JP2019039993A - Fluorescence observation filter and fluorescence observation microscope - Google Patents

Abstract

To provide a small and highly portable lensless fluorescence observation filter and a fluorescent observation microscope using the same, including fewer own fluorescent component, capable of removing excitation light component with high performance, and minimizing reduction in spatial resolution of a lensless optical system.SOLUTION: There is provided a fluorescence observation filter for lensless fluorescent imaging for observing fluorescent light generated by irradiating a sample with excitation light using an image sensor, which comprises: an interference filter 2 and an absorption filter 3 which are identical to each other in permeation characteristics of excitation light wavelength; and a light incident angle restriction layer 4 in which excitation light wavelength absorbers extending in a thickness direction are dispersedly arranged. The fluorescence observation filter is provided by stacking the interference filter 2 and the absorption filter 3 with the light incident angle restriction layer 4 interposed therebetween. The observation sample is arranged immediately above the interference filter 2, and an image sensor 5 is arranged immediately below the absorption filter 3.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a fluorescence observation filter that does not use a lens and a fluorescence observation microscope having a lensless optical system.

  Conventionally, fluorescence observation technology has been widely used in the fields of life science and medical examination such as biological measurement, cell measurement, and chemical analysis. In the field of living body observation and cell observation, various phenomena can be observed by expressing fluorescence only at a specific site by staining or genetic modification. In particular, in chemical analysis, specific molecules can be detected by combining an antigen-antibody reaction or a fluorescent substrate with an enzyme reaction. Fluorescence observation is widely used for detecting proteins and viruses. Yes.

However, a fluorescence observation microscope that performs fluorescence observation has a point that an optical microscope using a lens can exhibit high performance compared to a microscope that does not use a lens. There is a problem of high cost.
Hitherto, high-precision fluorescence observation technology has been developed and put into practical use, but one of the problems is that the fluorescence observation microscope is large and expensive. In order to prevent the spread of infectious diseases, there is a demand for a small and simple fluorescence observation microscope that can be used even in an environment where medical facilities are not provided.

  As a small-sized and simple fluorescence observation microscope, there is known a configuration in which an observation sample is arranged immediately above an image sensor, with a simple configuration using a lensless optical system that does not use a lens in the optical system. In that case, as shown in FIG. 13, an observation sample 7 such as a cell to be observed or a bead modified with an antibody is arranged immediately above the image sensor device 5, and excitation light 8 is irradiated from directly above the observation sample 7. Then, the fluorescence of the fluorescent material 7a of the observation sample 7 is captured by the two-dimensional image sensor 5a. An excitation light removal filter 1 that removes the excitation light 8 is provided between the two-dimensional image sensor 5 a and the observation sample 7. The excitation light removal filter 1 removes the excitation light transmitted through the observation sample 7 and transmits the fluorescence of the fluorescent material 7a, thereby increasing the fluorescence sensitivity in the two-dimensional image sensor 5a.

As described above, in a fluorescence observation microscope of a lensless optical system, an excitation light removal filter is used as in a general fluorescence microscope, but only an interference filter is often used as this excitation light removal filter ( For example, see Non-Patent Document 1.) Further, when the interference filter is used under appropriate conditions, the interference filter can make the transmittance ratio of excitation light and fluorescence about 10 ⁵ or more.
As described above, although the interference filter has high performance, it has angle dependency. As shown in FIG. 14, the excitation light 8a incident perpendicularly to the filter surface of the interference filter 2 is reflected without passing through the interference filter 2 (8d). However, when the incident angle is inclined, the excitation light 8b interferes. It passes through the filter 2 (8e). Further, when the excitation light 8c hits the observation sample 7, a part of it is reflected (8f), but a part is scattered and the incident angle is inclined, so that the scattered light 9 passes through the interference filter 2. The scattered light 9 of the excitation light 8c becomes a factor that lowers the sensitivity of the image sensor, and the fluorescence detection performance is deteriorated. In lens optical systems, this problem can be avoided. However, in lensless optical systems, in most cases, the excitation light is scattered by the observation object itself, and a light component that is transmitted by appearing obliquely into the interference filter appears. There is.

In addition, a technique for avoiding transmission of scattering components by using an absorption filter instead of an interference filter as an excitation light removal filter in a fluorescence observation microscope is also known (see, for example, Non-Patent Document 2).
However, in the absorption filter, as shown in FIG. 15, due to the constituent materials of the absorption filter, a part of the energy of the excitation light 8 absorbed becomes autofluorescence 10, and the autofluorescence 10 has fluorescence and wavelength derived from the observation sample. It overlaps and cannot be distinguished in the image sensor and is observed together with the fluorescence derived from the observation sample. When the auto-fluorescence 10 is generated directly above the image sensor, the fluorescence intensity becomes non-negligible, which becomes noise and lowers the S / N ratio. In addition, since the autofluorescence 10 is radiated | emitted isotropic, if it is an optical system which can be separated from an image sensor, it can avoid.
Thus, since the absorption filter absorbs the energy of the excitation light, autofluorescence is generated, so that the excitation light can be removed, but the fluorescence observation performance is degraded by the generation of fluorescence having the same wavelength as the observation target. There is a problem.

