Examination device, slit lamp microscope, and examination method

Abstract

Provided is an examination device comprising: a laser irradiation unit that irradiates an eye being examined with a pulsed laser beam; and a fluorescence detection unit that is provided with a detector for detecting autofluorescence which is from floating cells in the eye being examined and which occurs due to irradiation of the eye being examined with the pulsed laser beam, wherein the radiant exposure of the pulsed laser beam with which the floating cells in the eye being examined are irradiated is not more than 10 J / cm2. Also provided is a slit lamp microscope comprising the examination device.

Description

Inspection device, slit lamp microscope, and inspection method

[0001] The present disclosure relates to an examination device, a slit lamp microscope, and an examination method.

[0002] A phenomenon known as autofluorescence occurs when biological tissue absorbs light and emits spontaneous light when irradiated with light. Methods for non-invasively identifying biological tissues using this phenomenon have been studied. Patent Document 1 discloses an ophthalmologic imaging device that captures images of the fundus showing the autofluorescence of lipofuscin, a waste product that accumulates in the fundus. Non-Patent Document 1 also discloses that, when normal cells and malignant lymphoma cells are irradiated with a continuous wave of a fixed wavelength, the malignant lymphoma cells exhibit a higher intensity of autofluorescence.

[0003] Japanese Patent Application Laid-Open No. 2022-106243

[0004] Daisuke Nakamura et al., "Intraocular Cell Fluorescence Measurement for Noninvasive Diagnosis of Ocular Inflammatory Diseases," Laser Society of Japan, Laser Society Research Meeting Report, September 2022, Laser Society 567th Research Meeting, pp. 25-28

[0005] In conventional non-invasive ophthalmologic examinations, it has been difficult to determine whether cells in the eye are normal, tumor, or inflamed by observing the fundus with the naked eye and analyzing images. Therefore, invasive examinations have been necessary to perform highly accurate examinations. However, invasive examinations involve directly collecting cells by inserting a syringe into the aqueous humor in the eye, placing a significant burden on the patient's mind and body. Furthermore, due to this burden on the patient, it takes a long time to obtain test results, and if tumor cells are present, there is a concern that the patient's condition may worsen before the test results are obtained. Therefore, the present disclosure provides an examination device and examination method that can reduce the burden on the patient and quickly examine the subject's eye. It also provides a slit lamp microscope equipped with such an examination device.

[0006] One aspect of the present disclosure provides a fluorescence detection system including: a laser irradiation unit that irradiates a subject's eye with pulsed laser light; and a fluorescence detection unit that includes a detector that detects spontaneous fluorescence from floating cells in the subject's eye that is generated by irradiating the subject's eye with the pulsed laser light, wherein the radiation exposure amount of the pulsed laser light irradiated onto the floating cells in the subject's eye is 10 J / cm ２ An inspection device is provided, which is as follows.

[0007] The laser irradiation unit of the above-mentioned inspection device applies a radiation exposure of 10 J / cm to the floating cells in the subject's eye. ２ The test device irradiates a pulsed laser beam having a pulse width of 1000 s or less. By using pulsed laser beams and shortening the pulse width, damage to suspended cells can be reduced while increasing the irradiance per pulse to obtain strong autofluorescence. Therefore, when such pulsed laser beams are irradiated onto the subject's eye, damage to suspended cells in the subject's eye can be reduced, and the burden on the patient undergoing the examination can be reduced. Furthermore, when the subject's eye is irradiated with pulsed laser beams, the suspended cells in the subject's eye generate autofluorescence specific to their type. The fluorescence detection unit of the test device detects the autofluorescence from the suspended cells. Therefore, the test device is non-invasive, can reduce the burden on the patient, and can quickly examine the subject's eye.

[0008] One aspect of the present disclosure provides a slit lamp microscope including the above-described examination device. The slit lamp microscope is non-invasive, reducing the burden on the patient and enabling rapid examination of the subject's eye.

[0009] One aspect of the present disclosure is a method for treating a patient with a radiation exposure of 10 J / cm ２ The present invention provides an inspection method comprising: an irradiation step of irradiating suspended cells in a test eye with pulsed laser light, and a detection step of detecting spontaneous fluorescence generated from the suspended cells by irradiating the suspended cells in the test eye with the pulsed laser light.

[0010] The above inspection method uses a radiation exposure dose of 10 J / cm ２ The suspended cells in the subject's eye are irradiated with the following pulsed laser light: the radiation exposure dose of the pulsed laser light is 10 J / cm ２By satisfying the following conditions, it is possible to obtain strong autofluorescence while reducing damage to suspended cells. Therefore, it is possible to reduce damage to suspended cells in the subject's eye when the subject's eye is irradiated with pulsed laser light, and to reduce the burden on the patient. The above examination method is non-invasive, can reduce the burden on the patient, and can quickly examine the subject's eye.

[0011] The present disclosure provides an examination device, a slit lamp microscope, and an examination method that can reduce the burden on the patient and quickly examine the subject's eye.

[0012] 1 is a diagram showing the configuration of an inspection device according to one embodiment. FIG. 2 is a diagram showing the configuration of an inspection device according to another embodiment. FIG. 3 is a diagram showing the configuration of an inspection device according to yet another embodiment. FIG. 4 is a diagram showing the appearance of a slit lamp microscope according to one embodiment. FIG. 5 is a diagram showing a modified example of the inspection device of FIG. 1. FIG. 6 is a diagram showing the intensity of autofluorescence when PBMC is irradiated with pulsed laser light having pulse frequencies of 50, 100, 500, 700, and 1000 kHz. FIG. 7 is a diagram showing the intensity of autofluorescence when OCI-Ly10 is irradiated with pulsed laser light having pulse frequencies of 50, 100, 500, 700, and 1000 kHz. FIG. 8 is a diagram comparing the fluorescence intensities of autofluorescence of PBMC, OCI-Ly10, and CA46 when the pulse frequency is 700 kHz. This is a graph comparing the fluorescence intensity of autofluorescence from PBMC, OCI-Ly10, and CA46 when the pulse frequency was 1000 kHz.

