Tissue lithography will enable physicians and researchers to obtain macromolecules with high purity (>90 percent) from desired cells in conventionally processed, clinical tissues by simply annotating the desired cells on a computer screen. After identifying the desired cells, a suitable lithography mask will be generated to protect the contents of the desired cells while allowing destruction of all undesired cells by irradiation with ultraviolet light. The DNA from the protected cells can be used in a number of downstream applications including DNA sequencing. The purity (i.e., macromolecules isolated form specific cell types) of such specimens will greatly enhance the value and information of downstream applications.
In this method, the specific cells are isolated on a microscope slide using photolithography, which will be faster, more specific, and less expensive than current methods. It relies on the fact that many biological molecules such as DNA are photosensitive and can be destroyed by ultraviolet irradiation. Therefore, it is possible to “protect” the contents of desired cells, yet destroy undesired cells. This approach leverages the technologies of the microelectronics industry, which can make features smaller than 1 μm with photolithography.
A variety of ways has been created to achieve identification of the desired cell, and also to designate the other cells for destruction. This can be accomplished through chrome masks, direct laser writing, and also active masking using dynamic arrays. Image recognition is envisioned as one method for identifying cell nuclei and cell membranes. The pathologist can identify the cells of interest using a microscopic computerized image of the slide, and appropriate custom software.
In one of the approaches described in this work, the software converts the selection into a digital mask that can be fed into a direct laser writer, e.g. the Heidelberg DWL66. Such a machine uses a metalized glass plate (with chrome metallization) on which there is a thin layer of photoresist. The laser transfers the digital mask onto the photoresist by direct writing, with typical best resolution of 2 μm. The plate is then developed to remove the exposed photoresist, which leaves the exposed areas susceptible to chemical chrome etch. The etch removes the unprotected chrome. The rest of the photoresist is then removed, by either ultraviolet organic solvent or overdevelopment. The remaining chrome pattern is quickly oxidized by atmospheric exposure (typically within 30 seconds).
The ready chrome mask is now applied to the tissue slide and aligned manually, or using automatic software and pre-designed alignment marks. The slide plate sandwich is then exposed to UV to destroy the DNA of the unwanted cells. The slide and plate are separated and the slide is processed in a standard way to prepare for polymerase chain reaction (PCR) and potential identification of cancer sequences.
This work was done by Lawrence A. Wade of Caltech and Emil Kartalov, Darryl Shibata, and Clive Taylor of the University of Southern California for NASA’s Jet Propulsion Laboratory.
In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to: Innovative Technology Assets Management JPL Mail Stop 202-233 4800 Oak Grove Drive Pasadena, CA 91109-8099 E-mail:
This Brief includes a Technical Support Package (TSP).
Tissue Photolithography
(reference NPO-47507) is currently available for download from the TSP library.
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Overview
The document is a Technical Support Package from NASA, detailing advancements in tissue photolithography, particularly for isolating single cells from tissue slices for genetic analysis. The primary focus is on methods that enhance the precision and efficiency of identifying potentially cancerous cells.
One of the key techniques discussed is the use of direct laser writing, which allows for the direct application of UV lasers to tissue slides. This method eliminates the need for physical masks, enabling precise targeting of unwanted cells while preserving the integrity of desired cells. The resolution achievable with this technique is approximately 2 microns or better, which is critical for effective cell selection.
Another innovative approach involves the use of fiber optics to create an active array of illuminators. By coupling a bundle of fiber optic cables to an LCD array, the output illumination area can be reduced, facilitating both light transduction and size reduction. This method leverages the properties of fiber optics to enhance illumination efficiency, which is essential for applications requiring high precision.
The document also explores the potential of micro- and nanolasers for generating dynamic arrays for selective UV exposure. These individually addressable lasers can be controlled via software, allowing for targeted activation without damaging the DNA of the cells. This approach is particularly advantageous as it enables the use of larger-wavelength light to pump UV nanolasers, thus minimizing potential damage to the genetic material.
Additionally, the document discusses alternative illumination methods, such as two-photon and multi-photon excitation. These techniques utilize larger wavelength light to achieve effective excitation at a fraction of the original wavelength, allowing for tighter focusing and improved resolution during exposure.
The overall goal of these advancements is to improve the accuracy and reliability of genetic analysis in tissue samples, particularly in the context of cancer research. By employing these innovative techniques, researchers can enhance the specificity of cell selection, ultimately leading to better diagnostic and therapeutic outcomes.
In summary, the document outlines cutting-edge methodologies in tissue photolithography that promise to revolutionize the way researchers isolate and analyze cells, particularly in the field of oncology, by combining precision laser techniques with advanced optical technologies.
