JPH0387804A - Scanning cofocal microscope - Google Patents

Abstract

PURPOSE: To mutually install a light source and a photodetector at free positions without requiring any fixed connection with other parts by using optical transmitting and separating means flexible for transmitting light among the light source, condenser and detector. CONSTITUTION: Light from a laser generator 1 is converged to one end of an optical fiber 4 by a lens 3 and connected through optical fibers 6 and 8 to a refraction factor matching medium 7 or an optical microscope 10 by a directional coupler 5. The light guided to the optical microscope 10 moves and scans in X and Y directions on a sample 20 based on the signal supplied from an electronic scanning signal generator 25 to transducers 21 and 22. Light emitted from the sample 20 is returned through the optical system of the microscope 10 to the optical fiber 8 and one part of light is sent from the coupler 5 through an optical fiber 31 to a photodetector 34. A signal from the photodetector 34 is sent from a processor 36 to a display screen 37 and displayed.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the field of microscopy, in particular scanning confocal microscopy.

(Prior technology) The principle of scanning confocal microscopy is based on Marvin Minski's
Marvin Minsky) US Patent No. 3, 0
No. 13.467. The basic principle is that the illumination of the observed specimen or object is limited to a single point observation area, and the observation or detection is limited to that illumination point area. A complete image is obtained by scanning the specimen or object under observation point by point across the field of view of the microscope.

Traditional confocal microscopes, including the one proposed in the Minski patent, have fixed optical systems that scan the specimen or object being viewed by moving it in a scanning pattern across the focal point of the illumination. Modern high-speed scanning microscopes employ beam scanning techniques rather than moving the specimen. Typically, such microscopes use a laser as the high-level illumination source and are equipped with a computer or video system for processing and storing the detected image signals.

Confocal microscopes have higher resolution and sharpness than conventional microscopes because out-of-focus signals and interference are significantly suppressed. These are commonly used to examine biological specimens by epifluorescence, where the reduction of out-of-focus interference is a major advantage.

Confocal microscopes are known to be manufactured by attaching a confocal imaging system to a microscope equipped with a condensing or focusing lens.

However, in any known confocal device, the condenser, the light source and the detector, and all the parts forming the optical path of the microscope need to be precisely positioned with respect to each other and are therefore mounted on a large common body structure. .

(Problems to be Solved by the Invention) The present invention provides a modified structure in which some of the above-mentioned parts can be positioned completely independently in space without the usual fixed, geometrical constraints on their relative positions and orientations. I will provide a. In particular, the invention allows the light source and photodetector to be mounted in any position without the need for fixed mechanical connections between each other and with other components. Other advantages of the invention will become apparent from the description below.

(Means for Solving the Problems) The present invention includes a light source that supplies a light beam, a light beam that is focused on an object to illuminate a point observation range of the object, and a light source that receives light emitted from the point observation range of the object. a concentrator, a detector for detecting light emitted from the object and received by the concentrator, and a detector for transmitting light from the light source to the concentrator and detecting the light emitted from the object and received by the concentrator; A scanning confocal epi-illumination microscope comprising a light transmission means for transmitting light to a point observation region, and a scanning means for causing relative movement between the object and the point observation range so that the point observation range traverses the object in a scanning pattern, the light transmission means comprising: It is characterized by comprising a flexible light transmission means for transmitting a light beam between a light source and a condenser, and a light separation means for separating light emitted from an object from the light beam.

The flexible light transmission means may consist of a first optical fiber extending from the light source to the light separation means and a second optical fiber extending from the light separation means to the detector. In this case, the optical separation means comprises an optical fiber coupler connecting the first and second fibers to a third optical fiber, transmitting the light beam from the light source to the collector and coupling the light emitted from the object from the collector. An optical path for transmission to a device may also be formed.

The scanning means may be configured to move the light beam sent from the third optical fiber to the condenser.

For example, the scanning means may consist of a transmission member which forms the optical path of the light beam from the third optical fiber to the concentrator and causes a scanning movement of the light beam. Alternatively, the scanning means may be configured to move the third optical fiber, thereby moving the light beam sent from the third optical fiber to the collector, resulting in a scanning movement thereof.

In another configuration, the light separation means may consist of a beam splitter provided between the light source and the flexible light transmission means or between the flexible light transmission means and the condenser.