On the other hand, in order to reduce the leakage light related to the detection capability of the fluorescence image detection device, there is known a lens optical device in which an interference filter and an absorption filter are arranged in series in the fluorescence traveling direction in the fluorescence side filter section. (See Patent Document 1). As shown in FIG. 16, the fluorescence observation filter used in this apparatus includes an excitation side filter 11 (F _ex ), a fluorescence side multilayer film interference filter 12 of the fluorescence side filter section (F _em ), and an absorption filter 13. Is done. Excitation light is irradiated to the observation sample 31 from the irradiation unit 30, and the fluorescence of the observation sample 31 is detected by the two-dimensional detector 38 via the imaging lens 32.
The interference filter 12 and the absorption filter 13 are arranged in series in the direction of travel of fluorescence and shield light in a wavelength band corresponding to excitation light irradiated on the sample while sufficiently transmitting light in a wavelength band corresponding to fluorescence. .
However, it is difficult to obtain sufficient observation performance only by the configuration in which the interference filter and the absorption filter are arranged in series in the direction of travel of the fluorescence, and the fluorescence side filter section (F _em in which the interference filter 12 and the absorption filter 13 are arranged in series ) Is followed by a lens optical system configuration using the imaging lens 32 to enhance the fluorescence observation performance.

Republished Patent No. 2010/032306

A. Hassibi et al., ISSCC2017, 4.2. C. Han et al., Anal. Chem. 83, 2356 (2013)

Only the filter configuration in which the interference filter and the absorption filter described above are arranged in series in the fluorescence traveling direction cannot sufficiently secure the fluorescence observation performance. In the case of a lensless optical system, the spatial resolution is lowered, and the lens optical system It is necessary to.
On the other hand, in order to realize a small and simple fluorescence observation microscope, there is a demand for a lens-less optical system and an apparatus configuration in which an observation sample is arranged immediately above an image sensor.

  In view of such circumstances, the present invention is a lensless fluorescence observation filter and a fluorescence observation microscope using the same, and has a low autofluorescence component and can remove excitation light components with high performance. It is an object of the present invention to provide a fluorescence observation filter and a fluorescence observation microscope that are small in size and excellent in portability.

  In order to solve the above-described problems, the fluorescence observation filter of the present invention is a fluorescence observation filter in lensless fluorescence imaging in which fluorescence when an observation sample is irradiated with excitation light is observed with an image sensor, and having an excitation light wavelength. An interference filter layer and an absorption filter layer having the same transmission characteristics, and a light incident angle limiting layer in which absorbers of excitation light wavelengths extending in the thickness direction are dispersedly arranged. In the fluorescence observation filter, the interference filter layer and the absorption filter layer are laminated via the light incident angle limiting layer, or in the fluorescence observation filter, the interference filter layer and the absorption filter layer are laminated, and the light incident angle limiting layer is laminated. Are stacked on the interference filter layer. An observation sample is disposed immediately above the interference filter layer or the light incident angle limiting layer, and an image sensor is disposed immediately below the absorption filter layer.

According to the fluorescence observation filter having the above configuration, the spatial resolution can be achieved even in a simple lensless optical system by simultaneously realizing high excitation light removal performance, reduction of autofluorescence of the filter itself, and removal of scattered components from the observation sample. High sensitivity can be realized.
The interference filter layer and the absorption filter layer to be laminated have the same transmission characteristics at the excitation light wavelength. Here, it is not necessary that the transmission characteristics are the same as each other, and it is only necessary that the transmission characteristics are substantially the same. For example, approximately the same wavelength transmission spectrum, for example, both filters are short-pass filters or approximately the same wavelength. It may be a bandpass filter for the band.

The interference filter layer and the absorption filter layer having the same transmission characteristics are stacked via the light incident angle limiting layer. In the light incident angle limiting layer, the absorber of the excitation light wavelength is distributed in the thickness direction, effectively absorbing the light traveling obliquely from the optical path entering at right angles to the filter surface, and the spatial resolution is reduced To achieve high sensitivity.
Alternatively, an interference filter layer and an absorption filter layer having the same transmission characteristics are laminated, and a light incident angle limiting layer is laminated on the interference filter layer.

In the fluorescence observation filter of the present invention, the refractive index of the light incident angle limiting layer is specifically smaller than the refractive index of the interference filter layer, and the absorber is a spacer between the interference filter layer and the absorption filter layer. Used as
Here, it is a preferable aspect that the spacer is provided in accordance with a boundary portion between the pixel sensors in the image sensor.

