Radiant energy – Photocells; circuits and apparatus – Optical or pre-photocell system
Reexamination Certificate
1999-06-23
2001-07-03
Lee, John R. (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Optical or pre-photocell system
C356S370000, C359S368000
Reexamination Certificate
active
06255642
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to methods and systems for obtaining high-resolution images of microscopic samples including, for example, biological specimens and novel materials.
Obtaining images of microscopic phenomena has long been a crucial aspect of research in biomedical and material science. To improve such research, there has been ongoing study to improve the resolution of imaging techniques along both axial and lateral directions. Existing techniques include far-field techniques such as conventional optical microscopy, confocal microscopy, and two-photon fluorescence microscopy, and near-field techniques such as near-field scanning optical microscopy (NSOM), scanning tunneling microscopy (STM), and atomic force microscopy (AFM).
SUMMARY OF THE INVENTION
The invention features an optical microscopy method and system that can obtain high-resolution images of a sample along both axial and lateral directions with relatively rapid acquisition times. The method and system involve coupling an evanescent standing wave into the sample and recording an image of optical radiation emitted from the sample in response to the evanescent standing wave for each of multiple positions of the standing wave. The evanescent standing wave can be produced by totally internally reflecting two counter-propagating beams from an interface of an optically dense substrate adjacent the sample. Selecting portions of each of the recorded images corresponding to the peaks of the evanescent standing wave and combining the selected portions can produce a high-resolution image. The lateral resolution is comparable to the half-width of the peaks in the evanescent standing wave, which is inversely related to the refractive index of the optically dense substrate. We refer to the technique of the invention as Standing Wave Total Internal Reflection (SWTIR) Imaging.
In general, in one aspect, the invention features a microscopy system for imaging a sample. The system includes a substantially transparent optical block having an interface for positioning the sample adjacent the interface; a light source which during operation overlaps at least two optical beams at the interface; and a detector. During operation, the light source directs the beams into the block towards the interface at incident angles that cause the beams to reflect from the interface and establish an evanescent standing wave of electromagnetic energy that extends away from the interface and into the sample. During operation, the detector records an image of the sample based on optical radiation emitted from the sample in response to the evanescent standing wave.
The microscopy system can include any of the following features. The evanescent standing wave can have a period less than the wavelength of the optical beams. The at least two beams can include four beams and the evanescent standing wave can extend along both lateral dimensions. The light source can include a laser and a mirror, wherein the laser directs a first of the at least two beam to the interface, the interface reflects the first beam to define a reflected beam, and the mirror retroreflects the reflected beam back to the interface to define a second of the at least two beams. The optical block can be homogeneous or it can include multiple layers and/or multiple coatings. Suitable materials for the optical block can include fused quartz, gallium phosphide, tellerium oxide, and flint glass. The detector can include a microscope objective, relay optics, and a CCD camera. The microscope objective can be positioned on the same side of the optical block as the interface, or on the opposite side.
Furthermore, the microscopy system can include a controller which during operation causes the optical source to vary the incident angles of the optical beams and thereby vary the depth to which the evanescent standing wave extends into the sample.
The microscopy system can also include a controller which during operation causes the light source to translate the position of the evanescent standing wave established by the reflected beams, causes the detector to record an image of the sample for each of multiple positions of the standing wave, and determines a high-resolution image of the sample based on the multiple images recorded by the detector. During operation the controller can further cause the optical source to vary the incident angles of the optical beams, determine a high-resolution image of the sample for each of the incident angles, and construct an axially resolved high resolution image of the sample based on the determined high-resolution images.
In general, in another aspect, the invention features a method for determining a high-resolution image of a sample including: coupling an evanescent standing wave of electromagnetic energy into the sample; recording an image of the sample based on radiation emitted from the sample in response to the evanescent standing wave for each of multiple positions of the standing wave; and constructing the high-resolution image based on the multiple recorded images.
The method can include any of the following features. The constructing step can include: selecting from each recorded image portions of the recorded image; and combining the selected portions to construct the high-resolution image. The selected portions for each recorded image can be intensity values corresponding to the peaks of the standing wave. The evanescent standing wave can extend along one or two dimensions. The radiation emitted from the sample can be fluorescence or scattered radiation.
Furthermore, the coupling step can include: positioning the sample adjacent an interface of an optical block; and totally internally reflecting at least two counter-propagating beams from the interface. The coupling step can also include coupling a first one-dimensional evanescent standing wave along a first axis in the sample plane and separately coupling a second one-dimensional evanescent standing wave along a second axis in the sample plane, in which case the recording step includes recording images for each of multiple positions of each standing wave.
In another aspect, the invention features a method for determining an axially resolved high-resolution image of a sample. The method includes determining a lateral high-resolution image of the sample using the first-mentioned method for each of multiple penetration depths of the evanescent standing wave; and constructing the axially-resolved high resolution image from differences between the determined lateral high-resolution images.
Embodiments of the invention have many advantages. For example, the resolution along the lateral direction can be better than &lgr;/13, where &lgr; is the wavelength of the excitation light producing the evanescent standing wave. Such lateral resolution can be further improved by using nonlinear imaging modalities such as two photon excitation and pump-probe. The imaging light is confined along the axial direction to within the skin depth of the evanescent wave, e.g., on the order of 50 to 100 nm, thereby eliminating out of focus light that would wash out the in-plane resolution.
Furthermore, images can be constructed that section the sample along the axial direction by processing multiple images in which the evanescent standing wave penetrates into the sample to different depths. As a result, high-resolution images can be constructed along three dimensions.
The lateral image acquisition rates can also be very fast, comparable to, or faster than, video rates. Moreover, the optical technique is non-invasive, requiring no mechanical contact between the sample and a probe. Thus, the technique can be easily applied to soft biological samples.
Other features and advantages will be apparent from the following detailed description and from the claims.
REFERENCES:
patent: 4297032 (1981-10-01), Temple
patent: 4584484 (1986-04-01), Hutchin
patent: 4621911 (1986-11-01), Lanni et al.
patent: 5394268 (1995-02-01), Lanni et al.
patent: 5538850 (1996-07-01), King et al.
patent: 5633724 (1997-05-01), King e
Cragg George
Dong Chen-Yuan
Kwon Hyuk-sang
So Peter T. C.
Fish & Richardson P.C.
Lee John R.
Massachusetts Institute of Technology
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