This invention relates to microscopy, and particularly to scanning microscopes for confocal fluorescence microscopy.
In fluorescence microscopy, a sample to be examined is treated with one or more dyes that selectively attach to parts of the sample and, when illuminated with excitation light of one wavelength, fluoresce at light of another, emission wavelength. The fluorescent light is examined microscopically to identify the parts of the sample to which the dyes have attached. Typically, in modem fluorescence microscopy, the illumination is provided with a laser, which provides relatively intense light over a narrow spectrum to excite particular dyes selectively.
It is advantageous in fluorescence microscopy to use a scanning microscope that images only one point or pixel of the sample at a time. Where the image is to be digitized for computerized enhancement or analysis, this permits descritization of the sample by use of a relatively simple detector and relatively simple optics. Also, scanning microscopy makes confocal microscopy possible, which provides higher resolution than would otherwise be available. As discussed hereafter, scanning microscopy may be accomplished either by moving the scanning spot using a fixed objective lens, by moving the optical system itself, or by moving the sample.
An early approach to scanning microscopy is illustrated in Petran et al. U.S. Pat. No. 3,517,980, which discloses the use of a fixed, extended light source focused on a Nipkow disc, having spiral apertures, which produces a point source of light that scans the object field of a fixed microscope objective. The light emitted from the sample is focused on a complementary aperture in the disc and thereafter propagated to an eyepiece to effectuate confocal microscopy. A similar approach is disclosed in McCarthy et al. U.S. Pat. No. 4, 863,252, which is directed to producing relative movement of the optics and sample stage for selecting the region and depth of the sample to be scanned.
More recently, scanning microscopes have employed a fixed point source and fixed objective, and cause the illumination light and the light emitted from the specimen to be scanned. For example, Hayashi U.S. Pat. No. 6,028,306 discloses a scanning microscope wherein the beams produced by a plurality of point sources are caused to scan in two dimensions by pivoting mirrors operated by galvanometers. More generally, Atkinson U.S. Pat. No. 6,108,127 describes the use of scanning optics to alter the path of a light beam through a fixed microscope objective.
The aforementioned types of scanning microscopes require highly corrected and expensive lenses to achieve high resolution and sensitivity. They also require complex and expensive optical systems mechanical scanning devices.
Another recent approach to scanning microscopy is illustrated by Boettner et al. U.S. Pat. No. 5,880,465, wherein the microscope objective itself is translated laterally in two orthogonal directions to scan the sample by moving the focal point of the objective. While this approach provides the advantage of reducing the effect of off-axis light beams by ensuring that the light emitted from the specimen is always on axis, the use of linear translation to cover the entire region to be scanned limits both the area that can be scanned and the speed at which scanning can occur, and can work only with low mass objectives which have limited optical performance.
Accordingly, there is a need for a scanning microscope that provides high resolution, high sensitivity and high scanning speed using relatively straightforward and inexpensive, but high performance, optics and mechanics.
The invention of the present application meets the aforementioned need by providing a scanning microscope that employs a combination of rotational and linear scanning to permit the use of a relatively simple optical elements and a relatively simple mechanical scanning system. In its general form the microscope comprises an objective lens for receiving light emitted from a sample in object space and propagating it to image space thereof, a collection lens that receives light from the objective lens and propagates it to a focal point in image space of the collection lens, and a motor, having an axis of rotation that is offset from and extending in substantially the same direction as the optical axis of the objective lens, for rotating the objective lens around the axis of rotation. This causes an optical pathway to be scanned across a sample in object space of the objective lens.
The sample is mounted on a stage. After each rotation of the objective lens, the stage and the optics are moved relative to one another with respect to the axis of rotation so that each subsequent scan covers a new part of the sample. Thus, the entire sample may be scanned. The stage and the optics may be moved relative to one another in another lateral direction as well, to move another sample, or sample portion, under the scanning area.
For fluorescence microscopy, a light source, typically a laser, is provided. A wavelength-selective beamsplitter directs the laser light toward the objective lens, while allowing fluorescence or reflected light emitted from the sample to pass through to the collection lens.
A photo detector is placed at the focal point of the collection lens in image space so as to convert the intensity of the light emitted from the sample at the focal point of the objective lens in object space to an electrical signal. Preferably, an aperture is placed at the image space focal point of the collection lens so as to provide confocal microscopy. Additional detectors may be used, combined with corresponding wavelength-sensitive beamsplitters to tap light from the collection lens, for measuring the intensity of various wavelengths of fluorescence light produced by excitation of the sample.
To achieve high resolution, the maximum numerical aperture of the objective lens may be used. A light-path-altering optical element is inserted between the light source and the objective lens so as to translate the light centered on the axis of rotation to light centered on the optical axis of the objective lens, thereby filling the entrance pupil of the objective lens despite rotation thereof.
Where the objective lens is fixed, a rotating light-path-altering optical element receives light from an off-objective-optical-axis point in object space of said objective lens and directs it along the objective optical axis, and vice versa.
Accordingly, it is a principal object of the present invention to provide a novel and improved scanning microscope and scanning microscopy method.
It is another object of the invention to provide a scanning microscope that achieves high resolution and sensitivity with relatively straightforward optics.
It is a further object of the invention to provide a scanning microscope that achieves high resolution and sensitivity with a relatively straightforward scanning mechanism.
It is yet another object of the invention to provide a relatively high speed scanning microscope.
The foregoing and other objects, features, and advantages of the invention will be more readily understood upon consideration of the following detailed description of the invention, taken in conjunction with the accompanying drawings.