WO2003019242A2

WO2003019242A2 - Line scanning confocal microscope

Info

Publication number: WO2003019242A2
Application number: PCT/US2002/027926
Authority: WO
Inventors: Roger C. Young
Original assignee: MUSC Foundation for Research and Development
Current assignee: MUSC Foundation for Research and Development
Priority date: 2001-08-29
Filing date: 2002-08-28
Publication date: 2003-03-06
Anticipated expiration: 2004-02-29
Also published as: WO2003019242A3; AU2002332802A1

Abstract

A scanning confocal microscope uses a focused line of light to scan an objective. The line of light for scanning is created by an appropriate source. A scanning device (12) produces a scanning effect with the light. The scanning, focused line of light is directed an objective, such as tissue, whose image is magnified by a microscope (14, 16, 18, 20). The line of light is scanned across the specimen by the scanning device so rapidly that the image it produces appears as if it were viewed directly. The rapid scan rate obviates the need for synchronization between the scanning device and image recorder (26).

Description

LINE SCANNING CONFOCAL MICROSCOPE

FIELD OF THE INVENTION

This invention relates to confocal optical microscopes generally, and is specifically directed to scanning confocal microscopes.

BACKGROUND

Scanning microscopes are commonly used with fluorescent techniques, such as fluorescence imaging of tissue. Confocal microscopy allows improved resolution in such imaging techniques. The confocal image may be viewed directly through the microscope, or the image may be captured by photo imaging devices, including analog and digital devices.

Conventional scanning microscopes are categorized into two types. 1) laser scanning and 2) mask or Nipkow rotating disk type. The laser scanning type scans the specimen by means of two galvanometers across the specimen, using a point of focused light for excitation, and detects the reflected or fluorescent light with a photodetector, usually a photomultiplier tube. The mask or rotating disk type normally uses incoherent (non-laser) light as incident light, but achieves confocal images using mechanical embodiments to mechanically remove out-of-focus light. The photodetector for^* a device of this type is usually a 2-dimensional CCD camera. Each type of scanning microscope has inherent advantages and disadvantages. The laser scanner is expensive, requiring a laser and two galvanometers, and is limited in speed by the scan rate of the galvanometer. A significant disadvantage of the laser is the limitation in choice of the wavelength of the incident light. The mechanical type scanning microscope has the advantage of using conventional (incoherent) illumination, but loss of light intensity is large, and the speed is limited by the rotation rate or light detection. In both of these confocal microscopes, a means for synchronizing the photodetector output with the incident light is usually required.

Microscopes in the prior art direct light through a pin hole to produce a point of light. This point of light may be scanned along a line by the microscope. However, such microscopes do not produce a focused line of light.

SUMMARY OF THE INVENTION

This invention is a scanning confocal microscope that uses a focused line of light to scan an objective. The line of light for scanning is created by an appropriate source. A scanning device produces a scanning effect with the light. The scanning, focused line of light is directed at an objective, such as tissue, whose image is magnified by a microscope. The device does not require the use of a laser to form the line of light, which reduces the production cost of the device. The line of light is scanned across the specimen by the scanning device so rapidly that the image it produces appears as if it were viewed directly. The rapid scan rate obviates the need for synchronization between the scanning device and image recorder.

DESCRIPTION OF THE DRAWING Figure 1 demonstrates the scanning confocal microscope of the present invention.

DESCRIPTION OF THE PREFERRED EMOBIDMENT

Referring to Fig. 1, light 2 radiating from an incoherent light source of circular cross sectional shape is passed through a convex lens 6, pinpoint or pinhole filter 8, and convex lens 10, to shape the beam into nearly parallel rays. Prior to entering the lenses, the light may be filtered, such as by a filter 4, to select the wavelength of the incident light. This light is passed onto a scanning device, such as a resonant galvanometer 12, having a mirror or other reflector to reflect light from the light source. The operation of the resonant galvanometer produces a scanning effect with the light that is reflected from the resonant galvanometer. Scanning should occur at a frequency that is substantially greater than 30 Hz, so that the image appears to be continuously illuminated. A resonant galvanometer will scan at 1kHz to 8 kHz, or greater, which is useful in practicing the invention.

After reflection by the galvanometer, the beam is passed into a microscope and through a series of optics that are defined by the particular microscope. The light is passed through the objective lens and focused onto the objective, which may be a tissue sample or specimen.

A cylindrical lens 14 is present. In the embodiment of Figure 1, the cylindrical lens is positioned so that light enters trie cylindrical lens immediately prior to the dichroic mirror 18 and objective 20. The light is transformed by the cylindrical lens 14 into a focused line of light having a finite length that is determined by the cylindrical lens selected. The line of light is well focused by the cylindrical lens, so that the line of light has no material width or thickness. The newly formed line of light is subsequently focused onto the objective by the operation of the microscope, such as by lens 16. This focused line of light rapidly scans the objective to produce an image that is reflected from the objective.