OPERATION AND EFFECTS OF THE INVENTION By using a flexible optical transmission means (usually an optical fiber), the light source (usually a laser) and the detector can be separated from a rigid mechanical Can be installed without connection. The nature of such devices has two important consequences. First, the production of a device that can be attached to an existing microscope using standard microscope optics and mechanical adjustments to yield a confocal imaging system in which the laser generator and detector can be placed well away from the microscope. enable. Second, it allows the design of confocal imaging systems using very compact remote head beads that serve specific purposes, such as as endoscopes or as implantable remote head microscopes in medicine.

(Example) FIG. 1 shows a scanning and focal epi-illumination microscope system of the present invention. This system consists of laser generator 1
The light beam 2 is focused by a lens 3 onto one end of a flexible optical fiber 4. The other end of the optical fiber 4 is extended to a directional coupler 5, which is a junction biconical Taber coupler or the like that separates light beams in opposite directions. The light traveling to the output leg 6 on the other side of the coupler 5 is absorbed by the index matching medium 7 with minimum Fresnel reflection, and the light traveling to the other leg of the coupler 5 is transmitted from the end 9 of the flexible optical fiber 8 to an optical microscope. It is transmitted to the optical system of IO.

The optical microscope 1O includes a base 11, a mechanically adjustable specimen support 12 attached to the base 11, and a microscope body 13 that houses components constituting an optical system. These optical components include a lens 14 that receives light diverging from the end 9 of the fiber 8, a pair of mirrors 16 and 17 that sequentially reflect the light that has passed through the lens 14, a focusing lens 19, and a focusing lens 18. The condensing lens 18 condenses light onto a spot or point observation area of the specimen 20 supported on the table 12.

The mirrors 16, 17 are configured to be movable by means of transducers 21, 22 on the basis of signals supplied from the electronic scanning signal generator 25 via the electrical connections 23, 24, so that the reflected beam is moved in the X and Y directions. move,
The illumination spot traverses the specimen in a scanning pattern. Such scanning means are used in known scanning confocal microscopes.

The condenser lens 18 focuses intense light onto the specimen to generate an illumination spot, and at the same time receives light emitted from the specimen. This light is returned to the optical fiber 8 via the optical system of the microscope IO. Depending on the type of specimen, this light can be reflected light, Raman scattered light or fluorescence. As used herein, the term "emit" has a broad meaning that includes any light that is transmitted back from an object through a concentrator. This light is focused back onto the end 9 of the optical fiber 8 and transmitted through the fiber 8 to the coupler 5, where a portion of the light is transferred to the fourth leg of the coupler 5 and to the flexible optical fiber 31, and The light is sent to a photodetector 34 via a filter 32 and a lens 33. The signal from photodetector 34 is passed through electrical connection 35 to
It reaches the signal processor 36 of the video display system which displays the image on a display screen. The signal from photodetector 34 is output from processor 36 on output line 3.
8 to the display screen 37, the mechanical scanning movement of the mirror 16.17 is transmitted to the display system via the connection between the electronic scanning signal generator 25 and the processor 36. Synchronized with electronic rask scan movement.

FIG. 2 shows a modified scanning confocal epi-illumination microscopy system of the present invention. This system is similar to the system shown in FIG. 1, but employs a beam splitter instead of the junction biconical taper coupler of FIG. 1 as a means of separating the output and return light. Many parts of this device are identical to those of the system of FIG. 1 and function similarly. These parts are designated by the same number with the addition of an A. In this example, the junction biconical taper coupler and the manifold optical fiber are replaced by a single optical fiber, at one end of which the light from the laser IA is focused by the lens 3A, and from the other end the light is directed to the lens of the microscope head IOA. 14A, travels along the optical path of the microscope head IOA, and illuminates the specimen 2OA as in the case of FIG.

The returned light captured by the condenser 18A returns to the lens 3A through the same optical path and fiber 41. This returned light is split by a beam splitter 42 provided between the laser source IA and the lens 3A, and a returned light beam 43 is deflected and detected by the photodetector 34A.

FIG. 3 shows another modified system of the invention in which the returned light is split by a beam splitter located within the microscope head. Components that are the same as the system of FIG. 1 are designated by the same number with the suffix B added. In this device, light from a laser source IB is passed through an optical fiber 5 by a lens 3B.
The light is focused on one end of the specimen 20B, sent to the optical system of the microscope head IOB, and focused on one point on the specimen 20B.

Light emitted from the same point on specimen 20B is captured by condenser 18B and returned through the optical system of the microscope. The microscope head IOB is provided with a beam splitter 52, which splits the returned light and sends it through a lens 54 to a second optical fiber 53.
The fiber 53 focuses the light on one end and transmits the light to the photodetector 34.
Send to B.