Further, as the light incident angle limiting layer, specifically, an air layer, a vacuum layer, or a porous material layer having many minute holes can be used. The absorber functions as a spacer, and maintains an interval between the interference filter layer and the absorption filter layer. By using an air layer as the light incident angle limiting layer, the light transmitted from the interference filter layer side is refracted by the component that passes through the interference filter layer and absorbed by an absorber dispersed in the air layer. Removal performance can be obtained. When fluorescence is observed directly after being inserted into a living body by providing an air layer, a vacuum layer, or a porous material layer as a light incident angle limiting layer between the interference filter layer and the absorption filter layer However, even when the excitation light is scattered in the living body and the angle distribution of the incident light of the fluorescence observation filter becomes wide, it can be dealt with.
For a configuration using an air layer or a vacuum layer as the light incident angle limiting layer, it is necessary to develop a method for peeling and transferring the interference filter layer and a sealing method for preventing the surrounding medium from entering the air layer. In addition, about peeling, it can implement using the method which applied the existing technique, such as peeling using a laser, and the utilization of the filter which pinched | interposed the sacrificial layer.

Here, by reducing the arrangement interval of the absorber, the thickness of the light incident angle limiting layer (any of the air layer, the vacuum layer, and the porous material layer) can be reduced.
In addition, the light incident angle limiting layer (air layer, vacuum layer, porous material layer) is reduced by at least one of shortening the excitation light wavelength or increasing the cutoff wavelength of the interference filter layer. Can be reduced in thickness.

In another aspect of the fluorescence observation filter according to the present invention, the light incident angle limiting layer is a fiber optic plate configured by bundling a plurality of optical fibers, and the absorber is a coating material for each optical fiber ( Included in the cladding).
The fiber optic plate is a plate formed by bundling optical fibers. Here, when the numerical aperture (NA) of the fiber optic plate is larger than 0.7, an interference filter layer and an absorption filter layer are mounted on both sides of the fiber optic plate, respectively, and the observation sample is arranged on the interference filter layer side. Is good. Thereby, most of the energy of the incident excitation light can be removed. Moreover, the rigidity of the entire filter can be increased by using a fiber optic plate as the light incident angle limiting layer. It can be applied in chemical analysis or the like with no restrictions on the filter size.
In the fluorescence observation filter of the present invention, a higher spatial resolution can be expected for bright field observation when the interference filter layer is on the outermost surface. Under conditions where there is a lot of light scattering, such as in living organisms, the scattered excitation light cannot be completely removed by the absorption filter layer alone. Therefore, the scattered excitation light is absorbed by the fiber optic plate placed between the interference filter layer and the absorption filter layer. To be removed.

As the interference filter layer, a generally used configuration can be used. For example, it is formed by laminating a large number of TiO ₂ and SiO ₂ layers. An absorption filter layer is disposed on the lower layer side of the interference filter layer. In general, it is difficult to directly stack an interference filter layer on the absorption filter layer, but the interference filter layer is disposed on the absorption filter layer by providing a fiber optic plate on the light incident angle limiting layer.

When the numerical aperture of the fiber optic plate is smaller than 0.4, the fluorescence observation filter is formed by laminating the interference filter layer and the absorption filter layer, and the fiber optic plate is laminated on the interference filter layer. It is preferable to place it directly above the fiber optic plate. By using a fiber optic plate having a low numerical aperture as the outermost surface, the strength of the surface of the fluorescence observation filter can be increased. Further, even under conditions such as in vivo where light scattering is large, excitation light can be sufficiently reduced and highly sensitive fluorescence observation can be performed.
When using a high spatial resolution fiber optic plate with a numerical aperture of 0.4 or more, the spatial resolution in bright field observation is reduced, but when the numerical aperture is smaller than 0.4, the fiber optic plate and the interference filter Even when the layers are stacked in the order of the absorption filter layer, the reduction in spatial resolution is small. Under conditions where light scattering is relatively small, such as cells and microscopic fluorescent droplets on a DNA chip, the numerical aperture of the fiber optic plate can be increased to achieve high spatial resolution.

In addition, when the numerical aperture of the fiber optic plate is 0.4 or more and 0.7 or less, the following arrangement 1) or 2) may be used.
1) In the fluorescence observation filter, an interference filter layer and an absorption filter layer are laminated via a fiber optic plate, an observation sample is disposed immediately above the interference filter layer, and an image sensor is disposed immediately below the absorption filter layer.
2) In the fluorescence observation filter, an interference filter layer and an absorption filter layer are laminated, a fiber optic plate is laminated on the interference filter layer, an observation sample is disposed immediately above the fiber optic plate, and an image sensor is directly below the absorption filter layer. Is placed. When emphasizing the strength of the surface of the filter for fluorescence observation, it is preferable to arrange the fiber optic plate on the outermost surface.