Alternatively, the cylindrical lens may focus the light into a line of light prior to the light striking and being reflected by the resonant galvanometer. The line of light exits the cylindrical lens and is directed to the resonant galvanometer in this alternate embodiment. The resonant galvanometer produces a scanning effect that is directed to the objective as described above.

Reflected or fluorescent light is then passed back through the microscope, through the dichroic mirror (but not the cylindrical lens), and onto an image-capturing device 26, which may be a 2-dimensional CCD camera.

The light may be filtered to select reflected or luminescent light by the use of filter 20. A focusing lens 22 may be provided. Out of focus light is rejected by a slit 24. Scanning by using a line of light produces luminescence that is sufficient for tissue viewing and image capturing, without the need of a laser. However, the line of light may be produced by devices other than as disclosed by the device of Figure 1 , and could be produced by a laser. The modifications required to convert a standard microscope to the line scanning confocal microscope as described in the device of Figure 1 are modest.

"Line of light" and "line scanning" as used herein should not be confused with line scanning as that term is sometimes used in the prior art. Line scanning as the term is sometimes used in the prior art is scanning by the sequential and progressive movement of a point of light along a line. The device and process of the present invention form a line of light, wherein the light is formed into a line that has a finite length, as defined by the cylindrical lens. The cylindrical lens focuses the line of light so that the line of light has no material width or thickness. The focused line of light produced and used according to the invention is not a single point of light that follows a predetermined line of travel. The line of light is a straight line having one dimension of finite length. This characteristic distinguishes the line of light from a point of light that is traveling, or is moved, along a line. The scanning device oscillates the line of light across the specimen or other objective at a rapid rate.

An advantage of the line scanning confocal microscope of the present invention is that the cost to convert a conventional fluorescent microscope to a confocal microscope is reduced, since the device is comprised of lenses and preferably a single galvanometer. The use of incoherent light as the illumination source obviates the wavelength limitations of the laser scanner.

The disadvantages of light loss and speed limitations of the mask or Nipkow disk type confocal microscopes are also overcome. A further advantage over both types is that the line scanning confocal microscope of the invention does not necessarily require synchronous output for the photodetector, meaning that no apparatus for synchronization, such as a computer, is necessarily required.

The line of light produces adequate light for image capturing using inexpensive CCD cameras, because it is rapidly and repetitively scanned across the specimen by the resonant galvanometer. Additionally, the image appears as a confocal image by the human eye since the rapid rate of scan is not able to be time-resolved by the brain. This is in direct analogy as to why frame rates of video greater than 30 frames per second appear to the human eye as seamless motion. The confocal image is not as sharp as a laser scan confocal image using two galvanometers, however is a significant improvement over wide-band fluorescence images. The standard method of conversion of wide-band to confocal images is computer deconvolution. Using deconvolution with the invention presented here, will allow confocal images as sharp as laser scanned images to be obtained at much less cost. The resulting image of the specimen is a confocal image with quality that is comparable to one obtained using laser scanning. This image is also amenable to deconvolution programs that will raise the quality of the image. Since the oscillations are hundreds to thousands of times faster than the means used to capture the images (human eye or video rate camera), these results are obtained without necessarily using methods to synchronize the oscillations of the focused line with the detector. The oscillations occur at a rapid rate that is sufficient to produce serial confocal images, without requiring the images to be synchronized for recording.

Claims

What is claimed is:

1. A method of forming an image using a microscope, comprising the steps of: providing light; forming a focused line of light from said light; and directing said focused line of light through at least one lens of a microscope, and onto a specimen; wherein said line of light is rapidly oscillated to scan said specimen.

2. A method of forming an image using a microscope as described in Claim 1 , where reflected light from said line of light is reflected from said specimen, and said reflected light is captured.

3. A method of forming an image using a microscope as described in Claim 1 or Claim 2, wherein said light is formed in generally parallel rays prior to being formed into a focused line of light.

A method of forming an image using a microscope as described in Claim 1 , Claim 2, or Claim 3, wherein said scanning of said light occurs before said light is formed into a focused line of light.

A line scanning confocal microscope comprising a light source, a scanning device that receives light from said light source and produces a scanning effect with said light, a line forming lens that forms a line of light from said light, and a magnification lens that receives said line of light.

6. A line scanning confocal microscope comprising a light source, means for light scanning that receives light from said light source, means for forming a line of light from said light, and magnification means that receives said line of light.

7. A line scanning confocal microscope as described in Claim 5 or Claim 6, wherein said line of light is directed at and strikes an objective, and further comprising a mirror for reflecting an image produced by said line of light striking said objective.

8. A line scanning confocal microscope as described in Claim 7, further comprising an image capturing device that receives said image and captures said image.

9. A line scanning confocal microscope as described in Claim 5, Claim 6, Claim 7 or Claim 8, further comprising at least one convex lens that receives light from said light source and shapes said light prior to said light being formed into a line of light.

10. A line scanning confocal microscope as described in Claim 8, fulher comprising a filter that is positioned between said mirror and said image capturing device.