This invention relates to fluorescent microscopy, and more particularly to the rapid selection of filters for measuring fluorescence at different wavelengths or Stokes shifts.
The phenomenon of fluorescence describes light emission that continues only during the absorption of the excitation light by a chromophore or other conjugated molecule, which is capable of emitting secondary fluorescence. Reflected light fluorescence microscopy is a favored technique in fluorescence microscopy. This mode of fluorescence microscopy is also known as incident light fluorescence, fluorescence, or scopic fluorescence. The universal reflected light vertical illuminator is interposed between the observation viewing tubes and the nosepiece carrying the objectives. A key feature of fluorescence microscopy is its ability to detect fluorescent objects that are sometimes faintly visible or even very bright relative to the dark (often black) background. In order to optimize this feature, image brightness and resolution must be maximized. Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with highly specific fluorophores.
While fluorescence is described, the invention can be used with any type of photoluminescence, provided that the material under observation exhibits a Stokes shift.
Fluorescence microscopy can be used for specimens, which exhibit autofluorescence, and for specimens enhanced by fluorochromes, also called fluorophores. The basic task of the fluorescence microscope is to permit excitation light to irradiate the specimen and then to separate the much weaker re-radiating fluorescent light from the brighter excitation light. Thus, only the emission light reaches the eye or other detector. The resulting fluorescing areas shine against a dark background with sufficient contrast to permit detection. The darker the background of the non-fluorescing material, the more efficient the instrument. Therefore efficient filters are desired.
When a fluorescing sample is observed with a fluorescence microscope. Ultraviolet (UV) light of a specific wavelength or set of wavelengths is produced by passing light from a UV-emitting source through the exciter filter. The filtered UV light illuminates the specimen, in this case a crystal of fluorspar, which emits fluorescent light when illuminated with ultraviolet light. Visible light emitted from the specimen, red in this case, is then filtered through a barrier filter that does not allow reflected UV light to pass. It should be noted that this is the only mode of microscopy in which the specimen, subsequent to excitation, gives off its own light. The emitted light re-radiates spherically in all directions, regardless of the direction of the exciting light.
For a given fluorochrome, the manufacturer indicates the wavelength for the peak of excitation/fluorescence intensity and the wavelength for the peak of emission/fluorescence intensity. To determine the emission spectrum of a given fluorochrome, the dye absorption maximum wavelength is found and the fluorochrome is excited at that maximum. The dye absorption maximum wavelength is usually the same as the excitation maximum. The absorption spectrum of a typical fluorochrome occurs where the relative intensity of absorption is plotted against relative wavelength. A monochromator is then used to scan the fluorescence emission intensity at successive emission wavelengths. The relative intensity of the fluorescence is measured at the various wavelengths to plot the emission spectrum. The excitation spectrum of a given fluorochrome is determined in a similar manner. The emission maximum is chosen and only emission light at that emission wavelength is allowed to pass to the detector. Then excitation is induced at various excitation wavelengths and the intensity of the emitted fluorescence is measured. There is usually an overlap at the higher wavelength end of the excitation spectrum and the lower wavelength end of the emission spectrum. This overlap of excitation and emission intensities/wavelengths must be eliminated in fluorescence microscopy. Elimination of the overlap of excitation and emission intensities/wavelengths is accomplished by means of appropriate selection of excitation filter, dichromatic beamsplitter, and excitation or barrier filter. Otherwise, the much brighter excitation light overwhelms the weaker emitted light and significantly diminishes specimen contrast.
The separation of excitation and emission wavelengths is achieved by the proper selection of filters to block or pass specific wavelengths. The design of fluorescence illuminators is based on control of excitation light and emission light by readily changeable filter insertions in the optical pathway on the way toward the specimen and then emanating from the specimen.
A dichroic material is one which absorbs light polarized in one direction more slowly than light polarized at right angles to that direction. Dichroic materials are to be distinguished from birefringent materials, which may have different refractive indexes for two electric vectors vibrating at right angles to each other but similar, if negligible, absorptions coefficients. The term dichroic is also used to denote the change in color of a dye solution with change in concentration, to denote a color filter that has two transmission bands in very different portions of the visible region and hence changes color when the spectral distribution of the illuminating source is changed, and to denote an interference filter that appears to be of a different color when viewed in reflected or transmitted light.
As used in this disclosure, xe2x80x9cdichroic beamspiitterxe2x80x9d refers to a mirror, which reflects most light below a predetermined wavelength and transmits light above that wavelength. Thus, in one sense the invention may use a dichroic filter, which is an interference filter that appears to be of a different color when viewed in reflected or transmitted light.
Dichroic filters are filters having or showing two colors. This can be obtained with doubly-refracting crystals that exhibit different colors when viewed in different directions or with solutions that show essentially different colors in different degrees of concentration.