In the fluorescence observation filter of the present invention, specifically, the interference filter layer is a dielectric multilayer film, and the absorption filter layer is a dye-added film.
Since the hybrid configuration includes an interference filter layer made of a dielectric multilayer film and an absorption filter layer to which a dye is added, the autofluorescence component can be reduced and the excitation light component can be removed with high performance.

  The fluorescence observation microscope of the present invention is a lensless optical system that does not use a lens by using the above-described fluorescence observation filter, and can observe a fluorescent substance in a wide field of view, and can achieve downsizing and high portability.

  According to the present invention, there are effects that the autofluorescence component is small, the excitation light component can be removed with high performance, the reduction in spatial resolution in the lensless optical system is minimized, the size is small, and the portability is excellent.

Illustration of the configuration image of the filter for fluorescence observation Cross-sectional image of filter for fluorescence observation Cross-sectional schematic diagram showing one embodiment of a filter for fluorescence observation System diagram of the experimental system Comparison results of fluorescence observation Illustration of fluorescence observation of fluorescein droplet Difference image before and after fading Cross-sectional schematic diagram showing another embodiment of a filter for fluorescence observation (Example 2) Explanatory drawing about filter characteristics for fluorescence observation (cut-off characteristics of interference filter) Explanatory drawing about filter characteristics for fluorescence observation (transmitted light angle of interference filter) Explanatory drawing on spacer spacing and air layer thickness Cross-sectional schematic diagram showing another embodiment of a filter for fluorescence observation (Example 3) Illustration of a lensless optical system fluorescence observation microscope Illustration of interference filter Illustration of absorption filter Explanatory drawing of the device of the lens optical system in which the interference filter and the absorption filter are arranged in series in the fluorescence traveling direction

  Hereinafter, an example of an embodiment of the present invention will be described in detail with reference to the drawings. The scope of the present invention is not limited to the following examples and illustrated examples, and many changes and modifications can be made.

  FIG. 1 is an explanatory diagram of a configuration image of the fluorescence observation filter of the present invention. Lens-less optics in which an observation sample 7 is arranged immediately above the image sensor device 5, and the excitation light 8 is irradiated from directly above the observation sample 7 to capture the fluorescence of the fluorescent substance 7a of the observation sample 7 by the two-dimensional image sensor 5a. In the system fluorescence observation microscope, the fluorescence observation filter 1 is provided between the two-dimensional image sensor 5 a and the observation sample 7 in order to remove the excitation light 8. In the fluorescence observation filter 1, an interference filter 2 and an absorption filter 3 having the same transmission characteristics of the excitation light wavelength are laminated via a light incident angle limiting layer 4. According to the filter 1 for fluorescence observation, the excitation light that passes through the observation sample 7 is removed, and the fluorescence of the fluorescent material 7a is transmitted, so that the sensitivity of the fluorescence in the two-dimensional image sensor 5a can be made higher.

  FIG. 2 shows a cross-sectional image of the fluorescence observation filter of the present invention. The fluorescence observation filter 1 is formed by laminating an interference filter 2 and an absorption filter 3 having the same transmission characteristics of the excitation light wavelength via a light incident angle limiting layer 4, thereby allowing the filters of the interference filter 2 and the absorption filter 3. Can compensate for the disadvantages. That is, the interference filter 3 has a drawback that it is difficult to prevent transmission of obliquely incident excitation light generated by scattering when the observation sample is irradiated with the excitation light 8. The absorption filter 3 can reduce the obliquely incident excitation light that is transmitted. In addition, the absorption filter 3 has a drawback that a part of the energy of the excitation light 8 absorbed as described above causes autofluorescence, but this is caused by excitation incident on the absorption filter 3 by the light incident angle limiting layer 4. Light 8 can be reduced. As will be described later, the light incident angle limiting layer 4 is formed by dispersing the absorbers having the excitation light wavelength extending in the thickness direction, reducing the excitation light that is transmitted obliquely, and entering the absorption filter 3. Is sufficiently reduced.

