The present invention relates to the monitoring of cellular activity through the use of excitable markers. More particularly, the invention relates to a system and method for using a plurality of fluorescent probes to monitor cellular activity.
Presently, fluorescence microscopy is one of the most widely used microscopy techniques, as it enables the molecular composition of the structures being observed to be identified through the use of fluorescently-labeled probes of high chemical specificity, such as antibodies. However, its use is mainly confined to studies of fixed specimens because of the difficulties of introducing antibody complexes into living specimens. For proteins that can be extracted and purified in reasonable abundance, these difficulties can be circumvented by directly conjugating a fluorophore to a protein and introducing this back into a cell. It is believed that the fluorescent analogue behaves like the native protein and can therefore serve to reveal the distribution and behavior of this protein in the cell.
An exciting, new development in the use of fluorescent probes for biological studies has been the development of the use of naturally fluorescent proteins as fluorescent probes, such as green fluorescent protein (GFP). The gene for this protein has been cloned and can be transfected into other organisms, This can provide a very powerful tool for localizing regions in which a particular gene is expressed in an organism, or in identifying the location of a particular protein. The beauty of the GFP technique is that living, unstained samples can be observed. There are presently several variants of GFP which provide spectrally distinct emission colors.
Conventionally, fluorescence microscopy only worked well with very thin specimens or when a thick specimen was cut into sections, because structures above and below the plane of focus gave rise to interference in the form of out-of-focus flare. However, this can be overcome by optical sectioning techniques, such as multi-photon fluorescence microscopy.
Multi-photon fluorescence microscopy involves the illumination of a sample with a wavelength around twice the wavelength of the absorption peak of the fluorophore being used. For example, in the case of fluorescein which has an absorption peak around 500 nm, 1000 nm excitation could be used. Essentially no excitation of the fluorophore will occur at this wavelength. However, if a high peak-power, pulsed laser is used (so that the mean power levels are moderate and do not damage the specimen), two-photon events will occur at the point of focus. At this point the photon density is sufficiently high that two photons can be absorbed by the fluorophore essentially simultaneously. This is equivalent to a single photon with an energy equal to the sum of the two that are absorbed. In this way, fluorophore excitation will only occur at the point of focus (where it is needed) thereby eliminating excitation of the out-of-focus fluorophore and achieving optical sectioning.
Often, multiple fluorophores are used, with each fluorophore having a different spectra, some of which may overlap. Typically, the ability to distinguish between the respective fluorophores is only possible where the excitation spectra are separated, or where the fluorescence lifetimes are distinct.
Another approach is to selectively excite different fluorophores by using various excitation photon wavelengths, each of which will approximate the wavelength of the absorption peak of a corresponding fluorophore. Such an approach is not practical for a number of reasons. Firstly, it is difficult to rapidly tune the excitation wavelength of the laser providing the excitation photons. Secondly, there is typically a very broad excitation spectrum, so that such an approach requires a significant amount of time.
Thus, the need exists for a system and method for efficiently monitoring a plurality of fluorescent probes, and to record emissions spectra relating to those probes for subsequent analysis. The present invention addresses these needs.
Briefly, the present invention is directed to a system and method for monitoring cellular activity in a cellular specimen. According to one illustrative embodiment of the invention, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into wavelength bands. The respective fluorescence bands are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.
According to another embodiment, the invention is directed to a system for monitoring cellular activity in a cellular specimen that contains a plurality of excitable markers. The system includes a laser microscope that is operative to excite the markers in a region of the specimen, so that those markers in the region radiate fluorescence. The system also includes a tunable filter that is operative to process the fluorescence and to pass a portion of the fluorescence wavelengths radiated by the markers. The system still further includes a detector that is operative to receive the processed fluorescence wavelengths.
In still another embodiment, the invention is directed to a system for monitoring cellular activity, including a two-photon laser microscope that is operative to excite the markers in a region of the specimen such that the markers in the region radiate fluorescence. The system also includes a detector that is operative to receive non-descanned fluorescence from the specimen.
These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered conjunction with the accompanying drawings wherein:
a-c depict emissions spectra for various fluorescent dyes; and
d depicts a measured spectrum from a specimen that contains plural different dyes.
Referring to
As shown in
The reflected light from dichroic mirror 18 is directed toward scanning optics, shown schematically at 20. The scanning optics may include a controllable deflection unit, or any other suitable structure that allows the reflected light to be redirected for scanning of the specimen 12 by the laser light, as is well known to those skilled in the art.
The directed light from scanning optics 20 is imaged by a microscope lens 22 onto or into the specimen 12. The laser light excites the fluorescent dyes in the region where the light is directed, such that those dyes fluoresce and emit light having respective emissions spectra.