[0013] Embodiments of the present disclosure are described below. However, the following embodiments are merely examples for explaining the present disclosure and are not intended to limit the present disclosure to the following content. The upper or lower limit of a numerical range specified in this disclosure may be replaced with any value shown in the examples. Furthermore, individually stated upper and lower limit values ​​may be arbitrarily combined. The symbol "to" used in a numerical range indicates a numerical range that includes the upper and lower limit. For example, "X to Y" indicates a numerical range "greater than or equal to X and less than or equal to Y." Unless otherwise specified, the materials or components exemplified in this disclosure can be used alone or in combination of two or more. In the description, identical elements or elements having the same function are designated by the same reference numerals, and redundant description is omitted. Furthermore, positional relationships such as up, down, left, and right used in the description are based on the positional relationships shown in the drawings unless otherwise specified.

[0014] FIG. 1 is a diagram showing the configuration of an inspection device according to one embodiment. The inspection device 100 includes a laser irradiation unit 70 and a fluorescence detection unit 80. The laser irradiation unit 70 has a laser generator 10. The fluorescence detection unit 80 has a relay lens 29 and a detector 30. A pulsed laser beam P generated by the laser irradiation unit 70 is irradiated onto an eye E to be inspected. When the pulsed laser beam P is irradiated onto the eye E, spontaneous fluorescence F is generated from floating cells within the eye E, and the detector 30 detects the fluorescence intensity of the spontaneous fluorescence F. The obtained fluorescence intensity can be displayed on a display unit (not shown), such as a monitor.

[0015] The autofluorescence generated from floating cells in the subject's eye E refers to fluorescence that is spontaneously generated from fluorescent proteins excited by pulsed laser light P. The fluorescence intensity of the autofluorescence varies depending on the type of fluorescent protein. Therefore, by examining the fluorescence intensity of the autofluorescence generated from floating cells, it is possible to non-invasively and quickly determine, for example, whether the floating cells are normal or abnormal. The subject's eye E may or may not contain floating cells. This examination device can also examine whether the subject's eye E contains floating cells.

[0016] The floating cells contained in the subject's eye E are not particularly limited and may include at least one selected from the group consisting of peripheral blood mononuclear cells (e.g., PBMC) and malignant lymphoma cells (e.g., OCI-Ly10 or CA46). Peripheral blood mononuclear cells are normal cells, while malignant lymphoma cells are cancerous, abnormal cells. The above cells may be activated by adding an activating agent such as PMA or ionomycin, or by stimulating with cytokines, antibodies, or other cells. When these cells are irradiated with pulsed laser light, the fluorescence spectrum of the autofluorescence emitted by peripheral blood mononuclear cells differs from that of malignant lymphoma cells. Therefore, irradiation with pulsed laser light allows for non-invasive and rapid discrimination between peripheral blood mononuclear cells and malignant lymphoma cells.

[0017] The laser generator 10 may be configured by combining a laser light source and a pulse generator, or may be configured by integrating these. For example, a semiconductor laser oscillator (Cobolt 06-MLD, manufactured by Cobolt) can be used as the laser light source. For example, a DG645 type digital delay pulse generator (manufactured by Stanford Research Systems) can be used as the pulse generator.

[0018] Pulsed laser light is a laser with the characteristics of a pulsed wave, which repeatedly emits a constant output, and differs from continuous wave, which always emits a constant output. When laser light is a pulsed wave, the output is repeatedly turned on and off at regular intervals, generally resulting in a smaller total energy amount than continuous wave. Therefore, when pulsed laser light P is irradiated onto the subject's eye E, the energy imparted to the subject's eye E is smaller than that of continuous wave, thereby sufficiently reducing damage to floating cells in the eye. Furthermore, since the laser light irradiated onto the subject's eye E is pulsed laser light, the fluorescent protein can be repeatedly irradiated with excitation light at regular intervals. This suppresses the attenuation of the fluorescent protein's spontaneous fluorescence and improves the accuracy of floating cell examination.

[0019] The average output power of the pulsed laser light P may be 1 to 100 W. The average output power of the pulsed laser light P may also be 1 to 1000 mW, 1 to 100 mW, or 10 to 50 mW. From the viewpoint of sufficiently detecting the fluorescence of the suspended cells, the average output power may be 1 mW or more. Furthermore, if there is no problem with the inspection accuracy, from the viewpoint of sufficiently reducing damage to the suspended cells, the average output power may be 1000 mW or less. The average output power is the time average of the output power of the pulsed laser light within a certain period of time.

[0020] The irradiance per pulse of the pulsed laser light P is 5 W / cm ２ The irradiance per pulse represents the time average of the radiation exposure amount per irradiation area while the pulse is being irradiated. From the viewpoint of further reducing damage to the suspended cells, the irradiance per pulse of the pulsed laser light P may be 1 W / cm or less. ２ or less, and may be 100 mW / cm ２ may be less than 1 mW / cm ２ From the viewpoint of shortening the inspection time and further promoting the generation of autofluorescence from suspended cells, the intensity may be 10 μW / cm ２ or more, and 100 μW / cm ２ or more, and 300 μW / cm ２ The range of the irradiance per pulse of the pulsed laser light P may be, for example, 10 μW / cm ２ ~5 W / cm ２ , 100 μW / cm ２ ~1 W / cm ２ , 300 μW / cm ２ ~100mW / cm ２ , or 300 μW / cm ２ ~1 mW / cm ２ may be.

