JP2003507766A - System and method for acquiring an image at a maximum acquisition speed while sequentially operating a microscope device asynchronously - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention relates to an automated (FIG. 5) microscope system (FIG. 1, item 100), a computer program product (FIG. 1, item 200 and FIG. 1, item 217) of a computer readable medium (FIG. 1, item 217). 245) and varying the various parameters of the acquired image at substantially the maximum speed of the camera (FIG. 1, item 130) as a sequence showing a continuous change in the acquisition parameters. Are displayed (FIG. 1, items 231 and 241) so as to acquire an image asynchronously with the camera while the external device of the camera (FIG. 1, item 130) is operating.

Description

DETAILED DESCRIPTION CROSS REFERENCE TO RELATED APPLICATION This application is filed on Aug. 13, 1999, US Provisional Application No. 60/148
, 819 claims priority. TECHNICAL FIELD The present invention relates to the field of image acquisition using an automated optical microscope system including a camera, in particular as a sequence for changing various parameters of the acquired image and showing continuous changes in the acquisition parameters. Image acquisition method and system as an apparatus external to the camera that operates asynchronously with the camera that displays various images. BACKGROUND ART To study living cells requires the use of an automated microscopy system controlled by application software. Taking a fluorescence microscope of cells and tissues as an example, take an image of cells stained with a fluorescent agent under the microscope.
Various methods have been conventionally known for extracting information on spatial and temporal changes occurring in these cells. Taylor et al. AMERICAN SCIENTIST 80 (1992
), P. 322-335, describes many of these methods and their applications.
These methods provide for the preparation of fluorescent reporter molecules to obtain, after processing and analysis, spatial and temporal resolution image measurements of the distribution, abundance, and biochemical environment of these molecules in living cells. Specially designed for.

Regarding automatic microscope systems, it has traditionally been the case that an automatic microscope system configures any of various types of cameras and an array of hardware devices in various device configurations according to a special research problem at hand. Well known. A standard textbook that is especially useful for displaying automated optical microscope hardware hardware systems and system construction is VIDEO MICROSCOPY, 2d Ed., By Inoue and Spring.
1997, especially Chapter 5 is hereby incorporated by reference. More generally,
John Ru is the description of automatic image acquisition and 3D image visualization.
ss is THE IMEGE PROCESSING HANDBOOK, 3d Ed., 1999, pp: 1-86,617-688,
See here.

Also, as is well known, an application software package controls and specifies a sequence, method and function of image acquisition and processing in which a device system operates, thereby supplementing a special device configuration and overlapping. It is a match. The acquisition and processing operations by which the software package is invoked again depend on the particular research task in hand. Sluder &Wolf's Salmon METHODS IN CELL B
IOLOGY, VOL. 56, ed. 1998, pp: 185-215 A High-Resolusion Multimode Digit
The al Microscope System chapter describes the application software for automating the microscope and controlling the camera, as well as the microscope, camera, Z
The design of the hardware system, including the axis focus device, is discussed.

Existing application software that automates microscope systems is capable of directing and controlling a host of operations, including: Recommended Standards (“RS”)-170 Video Equipment, CH
Image capture from NTSC and PAL video sources, exposure time, gain, analog-to-digital conversion time, bit per pixel setting for camera settings at each emission and / or excitation wavelength, analog- Driving the digitization of the acquired image from the digital converter, storing the acquired image in various formats such as TIFF, BMP and other standard file formats, driving the light illumination of the microscope, recording and one-time Provides a function to generate macros from user-specified sequences of programs that can be played back by clicking, arrange images in a stack, perform 3D construction, store the stack on disk, and improve the image quality. , Clear the image, perform the calculation, The imaged, the rate to the change in intensity in the graph, and the like for analyzing the image parameters such as the percentage of the concentration in the time direction of the ions, the specific processing for a group of related images called stack performed.

An example of widely used conventional application software for automating microscope systems is Universal Imaging Corporation, West Chester, PA.
Is a group of related application programs with different functions obtained from
There is Meta Imaging Series (TM). For example, general-purpose multipurpose image acquisition and processing
Users who want applications, MetaMorph (TM) application program
Would need to perform ratiometric analysis of intercellular ion measurements
Users will use MetaFluor (TM).