In one embodiment, laser 14 is a two-photon laser. As is well known, a two-photon laser microscope depends on the two-photon effect, by which the fluorescent dyes are excited not by a single photon, but rather by two relatively low energy photons that are absorbed contemporaneously by a fluorescent dye. Thus, the requirement for two coincident (or near coincident) photons to achieve excitation of the fluorescent dye means that only focused light reaches the required intensities and that scattered light does not cause excitation of the fluorescent dyes. As such, a two-photon laser microscope is inherently insensitive to the effects of light scattering in thick slices. Alternatively, the laser 14 may comprise some other multi-photon laser, or a single-photon laser.
The fluorescence radiated by the excited dyes is focused by the microscope lens 22, and passes through the scanning optics 20 and dichroic mirror 18. The fluorescence is then focused by a lens 24 and directed to a light guide 26 that delivers the focused light to a spectral analyzer 28. Spectral analyzer 28 receives the light and disperses the light into respective wavelength bands. Each band is detected by a corresponding detector, and the intensity of each band is recorded and processed to determine the ingredients of the received fluorescence, as is described in more detail below in connection with
Referring to
Aligned with exit opening 34 is a detector array 42 consisting of individual detectors 43. The detector array 42 may take many different forms, such as an array of photomultiplier tubes (PMTs), multiple window PMTs, position-dependant wire detectors, position/time-sensitive detectors, a photodiode array, an intensified photodiode array, charge-coupled devices (CCDs), intensified CCDs, an SIT or other video camera, or any other suitable optical-to-electrical transducer.
In any event, the detector array 42 is preferably a linear array, with the respective wavelength bands being incident upon a corresponding one of the detectors 43. Each detector 43 in the array receives the light incident upon it and generates a corresponding analog electrical signal. The electrical signals are then introduced to respective analog-to-digital converters (shown schematically at 44) which convert the incoming analog signals into corresponding digital signals. The digital signals are then delivered to a processor 46 that processes the digital signals to determine the constituents of the emissions spectra, as is described in more detail below.
Referring now to
Referring now to
System 60 is therefore suitable for use in connection with a non-descanned two-photon microscope. By diverting the radiated fluorescence before it passes through the scanning optics 20, a signal of increased intensity is received by spectral analyzer 28, as compared with a signal that passes through scanning optics 20 and dichroic mirror 18 before being received by a spectral analyzer or detector. Thus, the dwell time at each pixel can be reduced as a result.
Referring now to
Thus, in use of the various systems described above, laser light is directed by dichroic mirror 18, scanning optics 20, and focusing lens 22 to a region of specimen 12. The photons (either from a single-photon laser or from a multi-photon laser) excite the fluorescent dyes in the region, causing them to fluoresce. The entire emitted spectrum is received and processed simultaneously in certain of the illustrative embodiments to speed up the collection process. The spectral information is then processed to determine the amounts of each dye contributing to the emitted spectrum.
Referring now to
At step 104, processor 46 receives the measured imaging spectrum data from ADC 44 (see
In another embodiment, processor 46 is programmed to carry out principal component analysis (PCA) on the incoming data. As is well known in the art, PCA is a linear model which transforms the original variables of an emission spectrum into a set of linear combinations of the original variables called principal components, that account for the variance in the original data set. Suitable forms of PCA algorithms are disclosed in U.S. Pat. Nos. 5,991,653 to Richards-Kortum et al., and 5,887,074 to Lai et al., the disclosures of which are hereby expressly incorporated by reference.
From the foregoing, it will be apparent to those skilled in the art that the present invention provides an efficient and reliable system for receiving and processing emissions spectra in connection with fluorescence microscopy. The system processes the emissions spectra to determine concentrations of plural fluorescent dyes in a particular spectrum, even where the wavelength bands of the dyes overlap.
While the above description contains many specific features of the invention, these should not be construed as limitations on the scope of the invention, but rather as one exemplary embodiment thereof. Many other variations are possible. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal equivalents.
The present application is a continuation of U.S. application Ser. No. 10/817,297, filed Apr. 2, 2004, which is a continuation of U.S. application Ser. No. 10/159,703, filed May 28, 2002, which is a continuation of U.S. application Ser. No. 09/628,219, filed Jul. 28, 2000, which issued as U.S. Pat. No. 6,403,332, on Jun. 11, 2002, which claims priority to U.S. Provisional Application Nos. 60/146,490, filed Jul. 30, 1999 and 60/164,504, filed Nov. 9, 1999, the disclosures of which are incorporated herein by reference.
Number | Date | Country | |
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60146490 | Jul 1999 | US | |
60164504 | Nov 1999 | US |
Number | Date | Country | |
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Parent | 10817297 | Apr 2004 | US |
Child | 12335380 | US | |
Parent | 10159703 | May 2002 | US |
Child | 10817297 | US | |
Parent | 09628219 | Jul 2000 | US |
Child | 10159703 | US |