[0021] The irradiance per pulse is multiplied by the irradiation time (seconds) of the pulsed laser light and the total number of pulses to obtain the radiation exposure amount [J / cm ２The radiation exposure is defined as the total amount of energy per unit area exerted by the laser. In other words, the radiation exposure is the total amount of energy of the pulsed laser light irradiated per unit area. From the viewpoint of further reducing damage to the suspended cells, the radiation exposure of the pulsed laser light P is set to 10 J / cm ２ less than 5 J / cm ２ It may be less than 1 J / cm ２ or less, and may be 100 mJ / cm ２ or less, and may be 10 mJ / cm ２ or less, and may be 1 mJ / cm ２ From the viewpoint of further promoting the generation of spontaneous fluorescence from the suspended cells and further improving the accuracy of the examination, the radiation exposure amount of the pulsed laser light P may be 10 μJ / cm or less. ２ or more, and may be 100 μJ / cm ２ or more, and 300 μJ / cm ２ The range of the radiation exposure amount of the pulsed laser light P may be, for example, 10 μJ / cm ２ ~10 J / cm ２ , 100 μJ / cm ２ ~5 J / cm ２ , 300 μJ / cm ２ ~1 J / cm ２ , or 300 μJ / cm ２ ~1 mJ / cm ２ may be.

[0022] The radiation exposure amount of the pulsed laser light P is preferably less than the radiation exposure limit value from the viewpoint of further suppressing damage to the floating cells in the subject's eye E and further reducing the influence on the human body. The radiation exposure limit value is, for example, 180 mJ / cm when the frequency of the pulsed laser light P is 100 MHz. ２ may be.

[0023] The irradiation time of the pulsed laser light P may be 100 femtoseconds to 30 seconds. The longer the irradiation time, the greater the radiation exposure dose applied to the subject's eye E. Therefore, from the viewpoint of further suppressing damage to suspended cells, the irradiation time may be 30 seconds or less, 10 seconds or less, or 5 seconds or less. Furthermore, from the viewpoint of sufficiently detecting the fluorescence intensity, the irradiation time may be 0.001 seconds or more, 0.01 seconds or more, or 0.1 seconds or more. The range of the irradiation time may be, for example, 100 femtoseconds to 10 seconds, 0.001 to 10 seconds, 0.01 to 5 seconds, or 0.1 to 5 seconds.

[0024] From the viewpoint of sufficiently detecting the fluorescence of suspended cells, the pulse frequency of the pulsed laser light P may be 1 Hz or more, 100 Hz or more, 500 Hz or more, 1 kHz or more, 10 kHz or more, or 30 kHz or more. Furthermore, from the viewpoint of further reducing damage to suspended cells, the pulse frequency may be 100 MHz or less, 50 MHz or less, 10 MHz or less, 5000 kHz or less, or 2000 kHz or less. The pulse frequency may be in the range of, for example, 1 Hz to 100 MHz. The pulse frequency may be in the range of, for example, 1 Hz to 100 MHz, 100 Hz to 50 MHz, 1 kHz to 10 MHz, 10 to 5000 kHz, or 30 to 2000 kHz.

[0025] The pulsed laser beam P may include a plurality of types of pulsed laser beams having different pulse frequencies in a pulse frequency band of 1 Hz to 100 MHz, and the laser irradiation unit 70 may be configured to be able to irradiate the plurality of types of pulsed laser beams at different timings. １ and the second pulsed laser beam P ２ may also include:

[0026] Multiple types of pulsed laser light P １ , P ２When irradiating the laser beam, the pulse frequency of the laser generator 10 is switched to generate a plurality of types of pulse laser beams P having different pulse frequencies. １ , P ２ The eye E may be sequentially irradiated with a plurality of types of pulsed laser light (for example, a first pulsed laser light P １ and the second pulsed laser beam P ２ ) may be sequentially irradiated onto the subject's eye E. The pulse frequency that generates high fluorescence intensity varies depending on the type of fluorescent protein contained in the floating cells. Therefore, a plurality of types of pulsed laser light P having different pulse frequencies may be used. １ , P ２ By sequentially irradiating the subject's eye E with the laser beams, it is possible to identify more types of floating cells than when using pulsed laser light with a uniform pulse frequency.

[0027] Multiple types of pulsed laser light P １ , P ２ The difference between the maximum and minimum values ​​of the pulse frequency in may be 50 kHz or more, 100 kHz or more, or 200 kHz or more. When the difference between the maximum and minimum values ​​of the pulse frequency is in this range, the difference in fluorescence intensity due to the difference in pulse frequency becomes clear, and it is possible to identify many types of floating cells with greater accuracy. Note that the difference between the maximum and minimum values ​​of the pulse frequency may be 5000 kHz or less. Multiple types of pulse laser light P １ , P ２ is the first pulsed laser beam P １ and the second pulsed laser beam P ２ In the case where the plurality of types of pulsed laser beams P include the first pulsed laser beam and the second pulsed laser beam, the difference in pulse frequency between the first pulsed laser beam and the second pulsed laser beam may be within the above-mentioned numerical range. The plurality of types of pulsed laser beams may include three or more types of pulsed laser beams having different pulse frequencies. １ , P ２ The range of the difference between the maximum and minimum values ​​of the pulse frequency may be, for example, 50 to 5000 kHz, 100 to 5000 kHz, or 200 to 5000 kHz.