FIG. 3 is a schematic cross-sectional view showing an embodiment of the filter for fluorescence observation of the present invention. In the fluorescence observation filter shown in FIG. 3, a fiber optic plate 4 a configured by bundling a plurality of optical fibers is used as the light incident angle limiting layer 4 of the interference filter 2 and the absorption filter 3. The light incident angle limiting layer 4 uses a coating material (cladding) of each optical fiber as an absorber of the excitation light wavelength extending in the thickness direction.
By using the fiber optic plate for the light incident angle limiting layer, high mechanical strength can be secured in the fluorescence observation filter. In addition, the surface of the fluorescence observation filter can be flattened without substantially reducing the spatial resolution. The fiber optic plate having a high mechanical strength and a flat surface can easily manufacture the interference filter 2 and the absorption filter 3. That is, the interference filter 2 can be manufactured by directly applying a liquid agent to the surface of the fiber optic plate. Moreover, it is possible to produce the absorption filter 3 by applying a liquid agent to the back surface of the fiber optic plate. As described above, the fiber optic plate can be used as a substrate to which a liquid agent can be directly applied, and various mounting processes can be realized. For example, a thin film having a thickness of 5 μm is formed on the front and back surfaces of a fiber optic plate having a thickness of 2.54 mm, and the thin films are used as the interference filter 2 and the absorption filter 3. Furthermore, the fluorescent material 7a can be observed by placing the image sensor device 5 on the back surface and the observation sample 7 made of the fluorescent material 7a on the front surface and irradiating the excitation light 8 from above.

  In the image sensor device 5, the image sensor elements 5 b are spread in a planar shape in a lattice shape or other arrangement. The coating material (cladding) of each optical fiber of the fiber optic plate functions as an absorber of the excitation light wavelength, so that the obliquely incident excitation light transmitted from the interference filter 2 can be effectively reduced. That is, since the excitation light of oblique incidence is minimized with respect to the image sensor element 5b and the position of the fluorescence by the fluorescent material is propagated to the absorption filter 3 without being shifted by the fiber optic plate, A reduction in spatial resolution can be minimized. Alternatively, the obliquely incident excitation light transmitted from the interference filter 2 is repeatedly reflected by the coating material (cladding) of each optical fiber of the fiber optic plate, so that it is reduced to some extent. There is also an effect that can be minimized.

  As described above, the fluorescence observation filter of the present invention has high excitation light removal performance with respect to the excitation light irradiated on the observation sample. That is, when there is an observation sample immediately above the filter for fluorescence observation, when excitation light is directly incident, most of the excitation light is reflected by the interference filter and cannot be transmitted, but scattering components of the excitation light appear, and these scattering components Since the excitation light spreads in various directions, it is transmitted through the filter when obliquely incident on the interference filter at a certain angle or more. However, the transmitted obliquely incident excitation light component is absorbed and removed by the excitation light absorber provided in the lower light incident angle limiting layer. Therefore, there is almost no excitation light component reaching the absorption filter, and a small amount of excitation light component reaches the absorption filter, but the incident excitation light component is removed by the absorption filter itself. Thereby, high excitation light removal performance is realized. In the case of only the absorption filter, the self-fluorescence of the filter itself becomes a problem. However, in this configuration, most of the energy of the excitation light is removed by the interference filter and the light incident angle limiting layer. It is low enough.

Next, the results of the fluorescence observation will be described for the fluorescence observation filter using the fiber optic plate as the light incident angle limiting layer.
A green color filter resist pattern (“N” character pattern) was used as an observation sample. This resist emits slight green fluorescence when irradiated with blue light.

FIG. 4 shows the system configuration of an experimental system for fluorescence observation. A blue laser diode (LD) 51 having an oscillation wavelength of 450 nm was used as an excitation light source. The light intensity was adjusted by the polarizer 52 and the excitation filter 53. Then, in order to irradiate only the observation region and to adjust the irradiation range, the iris 55 was used to narrow the light irradiated to the observation sample 56.
In the fluorescence observation filter 57, a 510 nm long pass filter (LPF) was used as the interference filter, and a dye-added film that absorbs blue light was used as the absorption filter. A fiber optic plate (2.54 mmt) was used for the light incident angle limiting layer.
The fluorescence transmitted through the fluorescence observation filter 57 was imaged by the image sensor 58, and the captured image data was transmitted to the computer 59 to confirm the fluorescence observation image.

As a comparison of fluorescence observation, fluorescence observation was confirmed in the case of only the interference filter and in the case of only the absorption filter.
FIG. 5 shows a comparison result of fluorescence observation. First, FIG. 5A is an observation sample of a green color filter resist pattern (character pattern “N”), and FIG. 5B is a result of fluorescence observation in the case of only an interference filter. FIG. 5C shows the result of fluorescence observation when only the absorption filter is used. FIG. 5D shows the result of fluorescence observation using a fluorescence observation filter in which an interference filter and an absorption filter are stacked via a light incident angle limiting layer (fiber optic plate).
In the result of only the interference filter shown in FIG. 5B, the excitation light is scattered and transmitted through the surface scratches and the edge portions of the resist, and the surface scratches may be improved by the mounting method. Partial scattering has been a problem for most observation objects. Further, in the result of only the absorption filter shown in FIG. 5C, although the fluorescence of the resist portion can be observed brightly, the portion without the resist becomes bright and the contrast is lowered. This is due to the fluorescence of the absorption filter, and even if the film thickness is increased, the improvement is limited.
On the other hand, in the result of the fluorescence observation filter shown in FIG. 5D, the excitation light was removed to the extent that it was hardly detected in the used imaging system, and high contrast was obtained. From these results, it can be seen that the fluorescence observation filter of the present invention has high performance for performing weak fluorescence observation.

