1. Field of the Invention
The present invention relates to an immersion microscope objective for use in a laser scanning microscope that uses multi-photon excitation.
2. Description of the Related Art
A known microscopic fluorescence observation means uses multiphoton excitation in fluorescence observation. During multiphoton excitation, a fluorescent object is illuminated with a light beam having wavelengths of integral multiples of an inherent absorption wavelength so as to cause excitation nearly equivalent to that caused by light having a wavelength equal to the inherent absorption wavelength. Multiphoton excitation is a non-linear phenomenon. For example, in the case of two-photon excitation, the excitation occurs at a probability proportional to the square of the intensity of the excitation light.
On the other hand, excitation light collected by a microscope objective lens has a light intensity that is inversely proportional to the square of the distance from the focal plane. Microscope multiphoton excitation occurs only in the vicinity of the focal point and thus fluorescence is emitted only from the focal plane.
Because of the above, a confocal pinhole, which is provided in the detection system of a common confocal microscope in order to shield the detector from fluorescence emitted anywhere other than at the focal plane, is unnecessary when using a multiphoton excitation laser scanning microscope. Advantageously, fluorescence in samples is subject to less discoloration because the excitation occurs only at the focal plane.
In multiphoton excitation, infrared light (that has longer wavelengths than light ordinarily used for excitation) is generally used as the excitation light. Generally speaking, light having longer wavelengths tends to scatter less (i.e., Rayleigh scattering). On the other hand, infrared excitation light reaches deep inside scattering samples such as living samples. This allows the deep parts of a living body to be made observable, as those parts are otherwise impossible to observe using only visible light. Furthermore, infrared light is less photo toxic than ultraviolet and visible light. This allows for observation of living samples with minimum damage.
For example, serotonin (a substance found in the brain) is self fluorescent, having absorption wavelengths in the ultraviolet range. However, ultraviolet light is highly photo toxic and does not reach deep inside the brain. In such circumstances, multiphoton excitation laser scanning microscopes work effectively. The ultraviolet light and infrared light differ in wavelength by a factor of approximately three. Thus, the infrared light can be used to excite serotonin by three-photon excitation.
As described above, multiphoton excitation fluorescence observation has significant merits and currently is a very effective type of microscopic observation.
However, microscopic observation using multiphoton excitation has some technical difficulties. For example, for multiphoton excitation to occur, two or more photons at a time must collide with a molecule of the fluorescent object and be absorbed. In order for this to occur, a significantly high photon density must be created at the focal point of the microscope objective lens. In other words, a microscope objective lens having a large numerical aperture with aberrations that are properly corrected must be used. As the excitation light is infrared light, it is important that aberrations of the microscope objective lens be favorably corrected for the infrared light.
For observation of the deep parts of a sample, aberrations resulting from the refractive index of the sample cannot be ignored. In order to avoid a deterioration of the fluorescence efficiency due to such aberrations, it is desirable that a correction collar be provided that corrects for aberrations that vary according to the depth of observation in the sample.
On the other hand, even though the excitation light is strong, only a small intensity of fluorescent light emerges from a sample. Therefore, the emerging fluorescent light has to be detected with a minimum loss. Thus, the microscope objective lens should be composed of a minimum number of lens elements and lens groups so as to optimize the optical path to the detector.
Furthermore, ultraviolet light should be detected in the case of three-photon excitation. However, conventional optical glass has a low transmittance to ultraviolet light. Therefore, the microscope objective lens should be constituted by specific types of optical glass that have a high transmittance to ultraviolet light.
A microscope that uses multiphoton excitation is often used in conjunction with a technique known as patch clamping that is widely used in cellular biology studies. When used in this manner, a sufficient working distance has to be reserved between the leading end of the immersion microscope objective and the sample. In other words, a large working distance and a sufficient access angle at the leading end of the immersion microscope objective need to be provided.
The excitation light is infrared light and the emerging fluorescence lies within the visible and ultraviolet ranges. Therefore, the fluorescence to be detected is likely to be subject to Rayleigh scattering by the sample. Consequently, light emerging from one point may become diffused before it enters the immersion microscope objective. Therefore, it is desirable that the immersion microscope objective be provided with a large field of view so as to collect the scattered fluorescence without loss.
When the field of view of the immersion microscope objective is extended, the pupil diameter of the immersion microscope objective increases proportional to it. Even if it is assumed that an immersion microscope objective having a very large input pupil diameter is developed, the performance cannot be displayed unless the input laser beams of the confocal microscope device fill the pupil diameter in the input pupil position of the immersion microscope objective. Therefore, Japanese Laid-Open Patent Application No. 2008-040154 proposes a technique for adjusting the input beam diameter in such a way as to approximately match the pupil diameter of the immersion microscope objective with an input beam diameter. According to this technique, the confocal microscope device can adjust the input beam diameter in such a way as to approximately coincide with the pupil diameter of the immersion microscope objective. However, since the input beam diameter is adjusted before beams enter a laser beam deflection means (a two-dimensional scanning means), the maximum input beam diameter is defined by the laser deflection means.
In order to expand this, it is necessary to increase the device size of the laser beam deflection means such as a galvano-mirror and the like. However, the larger the size of the galvano-mirror becomes, the larger a deflection angle for scanning the specimen range becomes to reduce the scanning speed. In a resonant galvano-mirror provided with two galvano-mirrors near a position conjugate with the aperture position and capable of two-dimensional scanning, the distance must be extended in order to avoid the interference between the two galvano-mirrors. Thus, it deviates from the ideal position to increase illumination unevenness.
Therefore, it is important to combine an optimum immersion microscope objective with the maximum input beam diameter defined by the laser beam deflection means of the confocal microscope device.