[0028] Pulsed laser light P(P １ , P ２The pulse width per pulse of the pulsed laser light P(P) may be 100 fs or more, 100 ps or more, or 10 ns or more, from the viewpoint of sufficiently detecting the fluorescence of the suspended cells. Also, from the viewpoint of further reducing damage to the suspended cells, the pulse width per pulse may be 100 ms or less, 10 ms or less, 1000 ns or less, 500 ns or less, or 200 ns or less. The pulse width must be shorter than (1 / pulse frequency). The pulsed laser light P(P １ , P ２ The range of the pulse width per pulse of the laser diode 100) may be, for example, 100 fs to 100 ms, 100 ps to 10 ms, 10 ns to 1000 ns, 10 to 1000 ns, or 10 to 200 ns.

[0029] Pulsed laser light P(P １ , P ２ The wavelength of the light emitting element 1 is not particularly limited, and may be in the visible light range of 400 to 850 nm, or 400 to 700 nm.

[0030] The fluorescence detection unit 80 has a detector 30 and measures the fluorescence intensity of autofluorescence F generated from suspended cells. The detector 30 may be, for example, a spectrometer (LVM-200-S1, manufactured by Lambda Vision Co., Ltd.). The fluorescence detection unit 80 may include an optical fiber that guides the autofluorescence F to the detector 30.

[0031] The fluorescence detection unit 80 may include a spectroscopic element that transmits light in a specific wavelength range. The spectroscopic element may be a filter, a diffraction grating, or a prism. The filter may be, for example, a long-pass filter that transmits wavelengths of 450 to 850 nm. By using a long-pass filter, it is possible to measure the peak pattern of fluorescence intensity in the wavelength range of 450 to 850 nm for fluorescence having a wavelength of this range.

[0032] The fluorescence detection unit 80 may be provided with a relay lens 29 that focuses the spontaneous fluorescence F on the optical axis of the spontaneous fluorescence F. By providing the relay lens 29, the spontaneous fluorescence F can be focused, and the fluorescence intensity can be sufficiently detected by the detector 30.

[0033] Fig. 2 is a diagram showing the configuration of an inspection apparatus according to another embodiment. The inspection apparatus 110 in Fig. 2 includes a laser irradiation unit 71, a fluorescence detection unit 81, a mirror 12, an imaging unit 44, and an identification unit 90. The mirror 12 has a function of reflecting the pulsed laser light P irradiated from the laser irradiation unit 71 and changing the direction of travel of the light so that it is directed toward the subject's eye E. The mirror 12 also has a function of transmitting spontaneous fluorescence F generated from the subject's eye E and guiding the spontaneous fluorescence F to the fluorescence detection unit 81 located opposite the subject's eye E.

[0034] The laser irradiation unit 71 has a filter 60, a relay lens 52, a diaphragm unit 56, a cylindrical lens 53, a slit forming unit 54, and a relay lens 55 arranged in this order on the optical axis of the pulsed laser light P.

[0035] The filter 60 is an optical element that transmits only a specific wavelength of the pulsed laser light generated by the laser generator 10. The filter 60 may transmit a single wavelength, or may be a linear variable filter (LVF) whose wavelength selection characteristics (wavelength transmission characteristics) change depending on the location where light is irradiated. A combination of filters having multiple wavelength transmittances may also be used. By disposing the filter 60 on the path of the pulsed laser light P, the wavelength that passes through the filter 60 can be adjusted, and the pulsed laser light P from which only excitation light of a specific wavelength has been extracted can be irradiated onto the subject's eye E.

[0036] The filter 60 is configured to be movable automatically or manually. When moving the filter 60 automatically, the filter 60 is moved by an actuator 60A and a moving mechanism 60B. The actuator 60A is a device that generates a driving force and is configured, for example, by a pulse motor. The moving mechanism 60B moves the filter 60 using the driving force generated by the actuator 60A.

[0037] When manually moving the filter 60, the moving mechanism 60B is manually operated, and the filter 60 is moved by the driving force generated by the actuator 60A. Alternatively, the filter 60 may be moved to a desired position by manually moving a movable stage on which the filter 60 is mounted. Note that a diffraction grating or a prism may be disposed instead of the filter 60.

[0038] The relay lens 52 is used to relay the pulsed laser light P generated from the laser generator 10 to the cylindrical lens 53 .

[0039] The diaphragm unit 56 is configured so that the size of its light-transmitting portion can be changed. The diaphragm unit 56 can reduce reflection of illumination light by the cornea and the crystalline lens, and adjust the brightness of the pulsed laser beam P.

[0040] The cylindrical lens 53 is used to shape the pulsed laser light P into a sheet shape (planar shape). When the pulsed laser light P is irradiated onto the cylindrical lens 53, the pulsed laser light P is shaped into a sheet shape, and can be irradiated over a wide range of the subject's eye E. By shaping the pulsed laser light P into a sheet shape and irradiating a wide range of the subject's eye E, the subject's eye E can be directly observed or an image of the subject's eye E can be obtained.

[0041] The pulsed laser light P transmitted through the cylindrical lens 53 can be further slit by the slit forming unit 54. By slitting, the pulsed laser light P can be irradiated deep inside the subject's eye E, such as the fundus. The slit forming unit 54 is used to generate slit light from the pulsed laser light P. The slit forming unit 54 has a pair of slit blades. The slit width can be changed by changing the distance between these slit blades.

[0042] The slit blades are moved by an actuator 54A and a moving mechanism 54B. The actuator 54A is a device that generates a driving force and is configured, for example, by a pulse motor. The moving mechanism 54B changes the spacing of the slit blades based on the driving force generated by the actuator 54A.