In addition, while dichroic materials are described, the invention may be used with birefringent materials and other types of filters.
The functions of a dichroic element in a system used to analyze fluorescent labels in samples include directing light from a light source toward the sample under observation, and transmitting light reflected from the sample. By limiting the reflection to a predetermined color, it is possible to specifically detect reflected light within a predetermined waveband.
When using two or more fluorescent labels or fluorophores, it is necessary to provide dichroic beamsplitter mirrors for each of the fluorescent labels. It is further desired to detect these fluorescent labels separately. While the fluorescent labels are detected separately, there should not be a significant time difference between the detection of the different fluorescent labels. Optimally, it would be desirable to simultaneously detect the different fluorescent labels. If simultaneous detection is not practical, then the sequence of detecting the different fluorescent labels should be rapid enough that the end effect is similar to that of simultaneous detection.
In a particular type of fluorescence microscope, a dichroic beamsplitter mirror is used in combination with an excitation filter and an emission filter, sometimes referred to as a barrier filter. The excitation filter filters the excitation light to provide a monochromatic output, and the emission filter filters light transmitted through the dichroic mirror. The dichroic beamsplitter mirror and the emission filter are selected according to the Stokes shift of the fluorophores under observation.
Current fluorescent microscope designs employ an incident light or fluorescent design where a dichroic beamsplitter or chromatic mirror is mounted in a filter cube at a 45 degree angle to the excitation light path. The beamsplitter or chromatic mirror is used to reflect shorter excitation wavelengths of light onto the specimen while passing longer emission wavelengths to the eyepieces or camera. The beamsplitter has a cutoff wavelength or frequency which is between an excitation wavelength or frequency and an emission wavelength or frequency of the fluorescent material being sensed.
In addition to the dichroic beamsplitter, a monochromatic light source is provided at an excitation frequency, and a notch filter at an emission frequency is used at the sensing unit. The wavelengths of the monochromatic light source and the filter at the sensing unit generally correspond to the excitation wavelength and the emission wavelength of the fluorescent material being sensed. In the typical implementation the monochromatic light source is provided by using a notch filter to block emissions from a high intensity lamp at wavelengths above and below a selected excitation wavelength.
Many fluorescence microscopy applications require two or more fluorescent labels to be present in the specimen. Each label has its own excitation and emission spectra, and thus requires different excitation and emission filters, as well as a different dichroic beamsplitter.
To date two approaches to using multiple fluorophores have been employed:
1) Use dichroic beamsplitters that have multiple cut-off wavelengths. Thus a single dichroic can be used with multiple fluorophores. However because of bandwidth restrictions, total light throughput is reduced, thus creating longer exposure times when working with a camera. Longer exposure times translate into longer acquisition duty cycles, which is problematic in paradigms requiring repetitive high-speed data acquisition, such as screening applications and applications using living cells.
2) Motorized filter cube changers. Several commercially available microscopes employ motorized filter cube changers. These allow the use of a single dichroic for each fluorophore. However switching time is slow (12 seconds) which creates problems when using multiple fluorophores in paradigms requiring repetitive high-speed data acquisition. The slow switching time is the result of the significant mass of the motorized filter cubes. Increased speed of switching would require a substantial increase in power and control of reaction forces caused by the movement of the cubes during switching.
Dichroic beamsplitter mirrors are available with two or more cutoff wavelengths. This results in an ability to use such a beamsplitter with a first set of excitation and emission filters corresponding to a first cutoff wavelength to sense a first fluorophore, and to use the same beamsplitter mirror with a second set of excitation and emission filters with a second combined cutoff wavelength to sense a second fluorophore. This becomes difficult to accomplish if the beamsplitter mirror is provided in a filter cube.
According to the present invention, an optical system for a microscope, which includes an illumination source, microscope optics for obtaining a magnified image, and a beamsplitter changer is able to rapidly effect changes in Stokes shift wavelengths being detected. A plurality of dichroic beamsplitter elements (xe2x80x9cdichroic beamsplittersxe2x80x9d) are arranged about a center axis at a preferred angle such as 45xc2x0 to the lightpath of the microscope optics, or optical pathway. The beamsplitter elements are rotable about the center axis to allow positioning a selected one of the dichroic beamsplitter elements in a part of the light path between the illumination source and the microscope optics, with the light path resulting in reflection of light from the illumination source to a specimen under observation, and transmitting light received through the microscope optics from the specimen. When the selected dichroic element corresponds to a fluorescent material under observation, the dichroic beamsplitter reflects excitation light at an excitation wavelength corresponding to the fluorescent material under observation and the dichroic element transmits light shifted in wavelength by a Stokes shift of the fluorescent material under observation. Rapid positioning of the dichroic filter is achieved by a motor and motor controller, which rotates the filter about the center axis.