Fluorescence detection system

Abstract

Single molecular measurement is conducted with a high throughput. Fluorescence from labeled target molecules flowing a sample flow cell is measured by a line CCD element to realize single molecular measurement with a high throughput. Where, the number of photo detecting pixels of the line CCD element is smaller than a value obtained by dividing an exposure time by pixel transfer rate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a configuration diagram of a detecting system for realizing a conventional method;

FIG. 2 is an enlarged view of an area irradiated with laser in capillary;

FIG. 3 is an example of a configuration of a fluorescence detection system according to the present embodiment;

FIG. 4 is a schematic diagram of a sample flow cell;

FIG. 5 is a time table of exposure time in an example of an embodiment using a sample flow cell and line sensor;

FIG. 6 is a graph showing the relationship between average flow velocity and sensitivity;

FIG. 7 is an example of a configuration of a fluorescence detection system according to the present embodiment;

FIG. 8 is a schematic diagram of a sample flow cell;

FIG. 9 is an example of an image of fluorescent molecules;

FIG. 10 is a schematic cross section of a sample flow cell;

FIG. 11 a schematic cross section of a sample flow cell using low-refractive index thin film;

FIG. 12 a schematic cross section of a sample flow cell using a dielectric thin film;

FIG. 13 is a schematic diagram of a photo detecting CCD element;

FIG. 14 is a time table obtained for cases where the conditional expression (1) is satisfied;

FIG. 15 is a graph showing measurement results on change in SN ratio with respect to a normalized average velocity.

FIG. 16 is a graph of measurement results on relationship between counts of molecule and concentration;

FIG. 17 is a schematic diagram of a photo detecting CCD element;

FIG. 18 is an example of a configuration of a fluorescence detection system according to the present embodiment;

FIG. 19 is an example of a configuration of a fluorescence detection system according to the present embodiment;

FIG. 20 is a schematic diagram of a sheath flow cell; and

FIG. 21 is a top view of a sample flow cell.

Claims

1. A fluorescence detection system comprising: a flow cell including a flat channel greater in width than in height therein;a sample solution inlet which introduces sample solution into the flow cell while controlling a flow velocity thereof;an optical irradiation section which emits exciting light focused into a circular spot substantially perpendicularly to the direction in which the sample solution flows from the widthwise direction of the channel;a photo detecting device including a rectangular photo detecting section;an objective lens which forms a fluorescent image produced in the sample solution by irradiating the exciting light on the photo detecting section of the photo detecting device; anda two-dimensional fluorescent image generating means which coupling the outputs of the photo detecting device in time series to generate a two-dimensional fluorescent image; whereinM- and N-pieces of photo detecting pixels are arranged along the long and the short side of the rectangular photo detecting section of the photo detecting device respectively, where N is smaller than M and is an integer of 1 or more, and the long side is arranged along the direction in which the exciting light propagates inside the channel,a time Trans during which data of electric charges transformed from exposure detected by the M- and N-pieces of photo detecting pixels within an exposure time Texp are outputted to the outside of the photo detecting device is shorter than the exposure time Texp of the M- and N-pieces of photo detecting pixels.

2. The fluorescence detection system according to claim 1, wherein the N is two or more, data of electric charges transformed from exposure detected by each of the photo detecting pixels are added by N-pieces of photo detecting pixels themselves adjacent in the direction of the short side and outputted to the outside of the photo detecting device as M×1 pieces of one-dimensional arrangement data, and the two-dimensional fluorescent image generating means arranges the one-dimensional arrangement data in time series in the direction perpendicularly to the arrangement direction to generate a two-dimensional fluorescent image.

3. The fluorescence detection system according to claim 1, wherein the sample solution uniformly flows through the channel.

4. The fluorescence detection system according to claim 1, wherein the objective lens is 0.75 or more in numerical aperture.

5. The fluorescence detection system according to claim 1, wherein if the magnification of the objective lens is taken to be m, the center wavelength of the fluorescence is taken to be λ and a length of the photo detecting pixel perpendicular to the direction in which the sample solution flows is taken to be w, w/m≦λ is satisfied.

6. The fluorescence detection system according to claim 2, wherein the sample solution includes fluorescence labeled molecules and the system comprises counting means which counts the number of fluorescence labeled molecules based on the two-dimensional fluorescent image.

7. The fluorescence detection system according to claim 1, wherein the flow cell includes an inlet for sheath flow which flows while enveloping the sample solution.