[0043] The pulsed laser beam P may be irradiated onto the mirror 12 via the relay lens 55. By using the relay lens 55, the pulsed laser beam P can be condensed, and loss of light when reflected by the mirror 12 can be suppressed.

[0044] The fluorescence detection unit 81 has, in this order, an objective lens 31, a magnification changer 32, an aperture unit 33, a beam splitter 34, a relay lens 35, and a detector 30 on the optical axis of the spontaneous fluorescence F. A normal objective lens can be used as the objective lens 31.

[0045] The magnification changer 32 is configured to include multiple (for example, two) variable magnification lenses 32a, 32b. In this embodiment, multiple variable magnification lens groups are provided that can be selectively inserted into the optical axis of the spontaneous fluorescence F. These variable magnification lens groups are configured to provide different magnifications. The variable magnification lens groups arranged on the optical axis of the spontaneous fluorescence F are used as the variable magnification lenses 32a, 32b. This makes it possible to change the magnification (angle of view) when observing the subject's eye E with the naked eye or when capturing an image.

[0046] The beam splitter 34 splits the spontaneous fluorescence F into two. The spontaneous fluorescence reflected by the beam splitter 34 is guided to an image sensor 43 of an image capturing unit 44 via a relay lens 41 and a mirror 42. The image sensor 43 detects this reflected light and generates an image signal GS. The image signal GS can be displayed on a display unit (not shown) such as a monitor. In the examination device 110, the spontaneous fluorescence F can be detected by the detector 30 while the condition of the subject's eye E is confirmed on such a display unit.

[0047] The spontaneous fluorescence F that has passed through the beam splitter 34 is introduced into the detector 30 via a relay lens 35. The detector 30 detects the fluorescence intensity (fluorescence spectrum) of the spontaneous fluorescence F. Note that a spectroscopic element may be provided in the fluorescence detection unit 81, and the spontaneous fluorescence F may be introduced into the spectroscopic element to extract only specific wavelengths from the spontaneous fluorescence F, and the fluorescence intensity may be measured by the detector 30.

[0048] The inspection device 110 includes an identification unit 90. The identification unit 90 is connected to the detector 30 and can identify the type of floating cells in the subject's eye E based on the spectrum (fluorescence spectrum) of the autofluorescence F detected by the detector 30. The identification unit 90 can record spectral information of the autofluorescence F obtained by irradiating the subject's eye E with pulsed laser light P having a specific pulse frequency. In the present disclosure, the spectrum (fluorescence spectrum) represents the transition of fluorescence intensity between consecutive fluorescence wavelengths, with the horizontal axis representing the fluorescence wavelength and the vertical axis representing the fluorescence intensity.

[0049] The identification unit 90 may, for example, identify the type of floating cells in the subject's eye E by determining whether the fluorescence intensity at a given wavelength in the recorded spectral information is equal to or greater than a threshold value. Alternatively, the type of floating cells in the subject's eye E may be identified by comparing the shape of the spectrum of a specific floating cell stored in advance with the shape of the detected spectrum. Identification may be performed automatically by computer-based data processing. By having such an identification unit 90, the inspection device 110 can automatically identify the type of floating cells and more quickly inspect the subject's eye E.

[0050] The identification unit 90 may record multiple types of spectral information obtained by irradiating the subject's eye E with multiple types of pulsed laser light having different pulse frequencies, and identify the type of suspended cells based on the multiple types of spectra. Changing the pulse frequency may change the shape of the spectrum of the autofluorescence of suspended cells. The degree of such shape change may vary depending on the type of suspended cells. Therefore, based on multiple types of spectra, the type of suspended cells can be identified in more detail. For example, it is possible to not only identify tumor cells from normal cells, but also to identify the type of tumor cells or to distinguish tumor cells from inflamed normal cells.

[0051] The identification unit 90 may be configured to identify the type of suspended cells using statistical data or a machine learning model created in advance based on the acquired spectral information. The statistical data may include, for example, a correlation equation obtained by statistically processing the fluorescence wavelength and fluorescence intensity of the acquired spectral information. The relationship between the spectral shape and pulse frequency may vary depending on the suspended cell. Therefore, identification accuracy can be improved by preparing statistical data for each suspended cell.

[0052] The machine learning model may be constructed using, for example, training data that associates identified suspended cells with the pulse frequency of the irradiated pulsed laser light and the fluorescence intensity and wavelength of the autofluorescence. In this case, training data may be prepared for each suspended cell and used for learning. The machine learning model may have an algorithm for improving the accuracy of identification based on the discrepancy between the predicted and actual values ​​of the autofluorescence fluorescence intensity.

[0053] The identification unit 90 may be configured as a normal computer, and may include, for example, a storage unit that records the statistical data or the machine learning model, a calculation unit that compares the detected spectrum with the statistical data or performs calculations using the detected spectrum to calculate a prediction result, and a display unit that displays the prediction result.

[0054] Fig. 3 is a diagram showing the configuration of an inspection apparatus according to yet another embodiment. In the inspection apparatus 120 of Fig. 3, the imaging unit 44 in the inspection apparatus 110 of Fig. 2 is replaced with an observing eye Eo. In this case, the spontaneous fluorescence F reflected by the mirror 42 is guided to the observing eye Eo via a relay lens 35, a prism 36, and an eyepiece 37. The prism 36 includes two optical elements 36a and 36b, which translate the light upward. With this configuration, instead of outputting an image, the subject's eye E, which emits spontaneous fluorescence F, can be directly observed with binoculars.