Figures 4.5A and 5B show yet another modified system of the invention. This system is very similar to the system of FIG. 1, and like parts are designated with like numbers plus a C. The difference from the system in FIG. 1 is that scanning is performed here by moving the tip 9C of the optical fiber 8C that transmits light from the laser generator IC to the microscope head IOC. The fiber tip is moved by a movement generator 61.
by. This movement generator 61 is a scanning pattern generator 2
Conveniently, it comprises an electromechanical transducer receiving electrical signals from 5C. The transducer may be of any type that provides suitable scanning motion in the X and Y directions. A typical example of such a device is shown in FIGS. 5A (side view) and 5B (top view).

In the fiber movement generator 61 shown in FIGS. 5A and 5B, scanning movement of the fiber tip is performed by a combination of electromagnetic induction resonance vibration and hydraulic motion.

The permanent magnet 62 attached to the flexible piece 63 is
Generated by the scan control unit 25C and connected to the lead wire 65
is periodically attracted to the electromagnet 64 according to the electric pulses transmitted to the optical head by the electromagnet 64.

As a result, the optical fiber 8C extending along the flexible piece vibrates and scans in one direction (for example, the X direction). To ensure synchronization of the electronic image scan with the mechanical scan, a piezoelectric sensor 66 provides position feedback to the image processor 36C via a lead 67. Other scanning axial movements can be achieved by supplying or discharging liquid to or from the cylinder 68 via the supply tube 70, by actuating the piston 69 to which the entire electromagnetic scanning unit is attached to produce slower sub-linear movements. It will be done. As a result, the tip of the fiber moves in the Y direction.

It will be appreciated that in all the embodiments described so far, it is possible for the light source, detection and display unit to be located remote from other components such as optomechanical components normally incorporated into optical microscopes. These systems therefore enable the production of laser imaging devices that can be attached to existing microscopes to take advantage of standard microscope optics and mechanical adjustments. The ability to separate the optical head from the rest of the system using fiber optic connections also allows for miniaturization of the optical head, especially if scanning is performed by fiber movement. Therefore, the present invention can be applied to fields such as endoscopes that require a compact and remote head.

Figures 6-8 illustrate a system of the present invention incorporating an endoscopic head for examining soft tissue within a body cavity. This system is generally based on the system shown in FIG. 1, with identical parts designated by identical numbers with the addition of a D.

Here, the optical fiber 8 is used instead of the microscope 10 in FIG.
An endoscope head IOD connected to D is provided.

The scanning is provided in the endoscope head IOD and connects 23D
, 24D by the movement of the tip 9D of the fiber 8D by an electromechanical transducer 71 receiving signals from the scanning signal generator 25D via the scanning signal generator 25D. transducer 7
1 may be similar to transducer 61 in FIG.

The endoscope head IOD is used to perform a preliminary low-power exploratory examination of the entire soft tissue area 72 and then perform a high-power examination focused on a specific area. The endoscope head IOD is fi! It has an inner shell 73 made of J74.75, and an outer shell 76 that is slidable on the inner shell between the retracted protruding position shown in FIG. 7 and the protruding position shown in FIG.

The inner barrel 73 houses a collimator lens system 77 that receives light from the fiber 8D, and the light becomes a slightly converged beam and is projected onto a mirror 78 provided at one end of the inner barrel 73. When the outer shell 76 is in the retracted position shown in FIG. 7, the reflected light passes directly through the transparent boat 79 to the portion of tissue 72 to be examined.

The outer barrel 76 is provided with a cylindrical spigot 81 that projects outward, and a focusing lens system 82 is housed therein.

The spigot 81 forms a cup 80 whose proximal end is closed by a lens system 82 . When the outer barrel 76 moves to the protruding position shown in FIG. 8, the light beam from the collimator lens system 77 is reflected by the mirror 78 and projected onto the focusing lens system 82. A pair of flexible tubes 83
．． 84 extends along the exterior of the endoscope head IOD,
The spigot 81 communicates with the cup 80 through an opening in the side thereof. These tubes 83 and 84 apply suction force inside the cup to suck the soft tissue 72 to be examined into the cup, as shown in FIG. Supply cleaning fluid to wash.