Despite the list of operations that the above-mentioned conventional application software allows an automated microscope system to do, the conventional application software is able to obtain a group of images and at the same time focus position and radiation and / or Alternatively, drive an automated microscope system to obtain acquisition parameters such as the excitation wavelength, and to vary the parameters so that playback is performed on the acquired group of images as a sequence showing a continuous variation of these parameters. I haven't done it. That is, conventional application software cannot operate an external device that controls image acquisition parameters asynchronously with the camera to obtain a group of images to display as a sequence of continuously changing system parameters.

[0006] Acquiring groups of images asynchronously as biological phenomena occur so that the images can be played back as a sequence displaying continuous changes in certain parameters of the microscope system is very important for the study of living cells. Is important to.
The importance of the present invention for cell studies can be compared to that of time-lapse photography for the study of macroscopic biological systems. But more clearly, the present invention
It is not only the method of time-lapse photography of the acquisition of images at the cellular level of biological structure and processing that is more than just questioning. By using conventional application software to process images of cell structure and mechanics, researchers are unable to observe a continuous stream of images showing uninterrupted changes in system parameters other than time. The present invention allows the investigator to change the position of the objective lens and parameters such as emission and / or excitation wavelengths during image acquisition, and during playback, the acquired set of images can be continuously changed. This change is displayed. Specific examples of the types of studies that would benefit from using the present invention include the movement of cell surface protein adsorption during cell- (immune cell) interactions from living T cells to B cells. Includes those that observe and evaluate software models of diffusion of chemicals entrapped in cells.

The following technical problems remain unsolved by conventional application software that automates optical microscopy systems. Especially in an optical microscope system, how to obtain an image by using the camera while the external device of the camera continuously changes the setting of various parameters of the image acquisition in the operation close to the maximum acquisition speed. It is a point. The present invention addresses this technical problem by a sequence in which the camera and the external device in an automated optical microscope system operate asynchronously, ie, independently of each other, during image acquisition and thus exhibit a continuous change in image acquisition parameters. It is solved by providing a computerized method that allows the camera to acquire an image to display as. The best form definition stack realized by the present invention. When this is used, the stack refers to the entire set of images acquired by the method of the present invention during one acquisition event, which leaves room for image processing.

Focal plane . When this is used, the focal plane is the vertical position at which the object of interest is observed and put into virtual focus. Z position . When this is used, the Z position refers to the focal plane, which is the vertical position at which the object of interest is observed and put into virtual focus. The Z position is an image acquisition parameter that can be changed by moving the objective locator or moving the stage mover of the optical microscope.

Image acquisition parameters . When this is used, the image acquisition parameters are
The Z position and the irradiation wavelength including either or both of the excitation wavelength and the emission wavelength are included.

Piezo focus . When this is used, piezofocuser is a shorthand term for piezoelectric objective lens displacers that operate to change the focus position at speeds in the range of 1-2 ms.

Asynchronous operation . When this is used, asynchronous operation allows the camera to simultaneously image while the external device, such as the objective locator, stage mover, and / or wavelength tunable device, varies certain image acquisition parameters controlled by each. It means to perform acquisition operation.

Interframe time . If this is used, the interframe time is the time between the end of the imaging of one frame and the beginning of the imaging of the next frame. In a raster scan video camera, this is the time between the raster scan of the digital scan line again in one frame and the raster scan of the digital scan line in the next frame. For a full frame CCD camera, this is the full frame readout time. In the case of a frame transfer CCD camera, this is the frame transfer shift time. Overview of the Invention The present invention provides a method, a computer-readable medium, and an automated optical microscopy system that captures an image at a substantially maximum acquisition rate of the camera while a device outside the camera varies the acquisition parameters. The stack of images so obtained is displayed as a sequence of images showing a continuous change in image acquisition parameters. The present invention drives an external device to change the image acquisition parameters while the camera captures each frame of a set of frame images, and the external device waits for the camera to finish capturing the frame. Instead of having to wait, the camera and the external device work asynchronously. In other words, the present invention allows the camera to capture an image from external equipment, such as the focus position of the microscope objective, the emission and / or excitation wavelength, or the position of the microscope stage, which they control. While driving, drive so that it can change.