[0055] The above-described inspection devices 100, 110, and 120 detect spontaneous fluorescence from floating cells in the subject's eye E in the fluorescence detection units 80 and 81. Therefore, the inspection devices 100, 110, and 120 are non-invasive, can reduce the burden on the patient, and can quickly inspect the subject's eye E. Furthermore, when the subject's eye E contains floating cells, the type of floating cells can be quickly identified.

[0056] Although several embodiments of the inspection device have been described, the inspection device of the present disclosure is not limited to the above-described embodiments. The contents of each embodiment are mutually applicable. For example, some elements of the inspection device 110 of FIG. 2 or the inspection device 120 of FIG. 3 may be applied to the inspection device 100 of FIG. 1. The subject eye to be inspected may be a human eye or the eye of a non-human animal. The inspection device of the present disclosure may be used as is or may be incorporated into another device. Examples of such other devices include a slit lamp microscope and a scanning laser ophthalmoscope.

[0057] FIG. 4 is a diagram showing the appearance of a slit lamp microscope according to one embodiment. The slit lamp microscope M includes a base 102, a laser irradiation unit support arm 103, a fluorescence detection unit support arm 104, a forehead rest 105, a chin rest 106, an illumination operation handle 107, a photography operation handle 108, a display unit S, and an inspection device 110. The inspection device 110 includes a laser irradiation unit 71, a fluorescence detection unit 81, and an image capture unit 44. The illumination operation handle 107 can operate the laser irradiation unit support arm 103. The illumination operation handle 107 can also operate, for example, the filter 60 and the slit width of the slit forming unit 54, which are installed on the optical axis of the pulsed laser light P, as well as the width of the aperture unit 56, as shown in FIGS. 2 and 3 . The photography operation handle 108 can operate the fluorescence detection unit support arm 104. The photographing operation handle 108 can operate, for example, the width of the diaphragm portion 33 shown in FIGS. 2 and 3, the timing of photographing the subject's eye E, the shutter opening and closing time, the photographing magnification, and the like.

[0058] The subject places their head by placing their forehead on the forehead rest 105 and their chin on the chin rest 106. This allows the pulsed laser light P generated from the laser irradiation unit 71 to be irradiated onto the subject's eye E. By irradiating the subject's eye E with the pulsed laser light P, the autofluorescence F generated from the subject's eye E can be detected by the fluorescence detection unit 81. The detected fluorescence spectrum, a captured image of the subject's eye E, and the like may be displayed on the display unit S. The inspection device 110 may be replaced with the inspection device 100 in FIG. 1 , the inspection device 120 in FIG. 3 , or a modified example thereof (for example, the inspection device 101 in FIG. 5 , which will be described later).

[0059] The slit lamp microscope M is non-invasive, reduces the burden on the patient, and can quickly examine the subject's eye E. If the subject's eye E contains floating cells, the type of floating cells can be quickly identified.

[0060] In one embodiment, the method for inspecting cells includes a step of exposing the cells to radiation at a dose of 10 J / cm ２ The present invention includes an irradiation step of irradiating suspended cells in the subject's eye with pulsed laser light, a detection step of detecting autofluorescence generated from the suspended cells by irradiating the suspended cells in the subject's eye with the pulsed laser light, and an identification step of identifying the type of suspended cells based on the spectrum of the autofluorescence. This inspection method is non-invasive, reduces the burden on the patient, and enables rapid identification of the type of suspended cells. The inspection method may be performed using, for example, any of the inspection devices 100, 110, and 120, or a modified version thereof (e.g., the inspection device 101 shown in FIG. 5 , which will be described later). That is, the irradiation step may be performed using laser irradiation units 70 and 71, the detection step may be performed using fluorescence detection units 80 and 81, and the identification step may be performed using an identification unit 90. The descriptions of the inspection device and slit lamp microscope can be applied to each step of the inspection method.

[0061] For example, the pulsed laser light P irradiated onto the subject's eye E in the irradiation step may include a plurality of types of pulsed laser light having different pulse frequencies in a range of 1 Hz to 100 MHz. １ , P ２ may be irradiated onto the subject's eye E at different times.

[0062] The above-described inspection method can reduce damage to floating cells in the subject's eye E when the subject's eye E is irradiated with pulsed laser light P, thereby reducing the burden on the patient. The above-described inspection method is non-invasive, can reduce the burden on the patient, and can quickly inspect the presence or absence of floating cells in the subject's eye. Furthermore, if floating cells are present in the subject's eye, the type of floating cells can be quickly identified.

[0063] Although several embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiments. For example, as shown in FIG. 5 , the inspection device 101 sets the radiation exposure amount of the pulsed laser light P irradiated onto the floating cells in the subject's eye E at 10 J / cm 2 . ２ The laser irradiation device may include a controller CR that controls the following: The controller CR adjusts the laser irradiation conditions of the laser irradiation unit 70 to adjust the radiation exposure amount of the pulsed laser light P irradiated onto the suspended cells in the subject's eye E. The laser irradiation conditions are not particularly limited, and examples include the irradiation time of the pulsed laser light P, the pulse frequency of the pulsed laser light P, the pulse width per pulse, the energy per pulse, and the slit width. A plurality of laser light sources and pulse generators may be used. Furthermore, the above-described embodiments and modified examples may be combined as appropriate.