Next, in order to confirm the performance of the filter for fluorescence observation of the present invention, a fluorescein solution having a molar concentration of 10 μM (micromolar) was dropped on the letter pattern of “N” and “A”, and the fluorescence observation was performed. . Although fluorescein is a dye and is known to emit fluorescence, a molar concentration of 10 μM is very low, and thus fluorescence observation is usually difficult. Therefore, if the fluorescence of fluorescein having a molar concentration of 10 μM can be observed, it indicates that the fluorescence observation performance is high.
The experimental results of the fluorescence of fluorescein having a molar concentration of 10 μM will be described with reference to FIGS. In the experiment, a glass substrate with a large number of fine fluorescein droplets having a diameter of 5 μm arranged at a pitch of 30 μm was used, and a quartz glass plate was used for the top cover.

FIG. 6 is an explanatory view of fluorescence observation of a fluorescein droplet. (1) is immediately after preparing a 10 μM fluorescein droplet, (2) is after 2 hours of excitation light irradiation, and (3) is after 2 hours of irradiation. The fluorescence image of the observation region (region of “NA” character) is shown. It was confirmed that the fluorescence faded with the lapse of the excitation light irradiation time. In this experiment, laser light of 450 nm was used as the excitation light. However, when the excitation light is uniformly irradiated as in this experiment, the excitation light of a light source with low monochromaticity such as a light emitting diode (LED) is used. The uniform irradiation is possible, and a clear fluorescent image can be obtained.
FIG. 7 shows an image (before fading, after fading, and a difference image) in which noise is reduced and contrast is improved by observing an average image of 100 frames for each observation region. 7A is an image before fading, FIG. 7B is a difference image, and FIG. 7C is an image after fading after irradiation for 2 hours. The image in FIG. 7 (3) is not fluorescein, but is considered to be a fluorescence image from a substrate for forming a droplet pattern. That is, the difference image in FIG. 7 (2) shows a fluorescent component almost exclusively of fluorescein. Since the character pattern “N” and “A” formed as the arrangement pattern of the droplet array can be clearly discriminated, the excitation light removal performance is high enough to detect a minute 10 μM fluorescein droplet. I understand. Note that the character patterns “N” and “A” are upside down (upside down). It can be expected that the sensitivity of fluorescence observation will be further increased by combining sensor sensitivity enhancement and noise reduction techniques.

  FIG. 8 is a schematic cross-sectional view showing another embodiment of the filter for fluorescence observation of the present invention. In the fluorescence observation filter of this embodiment shown in FIG. 8, the air layer 4 b is used as the light incident angle limiting layer 4 of the interference filter 2 and the absorption filter 3. Since the refractive index of the air layer 4b is smaller than the refractive index of the interference filter 2, it is possible to refract the component that passes through the interference filter out of the light incident from the interference filter side and absorb it by the spacer 40 disposed in the air layer. it can. That is, in the fluorescence observation filter of this embodiment, among the excitation light, the excitation light 8a having a vertically incident component is reflected by interference by the multilayer film of the interference filter 2, and the excitation light having an oblique incident component that cannot be covered by the interference filter ( 8b and 8e) remove the spacer 40 by functioning as an absorber of excitation light. Further, the excitation light 8d having a large oblique incident angle is totally reflected at the interface with the air layer 4b. Further, the scattering component of the excitation light 8 c due to the fluorescent material 7 a is removed by the absorption filter 3. Thereby, high excitation light removal performance is realized. Further, by providing the spacer 40 in accordance with the boundary portion between the image sensor elements 5b in the image sensor device 5, the resolution of the image sensor elements can be maintained.

  In the fluorescence observation filter of the present embodiment, the component of the obliquely incident excitation light that cannot be covered by the interference filter 2 is totally reflected or refracted at the interface with the air layer (air gap) 41 and removed by the absorber spacer 40. To do. The scattering component of the excitation light is removed by the absorber spacer 40 and the absorption filter 3. Depending on the incident angle of the excitation light, there are those that can be reflected by interference by the multilayer film of the interference filter 2, and those that can be totally reflected at the interface with the air layer (air gap) 41. The excitation light having an oblique incident angle that passes through the interference filter 2 is present between “” and “total reflection”. In the following, the range of the oblique incident angle that passes through the interference filter 2 is the “transmission angle band”, the range of the incident angle that reflects by interference is the “interference filter reflection band”, and the range of the incident angle that totally reflects beyond the critical angle. This will be called “total reflection band”.

