the cell in the flow path.