[0064] The present disclosure includes the following several embodiments: [1] A method for detecting autofluorescence of a cell suspended in the subject's eye, comprising: a laser irradiation unit that irradiates a subject's eye with pulsed laser light; and a fluorescence detection unit that includes a detector that detects autofluorescence from the cell suspended in the subject's eye that is generated by irradiating the subject's eye with the pulsed laser light, wherein the radiation exposure of the pulsed laser light irradiated onto the cell suspended in the subject's eye is 10 J / cm. ２An inspection device as set forth below. [2] The inspection device according to [1], wherein the pulsed laser light includes a plurality of types of pulsed laser light having different pulse frequencies in a band of 1 Hz to 100 MHz, and the laser irradiation unit is configured to be able to irradiate the plurality of types of pulsed laser light at different timings to the subject's eye. [3] The inspection device according to [1] or [2], wherein the laser irradiation unit includes a cylindrical lens on the optical axis of the pulsed laser light that shapes the pulsed laser light into a sheet. [4] The inspection device according to any one of [1] to [3], wherein the fluorescence detection unit includes a spectroscopic element on the optical axis of the autofluorescence, and the detector detects a specific wavelength region of the autofluorescence. [5] The inspection device according to any one of [1] to [4], wherein the inspection device includes an imaging unit that outputs an image of the subject's eye. [6] The inspection device according to any one of [1] to [5], wherein the suspended cells include at least one selected from the group consisting of peripheral blood mononuclear cells and malignant lymphoma cells. [7] The inspection device according to any one of [1] to [6] above, wherein the pulse width per pulse of the pulsed laser light is 100 fs to 100 ms. [8] The inspection device according to any one of [1] to [7] above, which is provided with an identification unit that identifies the type of suspended cells based on the spectrum of the autofluorescence. [9] A slit lamp microscope equipped with the inspection device according to any one of [1] to [8] above.

[10] A radiant exposure dose of 10 J / cm ２ An inspection method comprising: an irradiation step of irradiating suspended cells in an eye to be inspected with pulsed laser light having a pulse frequency of 1 Hz to 100 MHz or more and a detection step of detecting autofluorescence generated from the suspended cells by irradiating the suspended cells in the eye to be inspected with the pulsed laser light.

[11] The inspection method according to

[10] , wherein the pulsed laser light includes a plurality of types of pulsed laser light having different pulse frequencies in a pulse frequency band of 1 Hz to 100 MHz, and the irradiation step irradiates the eye to be inspected with the plurality of types of pulsed laser light at different timings.

[12] The inspection method according to

[10] or

[11] , further comprising an identification step of identifying the type of the suspended cells according to the spectrum of the autofluorescence.

[0065] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0066] (Examples 1 to 5) [Preparation of Suspended Cells] Peripheral blood mononuclear cells (PBMCs) collected from healthy volunteers were prepared as normal cells, and OCI-Ly10 and CA46 were prepared as malignant lymphoma cells. OCI-Ly10 is not commercially available and was distributed among researchers. CA46 was purchased from the American Tissue Culture Collection. The cell density for both cells was 2 x 10 ６ Each cell was diluted 10-fold with phosphate buffered saline (PBS) containing 0.5% bovine serum to obtain three types of cell samples.

[0067] [Irradiation of pulsed laser light] A cell sample was placed in a quartz cell and fixed with a sample holder. The fixed cell sample was irradiated with pulsed laser light for 30 seconds. The pulsed laser light was obtained by pulsing a laser generated by a semiconductor laser oscillator (Cobolt 06-MLD, manufactured by Cobolt) with a DG645 digital delay pulse generator (manufactured by Stanford Research Systems). The average output of the pulsed laser light was fixed at 100 mW, the wavelength at 485.1 nm, and the pulse width at 100 ns, and the pulse frequency was set to 50, 100, 500, 700, or 1000 kHz. In addition, since the diameter of the irradiated portion of the cell sample was 2000 μm, the area irradiated with the pulsed laser light was (10 ２ π×10 －４ ) cm ２ It was decided.

[0068] When the average output is 100 mW, the pulse width per pulse is 100 ns, and the irradiation time is 30 seconds, the radiation exposure [J / cm ] applied to suspended cells at a pulse frequency of 50 kHz is ２ ] is 0.1 × (100 × 10 －９ ) x (50 x 10 ３ ) x 30 / (10 ２ π×10 －４ ) J / cm ２If we calculate this with π = 3.14, we get 5 × 30 / (10 ２ π)≒0.477J / cm ２ The radiation exposure amount at each pulse frequency was calculated in the same manner. The results are shown in Table 1. In all of the Examples, no damage to the suspended cells due to irradiation with pulsed laser light was observed.

[0069] In each example, the irradiance per pulse applied to the suspension cells was 100 mW / (10 ２ π×10 －４ )=3.18W / cm ２ It was calculated that:

[0070]

[0071] [Measurement of Fluorescence Intensity of Each Cell Sample] The cell sample was irradiated with pulsed laser light for 30 seconds, and the fluorescence generated from the cell sample was collected using a relay lens and measured using a detector. The detector used was an "LVM-200-S1" (manufactured by Lambda Vision Co., Ltd.). The exposure time was 30 seconds. The fluorescence was then separated using a long-pass filter (22 mm CA SCHOTT GG495 C-MOUNT Longpass Glass Color Filter, manufactured by Edmund Optics), and the fluorescence intensity was measured at fluorescence wavelengths of 480 to 850 nm.

[0072] The fluorescence intensity of each cell was calculated by subtracting the fluorescence intensity of the control (PBS containing 0.5% bovine serum without cells). The relationship between the fluorescence wavelength and fluorescence intensity at each pulse frequency for the cell sample containing PBMC is shown in Figure 6. The relationship between the fluorescence wavelength and fluorescence intensity at each pulse frequency for the cell sample containing OCI-Ly10 is shown in Figure 7. The relationship between the fluorescence wavelength and fluorescence intensity at each pulse frequency for the cell sample containing CA46 is shown in Figure 8.