The method of the present invention includes the following steps. a) an automatic microscope, a camera, and a device external to the camera, wherein the focal plane, the excitation wavelength and / or the emission wavelength, and the image acquisition parameters of the computer are varied so that the image is acquired asynchronously with the camera. Configuring an image acquisition system consisting of driving an external device b) acquiring images at a speed substantially close to the maximum image acquisition speed of the camera,
Store the captured image as digital data.

C) Operating at least one said external device and changing at least one image acquisition parameter during image acquisition and storage. The method of the present invention can be used under a wide range of microscope modes, including bright field, fluorescence, dark field, phase contrast, interference and differential interference contrast (DIC). One of the embodiments of the method of the present invention is a set of instructions existing in the information processing system. Another embodiment of the method of the invention is a collection of instructions residing on a computer-readable medium.

[0015]   A group of images, called a stack, has a variable focus position.
A sequence of groups of images obtained in this way showing a continuous change of the Z position and
It is a feature of the present invention that the information is acquired and displayed. Image stack
The wavelengths of the radiation and / or the excitation wavelength are tuned so that a group of images
/ Or acquired to be displayed as a sequence showing a continuous change in excitation wavelength
What is provided is another feature of the present invention. Image stack has focus position and radiation
And / or various acquisition parameters, such as excitation wavelength, are changed together,
Groups of images show continuous changes in various selected acquisition parameters.
It is a further feature of the present invention that it can be acquired, as displayed as a sequence.
is there. Another feature of another embodiment of the invention is that the image is captured at regular time intervals.
The stack is a stack of images where various image parameters can be changed simultaneously.
As a sequence changes continuously in the time direction, and the acquisition parameter changes continuously.
Is to be acquired so that it is displayed as   The advantage of the present invention is that the stack of images is
Alternatively, the structure is such that a chemical substance is introduced into a cell, or a biological condition such as cell division.
It is to enable a three-dimensional depiction during the event. Further benefits are images
Multiple stacks of are used during image acquisition parameter changes and at selected times.
To enable a three-dimensional depiction of a biological event of interest over an interval. General system configuration   1-4 and the following discussion describe an exemplary optical microscopy system of the present invention.
It is intended to be. The system of the present invention is an information processing system for performing the method of the present invention.
It consists of an automatic optical microscope system consisting of a processing subsystem. This method is
Of the camera while an external device of the microscope on the system is changing the image acquisition parameters.
It consists of acquiring images at maximum acquisition speed.

Those skilled in the art will appreciate that the present invention can be implemented in various information processing configurations, including personal computer systems, microprocessors, handheld devices, multiprocessor systems, minicomputers, mainframe computers, and the like. Furthermore, according to the person skilled in the art, the present invention allows the problem to be handled by a local or remote processing device connected via a communication network or connected to a server connected via the internet where the software method is accessed and used. It will be appreciated that it is implemented by a distributed computing environment.

Furthermore, according to those skilled in the art, specific research goals and needs, in particular for automated light microscopy systems, as these goals relate to the imaging of living cells during biological events. It will be appreciated that the configuration used and the particular device used. The elements of the image processing system using the program module of the present invention include a microscope, a camera, means for image processing according to the instructions of the program module of the present invention, and at least one image processing outside the microscope. A means for changing the parameters is provided.

FIG. 1 is a block diagram of an exemplary system of the present invention, which includes an optical microscope subsystem 100 and an information processing subsystem 200. In the figure, the microscope subsystem 100 is described in terms of its most general parts and should not be construed as limiting.

The exemplary microscope subsystem 100 comprises an optical microscope 160, shown here as a vertical microscope for illustration purposes. The external device of the microscope 160 includes the epi-illumination member 104, the emission wavelength tunable device 120, and the camera 130. Microscope 160 includes members 106-110 including transmitted light source 106-e.g.,
The transmission light irradiation arm 102 includes a halogen lamp, a transmission light shutter 108, and a condenser 110. The microscope 160 also includes an eyepiece lens 122, a stage 126, and an objective lens 124.

The epi-illumination member 104 includes, for purposes of illustration, an epi-illumination antigen 112 that includes a fluorescent lamp 112, a shutter 114 and an excitation wavelength tunable device 116.
The members 112 to 116 are included. The camera 130 is connected to both the microscope subsystem 100 and the information processing system 200.