  FIG. 9 shows a graph showing the correlation between the incident angle of the excitation light and the cutoff wavelength in the interference filter. For example, as a characteristic of the interference filter, assuming that a long-pass filter having a function of passing a long wavelength side with a wavelength of 510 nm or longer and having a function of cutting a short wavelength side with a wavelength of less than 510 nm is obtained from the graph of FIG. If the wavelength is 470 nm, the interference filter can cut only the excitation light component up to the incident angle of 0 to 34 (deg.), And cannot cut the oblique incident component whose incident angle exceeds 34 (deg.). Recognize.

FIG. 10 shows a graph showing the correlation between the incident angle of the excitation light and the transmitted light angle in the interference filter. In FIG. 10, an excitation light incident angle range “A” of 0 to 34 (deg.) Is an incident angle range “interference filter reflection band” that can be reflected by interference. As shown in FIG. 10, when the incident angle of the excitation light is further increased and reaches a critical angle of 49 (deg.), Total reflection occurs. That is, the range “C” of the excitation light incident angle of 49 (deg.) Or more is the “total reflection band”. It is assumed that the refractive index of the interference filter is equivalent to that in water (refractive index n = 1.33), and a critical angle 49 (deg.) Is derived.
The range “B” of the incident angle 34 to 49 (deg.) Of the excitation light is the range “transmission angle band” of the oblique incident angle that passes through the interference filter. The transmitted light angle at the incident angle 34 (deg.) Of the excitation light is 48 (deg.), And the transmitted light angle at the incident angle 49 (deg.) Of the excitation light (total reflection) is 90 (deg. ). The incident angle 34 (deg.) At which the excitation light begins to transmit is called the transmission start angle. Thus, there is an excitation light component with an oblique incident angle that passes through the interference filter, and it is necessary to remove this excitation light component.

In the fluorescence observation filter of this embodiment, the component of the obliquely incident excitation light not reflected by the interference filter 2 is refracted at the interface between the interference filter 2 and the air layer (air gap) 41 and removed by the absorber spacer 40. To do. The distance between the spacers 40 and the thickness of the air gap 41 in this case will be described with reference to FIG.
From FIG. 10, the excitation light component transmitted from the interference filter, that is, the excitation light having an incident angle of 34 to 49 (deg.) Is refracted at the interface between the interference filter and the air layer (air gap), and the transmitted light angle is 48 to 90 (deg.).
As shown in FIG. 11, assuming that the outgoing angle of the transmitted light component 8h of the excitation light 8g is θ, the interval (opening width) of the spacer 40 is w, and the thickness of the air gap 41 is d, the thickness d = w / tan θ. It can be expressed by the following formula. For example, in the case of FIG. 11, if all the transmitted light components of the excitation light transmitted from the interference filter are absorbed and removed by the spacer 40, the thickness d = w / tan 48, for example, if w = 10 μm. In this case, d = 9 μm.

  It can be seen from the above formula that the thickness d of the air gap can be reduced by reducing the spacer interval (opening width) w. In addition, since the interference filter reflection band is expanded by increasing the cutoff wavelength of the interference filter, the thickness d of the air gap can be reduced. In addition, since the interference filter reflection band is expanded by shortening the excitation wavelength, the thickness d of the air gap can be reduced. Furthermore, the thickness d of the air gap can be reduced by increasing the effective refractive index of the interference filter.

FIG. 12 is a schematic cross-sectional view showing another embodiment of the filter for fluorescence observation of the present invention. In the fluorescence observation filter 100 shown in FIG. 12, an interference filter 2 and an absorption filter 3 having substantially the same transmission characteristics of the excitation light wavelength are laminated, and a fiber optic plate 4a as the light incident angle limiting layer 4 is formed on the interference filter layer 2. Is laminated. A fluorescent material 7a as an observation sample is disposed immediately above the fiber optic plate 4a, and an image sensor device 5 is disposed immediately below the absorption filter layer 3.
As the fiber optic plate 4a, a plate having a numerical aperture (NA) of Low-NA smaller than 0.4 can be used. By using a fiber optic plate having a low numerical aperture as the outermost surface, the strength of the surface of the fluorescence observation filter can be increased. Even under conditions such as in vivo where light scattering is large, excitation light can be sufficiently reduced and highly sensitive fluorescence observation can be performed.

  Similarly to the conditions for reducing the thickness of the air gap in Example 2 described above, the numerical aperture (by reducing the excitation wavelength or increasing the cutoff wavelength of the interference filter) NA) can be relaxed, and a numerical aperture (NA) having a relatively high High-NA of about 0.7 can also be used. For example, although the excitation absorption wavelength of green fluorescent protein (eGFP) has a peak at 488 nm and the fluorescence wavelength is 509 nm, the excitation wavelength is shortened to 450 nm instead of 470 nm, and the cutoff wavelength of the interference filter is 510 nm. In this case, it is possible to select and observe High-NA by making the wavelength longer than 520 nm. However, the efficiency of fluorescence decreases by shortening the excitation wavelength or increasing the cutoff wavelength of the interference filter.