[0073] As shown in Figures 6, 7, and 8, when the pulse frequency was 50 and 100 kHz, malignant lymphoma cells showed higher fluorescence intensity than normal cells at fluorescence wavelengths of 500 nm or less. On the other hand, when the pulse frequency was 500, 700, and 1000 kHz, malignant lymphoma cells showed higher fluorescence intensity than normal cells across the entire measured fluorescence wavelength range.

[0074] Furthermore, the fluorescence intensity of each cell sample at a pulse frequency of 700 kHz is shown in Figure 9, and the fluorescence intensity of each cell sample at a pulse frequency of 1000 kHz is shown in Figure 10. As shown in Figure 9, at a pulse frequency of 700 kHz, the fluorescence intensity of malignant lymphoma cells was higher than that of normal cells at a fluorescence wavelength of around 550 nm. On the other hand, as shown in Figure 10, at a pulse frequency of 1000 kHz, the fluorescence intensity of OCI-Ly10 among malignant lymphoma cells was further improved at a fluorescence wavelength of around 550 nm, but the fluorescence intensity of CA46 decreased and became equivalent to that of normal cells.

[0075] Furthermore, when comparing the fluorescence intensity at a fluorescence wavelength of around 700 nm, the fluorescence intensity of malignant lymphoma cells was higher than that of normal cells at a pulse frequency of 700 kHz. On the other hand, at a pulse frequency of 1000 kHz, the fluorescence intensity of CA46, one of the malignant lymphoma cell lines, was higher than that of OCI-Ly10. Therefore, it was confirmed that the shape of the fluorescence spectrum of the fluorescence wavelength and fluorescence intensity differs for each suspended cell depending on the pulse frequency of the pulsed laser light. Therefore, it was confirmed that the use of pulsed laser light not only allows for the distinction between normal cells and malignant lymphoma cells, but also allows for the non-invasive and rapid identification of the type of malignant lymphoma cell.

[0076] According to the present disclosure, it is possible to provide an examination device, a slit lamp microscope, and an examination method that can reduce the burden on the patient and quickly examine the subject's eye.

[0077] 100, 101, 110, 120... inspection device, 70, 71... laser irradiation unit, 80, 81... fluorescence detection unit, 44... imaging unit, 90... identification unit, 10... laser generator, 30... detector, E... subject eye, Eo... observation eye, P... pulsed laser light, P １...first pulsed laser light, P ２ ...second pulsed laser light, F...autofluorescence, CR...control unit, 29, 41, 52, 55...relay lens, 31...objective lens, 37...ocular lens, 32...magnification variable lens, 32a, 32b...magnification variable lens, 33, 56...diaphragm unit, 34...beam splitter, 36...prism, 36a, 36b...optical element, 12, 42...mirror, 43...imaging element, GS...image signal, 53...cylindrical lens, 54...slit forming unit, 54A, 60A...actuator, 54B, 60B...moving mechanism, 60...filter, M...slit lamp microscope, 102...base, 103...laser irradiation unit support arm, 104...fluorescence detection unit support arm, 105...forehead rest, 106...chin rest, 107...lighting operation handle, 108...photography operation handle.

Claims

1. A method for detecting the autofluorescence of the suspended cells in the subject's eye, comprising: a laser irradiation unit that irradiates the subject's eye with pulsed laser light; and a fluorescence detection unit that includes a detector that detects autofluorescence from the suspended cells in the subject's eye that is generated by irradiating the subject's eye with the pulsed laser light, wherein the radiation exposure of the pulsed laser light irradiated onto the suspended cells in the subject's eye is 10 J / cm. ２ The following is the inspection equipment.

2. The examination device according to claim 1, wherein the pulsed laser light includes multiple types of pulsed laser light having different pulse frequencies in a pulse frequency band of 1 Hz to 100 MHz, and the laser irradiation unit is configured to be able to irradiate the multiple types of pulsed laser light to the subject's eye at different timings.

3. An inspection device according to claim 1 or 2, wherein the laser irradiation unit is provided with a cylindrical lens on the optical axis of the pulsed laser light that shapes the pulsed laser light into a sheet shape.

4. An inspection device according to claim 1 or 2, wherein the fluorescence detection unit is provided with a spectroscopic element on the optical axis of the spontaneous fluorescence, and the detector detects a specific wavelength range of the spontaneous fluorescence.

5. An examination device according to claim 1 or 2, comprising an imaging unit that outputs an image of the subject's eye.

6. The testing device according to claim 1 or 2, wherein the floating cells include at least one selected from the group consisting of peripheral blood mononuclear cells and malignant lymphoma cells.

7. The inspection device according to claim 1 or 2, wherein the pulse width of each pulse of the pulsed laser light is 100 fs to 100 ms.

8. An inspection device according to claim 1 or 2, comprising an identification unit that identifies the type of floating cells based on the spectrum of the autofluorescence.

9. A slit lamp microscope equipped with the examination device according to claim 1 or 2.

10. Radiation exposure is 10 J / cm ２ An examination method comprising: an irradiation step of irradiating suspended cells in a test eye with pulsed laser light, the pulsed laser light being as follows: and a detection step of detecting spontaneous fluorescence generated from the suspended cells by irradiating the suspended cells in the test eye with the pulsed laser light.

11. The examination method according to claim 10, wherein the pulsed laser light includes a plurality of types of pulsed laser light having different pulse frequencies in a pulse frequency band of 1 Hz to 100 MHz, and the irradiation step irradiates the subject's eye with the plurality of types of pulsed laser light at different timings.

12. An inspection method according to claim 10 or 11, further comprising an identification step of identifying the type of the floating cells according to the spectrum of the autofluorescence.

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