When the endoscope head IOD is first brought close to the tissue part to be examined, the focusing lens system 82 is moved out of the optical path as shown in FIG. 7, and the slightly converged light from the collimator lens 77 is spotted on the tissue part. To be projected, the outer body 7
6 is maintained in the retracted position. And the mobile generator 71
As a result of the movement of the tip 9D of the fiber 8D, the spot scans a relatively wide range. When a location to be inspected more closely is identified, the outer barrel 76 is extended as shown in FIG. 8, the focusing lens system 82 is positioned on the optical path, and one tube 8
By applying suction force to 3, the tissue becomes spigot 81.
into the end of the tube and kept properly fixed for high-power confocal microscopy. Irrigation fluid can be supplied to the tissue via the other tube 84 and aspirated and evacuated using a vacuum tube.

When the endoscope head IOD captures a relatively wide image of a distant object as shown in FIG. 7, confocal return may be poor. Light for modulation of the raster image can then be extracted from the tip 85 of a non-interfering fiber bundle 86 that extends along the endoscope head IOD to the photodetector. This system can also see distant objects without using a laser.

To do this, the incoherent fiber bundle 86 can transmit intense light to illuminate the object. The tip 9D of the scanning fiber 8D rasterizes the image space and captures the light.
It is returned to a photodetector and an image is generated from the photodetector's output signal.

Alternatively, it is also possible to directly illuminate the object with an incandescent bulb provided at the outer end of the endoscope head IOD.

FIG. 9 shows another system that is capable of distant object observation and high power confocal observation. Components that are the same as the system shown in FIG. 1 are designated by the same number with an E appended. The fiber 8E of this system is the optical head 15
0, and the light passes through the collimator lens 151 and the condensing lens 152. Object located in point observation area 153.

The body can be observed by confocal return light through the optical system to photodetector 34E. Fiber 155, which corresponds to fiber 6 described above, terminates in this embodiment at a tip 156 in a non-absorbing medium held in a movement generating head, similar to fiber 8E. Thus, the two fiber tips are scanned synchronously by the moving actuation system 166.

Light from the tip of fiber 155 is focused by another lens 157 to a distant spot 159 and scans object 159. A portion of the light emanating from scanning spot 158 enters the end of fiber optic bundle 162 and is transmitted to photodetector 34E.

The selection of which observation method to use is made using a plate 160 having an aperture 161. By moving this plate 160, light enters the photodetector 34E from either the end of the fiber 31E or the fiber optic bundle 159.

Scan drive signals are carried by leads 162 and 163, and position feedback signals are carried by leads 164 and 165.

FIG. 10 shows yet another modified system, in which parts that are the same as those in the system of FIG. 1 are designated by the same numbers with the addition of an F. In this system, the microscope head 10 is replaced by a small optical head IOF, which is used for tooth inspection, especially for detecting early caries holes in the dentin beneath the enamel. The optical head IOF includes a rigid housing 101 into which the end of an optical fiber 8F is inserted, and the tip 9F of the fiber 8F is driven by an electromechanical transducer 102 to perform the necessary scanning. The housing 101 includes a condenser lens 10
3, a lens 110 that forms an optical path between the fiber 8F and the condensing lens 103, and a mirror 105.

On the opposite side of the housing 101 from the condenser lens 103 is a "sandwich" layer 104 of a spongy or flexible material.
is glued and covered with a relatively hard plate 106.

The optical head IOF detects the tooth 1 on the opposite side of the tooth 107 to be inspected.
08 and the tooth 107 to be inspected is located above the condenser lens, and the opposite tooth 108 presses the plate 106. The sandwich layer 104 allows relative movement between the teeth without causing relative movement between the optical head IOF and the scanned tooth. A tube 109 may be provided for supplying an index matching gel or viscous fluid to the space between the objective lens and the tooth to be examined.

FIG. 11 shows yet another confocal microscope system of the present invention. This system uses a concave mirror instead of a lens system as a condenser.

Parts that are the same as those in the system of FIG. 1 are designated by the same numbers with a G appended. In the system of FIG.
The tip 9G of G is located near the support 111 of the specimen 112 to be examined. This fiber tip 9G and support stand 1
11 are arranged to face each other near the center of curvature of the concave mirror 113. The light sent from the fiber tip 9G is concave mirror 1
13 to the focus of the specimen 112. Specimen 112
The reflected light or fluorescence emitted from the fiber returns to the fiber tip 9G in a confocal manner and is sent to the photodetector 34G.

An electromagnetic transducer 114 is attached to the fiber tip 9G and receives signals from a scanning signal generator 25G to cause its scanning movement. It is clear that instead of this, scanning can be performed by moving the specimen stage 111 or by a combination of moving the specimen stage 111 and the fiber tip 9G.