  The present invention is useful for various inspection apparatuses using fluorescence observation, such as small-sized immunological inspection apparatuses, protein detection apparatuses, and virus detection apparatuses. Further, the present invention can be expected to be applied as an apparatus used for cell measurement (flow cytometry) and a sensor for biological measurement.

1,100 Fluorescence observation filter (excitation light removal filter)
2 Interference filter 3 Absorption filter 4 Light incident angle limiting layer 4a Fiber optic plate 4b Air layer 5 Image sensor device 5a Two-dimensional image sensor 5b Image sensor element 6 Fluorescence 7 Observation sample 7a Fluorescent substance 8, 8a-8j Excitation light 9 Scattered light DESCRIPTION OF SYMBOLS 10 Autofluorescence 11 Excitation side filter 12 Fluorescence side multilayer film interference filter 13 Absorption filter 15 Single wavelength light source 30 Irradiation unit 31 Observation sample 32 Imaging lens 38 Two-dimensional detector 40 Spacer 41 Air layer (air gap)
51 Laser diode (LD)
52 Polarizer 53 Excitation Filter 54 Mirror 55 Iris 56 Observation Sample 57 Fluorescence Observation Filter 58 Image Sensor 59 Computer

Claims (12)

A fluorescence observation filter in lensless fluorescence imaging that observes fluorescence when an observation sample is irradiated with excitation light with an image sensor,
An interference filter layer and an absorption filter layer having the same transmission characteristics of the excitation light wavelength, and a light incident angle limiting layer in which absorbers of the excitation light wavelength extending in the thickness direction are dispersedly arranged,
In the fluorescence observation filter, the interference filter layer and the absorption filter layer are laminated via the light incident angle limiting layer,
Or
In the fluorescence observation filter, the interference filter layer and the absorption filter layer are laminated, and the light incident angle limiting layer is laminated on the interference filter layer,
A fluorescence observation filter, wherein an observation sample is disposed immediately above the interference filter layer or the light incident angle limiting layer, and an image sensor is disposed directly below the absorption filter layer. The refractive index of the light incident angle limiting layer is smaller than the refractive index of the interference filter layer,
2. The fluorescence observation filter according to claim 1, wherein the absorber is used as a spacer between the interference filter layer and the absorption filter layer.   The fluorescence observation filter according to claim 2, wherein the spacer is provided in accordance with a boundary portion between pixel sensors in the image sensor.   The fluorescence observation filter according to claim 1 or 2, wherein the light incident angle limiting layer is one of an air layer, a vacuum layer, and a porous material layer.   5. The fluorescence observation filter according to claim 4, wherein the thickness of the light incident angle limiting layer is reduced by reducing the arrangement interval of the absorber.   The thickness of the light incident angle limiting layer is reduced by at least one of shortening the wavelength of the excitation light or increasing the cutoff wavelength of the interference filter layer. The fluorescence observation filter according to claim 4 or 5.   The light incident angle limiting layer is a fiber optic plate configured by bundling a plurality of optical fibers, and the absorber is included in a coating material (cladding) of each optical fiber. Item 2. The fluorescence observation filter according to Item 1.   When the numerical aperture of the fiber optic plate is larger than 0.7, the filter for fluorescence observation includes the interference filter layer and the absorption filter layer stacked via the fiber optic plate, and is directly above the interference filter layer. The fluorescence observation filter according to claim 7, wherein an observation sample is disposed, and an image sensor is disposed immediately below the absorption filter layer.   When the numerical aperture of the fiber optic plate is smaller than 0.4, the fluorescence observation filter has the interference filter layer and the absorption filter layer laminated, and the fiber optic plate is laminated on the interference filter layer, 8. The fluorescence observation filter according to claim 7, wherein an observation sample is disposed immediately above the fiber optic plate, and an image sensor is disposed directly below the absorption filter layer. When the numerical aperture of the fiber optic plate is 0.4 or more and 0.7 or less,
In the fluorescence observation filter, the interference filter layer and the absorption filter layer are laminated via the fiber optic plate, an observation sample is disposed immediately above the interference filter layer, and an image sensor is directly below the absorption filter layer. Arranged,
Or
In the fluorescence observation filter, the interference filter layer and the absorption filter layer are laminated, the fiber optic plate is laminated on the interference filter layer, an observation sample is disposed immediately above the fiber optic plate, and the absorption filter layer The fluorescence observation filter according to claim 7, wherein an image sensor is disposed immediately below the filter.   11. The fluorescence observation filter according to claim 1, wherein the interference filter layer is a dielectric multilayer film, and the absorption filter layer is a dye-added film.   A fluorescence observation microscope using the fluorescence observation filter according to claim 1.