8. A fluorescence detection system comprising: a flow cell including a flat channel greater in width than in height therein;a sample solution inlet which introduces sample solution containing fluorescence labeled molecules into the flow cell while controlling a flow velocity thereof;an optical irradiation section which emits exciting light focused into a spot substantially perpendicularly to the direction in which the sample solution flows from the widthwise direction of the channel;a photo detecting device including a rectangular photo detecting section;an objective lens which forms a fluorescent image produced in the sample solution by irradiating the exciting light on the photo detecting section of the photo detecting device; anda two-dimensional fluorescent image generating means which coupling the outputs of the photo detecting device in time series to generate a two-dimensional fluorescent image; whereinM- and N-pieces of photo detecting pixels are arranged along the long and the short side of the rectangular photo detecting section of the photo detecting device respectively, where N is smaller than M and is an integer of 1 or more, and the long side is arranged along the direction in which the exciting light propagates inside the channel,the photo detecting device outputs data of electric charges transformed from exposure detected by the M- and N-pieces of photo detecting pixels within an exposure time Texp to the outside of the photo detecting device as one unit, andif an average velocity of the sample solution flowing through an area in the channel irradiated with the exciting light is taken to be v, the length of the short side of the photo detecting pixel is taken to be 1p, the magnification of the objective lens is taken to be m, and the average of the number of photo detecting pixels is taken to be Ns when one fluorescent label is detected while spanning a plurality of photo detecting pixels to the long-side direction on the photo detecting device, the following condition is satisfied: (Ns×1p)/(m×Texp)≦v≦(N×1p) (m×Texp).

9. The fluorescence detection system according to claim 8, wherein the N is two or more, data of electric charges transformed from exposure detected by each of the photo detecting pixels are added by N-pieces of photo detecting pixels themselves adjacent in the direction of the short side and outputted to the outside of the photo detecting device as M×1 pieces of one-dimensional arrangement data, and the two-dimensional fluorescent image generating means arranges the one-dimensional arrangement data in time series in the direction perpendicularly to the arrangement direction to generate a two-dimensional fluorescent image.

10. The fluorescence detection system according to claim 8, wherein the sample solution uniformly flows through the channel.

11. The fluorescence detection system according to claim 8, wherein the objective lens is 0.75 or more in numerical aperture.

12. The fluorescence detection system according to claim 8, wherein if the magnification of the objective lens is taken to be m, the center wavelength of the fluorescence is taken to be λ and a length of the photo detecting pixel perpendicular to the direction in which the sample solution flows is taken to be w, w/m≦λ is satisfied.

13. The fluorescence detection system according to claim 9, comprising counting means which counts the number of fluorescence labeled molecules based on the two-dimensional fluorescent image.

14. The fluorescence detection system according to claim 8, wherein the flow cell includes an inlet for sheath flow which flows while enveloping the sample solution.

15. A fluorescence detection system comprising: a flow cell including a flat channel greater in width than in height therein;a sample solution inlet which introduces sample solution containing fluorescence labeled molecules into the flow cell while controlling a flow velocity thereof;an optical irradiation section which emits exciting light focused into a spot substantially perpendicularly to the direction in which the sample solution flows from the widthwise direction of the channel;a photo detecting device including a rectangular photo detecting section;an objective lens which forms a fluorescent image produced in the sample solution by irradiating the exciting light on the photo detecting section of the photo detecting device;a two-dimensional fluorescent image generating means which generates a two-dimensional fluorescent image by arranging one-dimensional arrangement data outputted from the photo detecting device in time series in the direction perpendicularly to the arrangement direction; andmeasuring means which measures the size and intensity of fluorescent image in the two-dimensional fluorescent image obtained by the above means; whereinM- and N-pieces of photo detecting pixels are arranged along the long and the short side of the rectangular photo detecting section of the photo detecting device respectively, where N is smaller than M and is an integer of 1 or more, and the long, side is arranged along the direction in which the exciting light propagates inside the channel,if there are charges generated by photo detection and photo detecting pixels adjacent to the upstream in the direction of the short side, each of the photo detecting pixels of photo detecting section sequentially transfers electric charges consisting both of an electric charge transferred from the photo detecting pixel adjacent to an upstream and an electric charge generated by the photo detection to the photo detecting pixel adjacent to the downstream in the direction of the short side at a predetermined period and outputs the charges outside the photo detecting device as M×1 pieces of one-dimensional arrangement data, andthe system further comprises flow velocity adjusting means which adjusts the flow velocity of the sample solution by the period and the sample solution inlet based on the size and intensity of fluorescent image in the two-dimensional fluorescent image measured by the measuring means.

16. The fluorescence detection system according to claim 15, wherein if an average velocity of the sample solution of the fluorescence labeled molecules flowing through an area irradiated with the exciting light is taken to be v, the length of the short side of the photo detecting pixel is taken to be 1p, the magnification of the objective lens is taken to be m, the period is taken to be Tf and data transfer velocity from the photo detecting device to the outside is f, the following relation is satisfied: v≈1p/(m×Tf)Tf≧M/f

17. The fluorescence detection system according to claim 15, wherein the sample solution uniformly flows through the channel.

18. The fluorescence detection system according to claim 15, wherein the objective lens is 0.75 or more in numerical aperture.

19. The fluorescence detection system according to claim 15, wherein if the magnification of the objective lens is taken to be m, the center wavelength of the fluorescence to be λ and a length of the photo detecting pixel perpendicular to the direction in which the sample solution flows to be w, w/m≦λ is satisfied.

20. The fluorescence detection system according to claim 15 comprising counting means which counts the number of fluorescence labeled molecules based on the two-dimensional fluorescent image.

21. The fluorescence detection system according to claim 15, wherein the flow cell includes an inlet for sheath flow which flows while enveloping the sample solution.