Optical system and method for inspecting fluorescently labeled biological specimens

This invention is generally in the field of optical measurement/inspection techniques and relates to an optical system and method for inspecting fluorescently labeled biological specimens.

One of the new emerging techniques used today in the research of molecular biology and genetics is fluorescent labeling of a biological specimen. According to this technique, fluorescent probes are used to mark the specific locations in a biological specimen aimed at detecting different genes, chromosomes, DNA strands, proteins, and bacteria.

In recent years, the fluorescent labeling based techniques have started to push their way into the diagnostic world, and it is anticipated that in the near future diagnostic assays based on fluorescent labeling will be used more and more routinely.

According to conventional techniques, the detection of fluorescent probes is done in research laboratories by using an “off the shelf” fluorescent microscope. The use of a fluorescent microscope was a logical choice, since this machine was readily available in most research labs. Furthermore, it was a familiar tool to all researchers, and had the benefit of being a multi purpose platform used for other lab applications as well.

However, the detection of fluorescent probes in a biological sample by means of the conventional fluorescent microscope suffers from several drawbacks associated with the following. Today, in diagnostic laboratories that use fluorescent techniques, an operator with genetic training typically manually operates a fluorescent microscope. The operator must manually select the correct objective and filters, manually scan the slide and search for good genetic material, focus on each image, analyze the fluorescent signals, and write down his analysis. The operator has to look through a binocular eyepiece during the entire process, which is a cumbersome and tiring process. Thus, an operator cannot work on the microscope for more than a few hours continuously, and not more than 8-10 hours daily. This of course limits the number of tests a lab can perform, thus limiting the lab\'s throughput significantly.

Furthermore, the laboratory, where this analysis is done, has to be in blackout conditions. This is associated with one of the major problems of using fluorescent labeling for routine diagnostic assays, consisting of keeping the fluorescent labeling “alive” long enough to finish the entire procedure, which typically includes scanning the sample on a slide, looking for region of interests (ROIs) in the sample (for example, a nucleus of the cell or a chromosome), focusing on the ROIs, taking an image thereof, refocusing on sub areas within the ROI (for example labeled genes), and taking images of the these sub areas as well. This procedure takes quite a while, since a large number of ROIs must be considered to achieve the high reliability required from an assay used for diagnostic purposes. For example, in prenatal FISH tests (fluorescence in situ hybridization) at least 100 good regions of interest (nuclei) are needed to be imaged for giving a reliable diagnosis from the test (“Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH)”, Prenat Diagn 2001; 21: 293-301. DOI: 10.1002/p. 57). FISH method is typically used to detect the absence or excess of a specific gene (e.g., elastin gene) from a chromosome, e.g., to detect the presence of down syndrome.

To detect 100 good enough regions of interest, one must scan several hundreds of fields on the sample. Working for so long on the sample raises the problem of bleaching. Bleaching of a sample causes the fluorescent probes to fade, thus making the reading of the sample impossible. This phenomenon, which occurs within minutes, is stimulated by light and oxygen. Operation with the conventional fluorescent microscope thus requires operation in the dark, and implies that other activities requiring light cannot be carried out at the same time and place, when fluorescent analysis is in process. As a result, all laboratory work has to be halted when fluorescent signals are analyzed, or a separate room has to be assigned for the fluorescent microscope. Furthermore, the necessity to work in a dark environment, affects the performance of the microscope operator. Working in the dark, is no doubt, a cumbersome task.

Other environmental hazards of the conventional techniques, such as heat, humidity, radiation, electromagnetic waves, also have undesired influence on some biological samples. With the conventional microscope and conventional technique, operating personnel are exposed to safety hazards due to UV light typically used to excite the fluorescent sample, but is harmful to people.

The use of a “semi-automatic” fluorescent microscope set-up has been proposed (BX51 Epi-Fluorescence Microscope commercially available from Olympus). In this set-up, a digital camera and a computer are added to the fluorescent microscope solely for archiving the images so as to enable reviewing the images at a later time. An “automatic” fluorescent microscope set-up (DM RXA2 Fluorescence Microscope commercially available from Leica) allows for integrating the “off-the-shelf” components such as a microscope, digital camera, scanning stage, and computer, in conjunction with a software package that controls the operation of these components. However, using the “off-the-shelf” components that were not designed specifically for fluorescent diagnostic tasks (to comply with the demands of the fluorescent-based diagnostic world) obviously decreases performance and increases costs of the optical system.

The use of fluorescent probes routinely for diagnostic purposes creates new standards and demands for a fluorescent detection system. Such a system must provide a complete and full solution for a fluorescent-based diagnosis. The system must be characterized by a high throughput capability, high levels of automation, a simple graphic user interface (GUI) that enables minimal user operating mistakes and thereby allows for layman operation of the system, a high level of reliability and accuracy, and last but not least, it must be economically beneficial.

The main idea of the present invention consists of solving the above problems by the encapsulation of a stage intended for supporting a fluorescently labeled sample under inspection, and an optical inspection (imaging) system. Such a capsule is made of a material preventing the penetration of light into the capsule from the outside thereof (i.e., non-transparent material), and preferably also preventing the penetration of electromagnetic waves (i.e., electrically conductive material). The capsule preferably also includes one or more environment control sensors, and is equipped with means enabling the adjustment of the corresponding environment conditions inside the capsule so as to meet the requirements of an optical inspection of fluorescently labeled samples.