The system of FIG. 11 has no fear of chromatic aberration.

Spherical aberration is small if the fiber tip 9G and the specimen 112 are close to each other. Correction of spherical aberration is made possible by making the concave mirror surface into a spheroid with two focal points coinciding with the fiber tip 9G and the inspection point of the specimen 112 with very slight mechanical deformation. Such deformation can be achieved by means such as mechanical tightening. It is also possible to provide a small cylindrical piece near the fiber tip to create astigmatism in the opposite direction and reconvert the image to a spot for the output and return beam.

FIG. 12 shows a confocal system in which multiple microscope heads can be used alternately.

This system is a modification of the system shown in FIG. 1, and identical parts are indicated by identical numbers with an H added. In this example, the output leg 6H of the coupler 5H is not connected to an absorber but is connected by a fiber 121 to the second microscope head IOH'.

This microscope head IOH' is the first microscope head IO
scanning mirror transducers 23H' and 24H connected to scanning signal generator 25H;
′ is provided.

According to the configuration shown in Fig. 12, while one microscope head is used for confocal observation, the other microscope head is set to focus on the specimen, and as soon as one microscope head is finished observing, the other microscope head is used for confocal observation. Focus observation is possible. In this way, the system can be maintained in substantially continuous operation.

The embodiments of the invention described above can be further modified and developed to utilize fiber optic technology. For example, although a single optical fiber is used to transmit the light beam in both embodiments, it will be appreciated that multiple fibers could be utilized. In that case, the depth of field can be increased by slightly shifting the tip of the fiber that receives the return light that is emitted from the object and generates the confocal image in the longitudinal direction of the fiber (light transmission direction). When scanning is performed by moving the fiber,
The entire fiber bundle can be attached to a fiber movement generator, such as a transducer, for movement as a unit.

If scanning is performed by other means such as mirrors or specimen movement, the offset fiber tip may be fixed relative to the condenser. Also, if multiple fibers are used, simultaneous scanning of different wavelengths becomes possible.

In another modification, the optical coupler of Figure 1 is split laterally at a midpoint to form an end that transmits light to the microscope head and focuses light emanating from the object to produce a confocal image. can do. In this case, FIG. 8 would be omitted and the end of the split coupler would replace the fiber tip 9.

In yet another embodiment, the splitter coupler has three converging limbs, and the additional converging limbs can provide light from laser sources of different wavelengths to provide multi-wavelength scanning.

The present invention is particularly useful for scanning confocal microscopes for testing purposes and confocal microscope systems equipped with small remote heads suitable for endoscopic examination of living tissue.

[Brief explanation of drawings]

Fig. 1 shows a confocal microscope of the present invention in which the separation of output light and return light is performed by a fiber optic directional coupler, and Fig. 2 shows a confocal microscope in which the separation of the light is performed between the light source and the flexible optical transmission means. FIG. 3 shows a modified microscope system in which light separation is performed by a beam splitter provided between a flexible optical transmission means and a condenser. , 4.5A and 5B show another confocal microscopy system similar to the system of FIG. 1 in which scanning is performed by moving the tip of an optical fiber of a flexible light transmission means, figures 6 to 8. Figure 9 shows a modified confocal microscope system employing a small remote head specifically used as an endoscope; Figure 9 is a diagram similar to Figures 6 to 8 using a modified endoscope head; Figure 10 shows a further system using a small optical head, especially used for dental examinations, and Figure 11 shows a system using a concave mirror instead of a lens system as the condenser. FIG. 12 shows a confocal system with two microscope heads. 1.1A, IB, IC, ID, IE, IF. IG, IH... light source. Night A. ': Mg, 2& Procedural Amendment October 11, 1990 1990 Patent Application No. 187003 2゜Name of Invention Scanning Confocal Microscope 3゜Relationship with the case

Claims (1)

[Claims] a light source that provides a light beam; a concentrator that focuses the light of the light beam onto an object to illuminate a point observation area of the object; and a concentrator that receives light emitted from the point observation area of the object; a detector that detects the received light; a light transmission means that transmits the light from the light source to the condenser; and a light transmission means that transmits the light emitted from the object and received by the condenser to the detector; a scanning confocal epi-illumination microscope comprising scanning means for effecting a relative movement of an object and a point observation field across the object in a scanning pattern, the light transmission means being flexible for transmitting a beam of light between a light source and a condenser; 1. A microscope comprising a light transmission means, and a light separation means for separating light emitted from an object from the light beam.