The technique of the present invention thus provides a unique platform that complies with the new demands arising from turning the latest research techniques in fluorescence into tomorrow\'s diagnostic tools. The system and method of the present invention makes up a Fluorescent Working Station (F-WOS) constructed and operated to provide optimal conditions for acquiring high-end fluorescent images (i.e., the combination of low light sensing, small size and high magnification, together with the enhanced image processing and control) from biological samples for research and diagnostic tasks, and to provide optimal conditions for the sample, fluorescent signals, operator, and all other personal working in the lab. By designing a complete system especially for fluorescent diagnostics, the high performance, high throughput, and low price system is obtained. It should be noted that fluorescent signals, for diagnostic tasks, are characterized by a low light (about 0.001 lux), capability of detecting small size fluorescent labels (0.1-0.4 microns), fast fluorescent intensity decay with time (bleaching), and environmental sensitivity. This means that in fluorescent imaging, working on the “edge” of technology is required, and it is thus crucial to address the fluorescent signal quality issues, as well as the imaging quality issues, for obtaining the best results.

The F-WOS of the present invention is the first fluorescent-based system that makes a significant effort in improving the quality of the fluorescent signals, and not just improving the quality of the acquired images. The F-WOS enables to easily load a fluorescent slide into the capsule, automatic scanning of the slide in the X,Y,Z planes at the appropriate resolution and speed, searching for the designated regions of interest on the fly, and optimal acquisition of the required images for research and diagnosis needs.

According to one aspect of the present invention, there is provided a system for imaging a fluorescently labeled sample, the system comprising a capsule, which is a closable structure made of a material isolating the inside of the capsule from its surrounding environment, and which has a support stage for receiving the sample and carrying it thereinside during the imaging; and an optical device at least partly accommodated inside the capsule and operable to illuminate the sample with incident radiation to excite a fluorescent response of the sample, detect the fluorescent response, and generate data indicative thereof.

Preferably, the capsule comprises one or more sensors for sensing the environment condition(s) inside the capsule to be controlled, and inlet and outlet means enabling to desirably affect the corresponding condition(s) inside the capsule. The capsule thus presents a “controlled environmental capsule” (CEC). The sensors suitable to be used in the capsule include at least one of the following: a temperature sensor, an ambient light sensor, an electromagnetic radiation sensor, oxygen or other gases\' sensor, and a humidity sensor. The CEC is designed to protect the biological media and the fluorescent probes therein from environmental hazards, and to provide them with optimal conditions during the imaging. For example, the capsule protects the sample from unwanted light in the room, high temperature, and the presence of oxygen, causing the sample to fade quickly (the Bleaching phenomena). Providing these optimal conditions for the fluorescent sample improves the quality of the fluorescent signals. The CEC also provides protection for the operating personal from hazardous conditions of the system such as the use of UV light. Furthermore, the encapsulation of the sample with the optical device enables installation of the working station in any room or laboratory, without the necessity to darken the room when working with the fluorescent signals, thus stopping all other activities in the laboratory at that time.

The optical device provides: means for selecting and guiding the excitation light to the sample, means for collecting and selecting the desired emitted (excited) light from the sample, means for forming the fluorescent image at a selected focal plane, and preferably also means for enlarging the images. All components are designed to obtain the best fluorescent images possible.

The optical device thus includes a light source system, an image formation system, and light directing/collecting optics. The light source uses one or more light sources of the kind generating excitation incident radiation to excite a fluorescent response of the sample. The optics used may include a light guiding means (filters) for selecting and guiding the desired excitation light to the sample, a beam shaping optics in the optical path of the exciting light, a light collecting optics for collecting light coming from the sample and selecting therefrom the desired fluorescent light, and an imaging optics to form the fluorescent image of the sample. The detection unit may include one or more detectors (e.g., with different specifications). For example, the detection unit may include a single imaging detector, but preferably includes at least two such detectors with different attributes. For example, one detector is a self-designed CMOS camera aimed at identifying pre-defined ROI(s) on-the-fly (in real time) and processing the images on the camera chip itself, thereby saving the need to send the images to an external computer, and the other detector is a cooled CCD camera aimed at providing high quality images for analysis and acquisition.

The device further includes a scanning system, which enables scanning the sample at different resolution and speeds at the X, Y and Z directions (3D scanning). The scanning system supports the ROI search and identification, preferably utilizing also auto-focusing abilities, and provides the F-WOS with the ability to automatically detect a large amount of fluorescent signals in a short period of time.

The system of the present invention utilizes a control unit (Computerized Central Control) that automatically controls and synchronizes the operation of the entire workstation). The control unit receives data from data indicative of the detected fluorescent response from the detector(s) to generate data indicative of an image of the sample, and preferably also receives data indicative of the environment condition(s) inside the capsule to analyze this data and operate the inlet and output channels of the system accordingly. The control unit is typically a computer device connectable to the capsule through wires or wireless communication, and includes inter alia a database utility for storing a specifically designed database, a data processing and analyzing utility preprogrammed with specially designed algorithms, and a display unit. The control unit preferably comprises appropriate communication means enabling “downloading” of the acquired images and all relevant data to the database. The algorithm packages especially designed for use in the F-WOS are responsible inter alia for the following: finding predefined regions of interest (ROI), focusing on these regions, analyzing the images in the ROI, selecting sub areas in the ROI for further acquiring and analysis, calculating the optimal parameters (i.e. roundness, overlapping, size and some others) for acquiring the different images, and giving a diagnosis evaluation based on the acquired images and predefined statistics. The same or an additional control unit is operable to automatically control and synchronize the operation of the entire workstation. The database utility enables the following operations: saving the acquired images, saving all other relevant information, conducting different search algorithms on the database, and options for adding new fields of data to each item in the database.

The fluorescent sample may be of the kind prepared in the FISH method, and may be used for obtaining diagnostic results, e.g., by using Aneuploidy methods.