The information processing subsystem 200 shown in FIG. 1 is depicted here as a personal computer 211, a central processing unit 213, a hard drive 215, a CD-ROM 217, a floppy (registered trademark) drive 219, Graphic adapter 221, Ethernet (registered trademark) network card 223, PCI card 225 for interfacing the computer 211 and camera 130, and a digital-analog converter [DAC] card for controlling the piezo electric objective lens arrangement device (340 in FIG. 3). 227 and, if included in the microscope subsystem 100, an exemplary computer 211 comprising a monochromator fluorescence illuminator (not shown) for the same purpose as the excitation tunable device (116 in FIG. 1, 316 in FIG. 3). hand There is. Peripheral devices of the information processing system 200 include a graphic display monitor 231 and a video monitor 241. The application software 245 enables control of the components of the microscope subsystem 100, in particular controlling the illumination, focus and camera, as well as image acquisition and processing. The method of the present invention operates as a program module of application software 245.

FIG. 2 is a pictorial representation of an exemplary hardware connection configuration between the information processing subsystem 200 and the exemplary embodiment of the optical microscope subsystem 100. FIG. 2 is an embodiment of a computer in the form of a personal computer 211. The rear side 251 of the computer 211 has various serial and parallel ports 251, 26
1, 271, 281, and 291 and physical connections 253, 263, 273, 283,
And 293, the computer 211 displays the optical microscope subsystem 100.
It is designed to communicate with and control various external devices. The ports and connections shown are the connection 253 connecting to the camera (130 in FIG. 1, 330 in FIG. 3) and the port 251 connected to the emission and / or excitation wavelength converter (116 and 120 in FIG. 1, 320 in FIG. 3). Port 261 to connection 263, external shutter (1 in FIG. 1).
14, a port 271 to a connection 273 connecting to 314) of FIG. 3, a port 281 to a connection 283 connecting a piezo electric objective disposition device (340 of FIG. 3), and a stage mover (336 of FIG. 3). Includes port 291 to connection 293.

FIG. 3 illustrates an optical microscope subsystem 3 used to implement the method of the present invention.
FIG. 50 is a schematic view of another embodiment of No. 00. The details of FIG. 3 are similar to the speed of the block diagram of the microscope subsystem 100 shown in FIG. 1, but FIG. is doing.

The microscope subsystem 300 of FIG. 3 comprises, for illustration purposes, a microscope 360, shown as an inverted microscope, with devices external to the microscope 320 including an epi-illumination member 304, an emission wavelength converter 320 and a camera 330. Microscope 360 includes a transmitted light illumination arm 320 that includes a halogen lamp 306 and a light source 306 illustrated as a shutter 308. The epi-illumination member 304 comprises an epi-illumination light source 312, illustrated here as a fluorescent lamp 312, an epi-illumination shutter 314 and an excitation wavelength converter 316.

The sample 332 to be observed has a microscope stage 326 as a mechanical stepper motor 336, which is vertically movable by a stage mover 336 shown in FIG. The piezo electric objective lens placement device 340 is
The lens 324 is moved vertically up and down to cover the objective lens 324 and bring the sample 332 into and out of the field focus. One or more objective lenses 324 are mounted on the rotating nosepiece 342.

The camera 330 is connected to the microscope subsystem 300 by a camera port 350. The emission wavelength converter 320 is arranged between the microscope 360 and the camera 330.

FIG. 4 is a pictorial representation of an embodiment of a microscope 460 used in the system of the present invention. The transmitted light irradiation arm 402 accommodates the transmitted light 406 and has a condenser 410.
Is included. The researcher can manually bring the sample 432 placed on the stage 426 into focus by looking through the eyepiece 422 and manipulating the stage mover 436, shown as coarse / fine focusing knob 436 in FIG. You can If necessary, a stepper motor (not shown) may be fitted with a focusing knob 436 to mechanize the stage motion.

An epi-illumination member (not shown) including an epi-illumination light source, a shutter, and an excitation wavelength converter (all not shown) may be attached to the microscope 460 behind the rotating nosepiece 442 and objective lens 424. A piezo electric objective lens arrangement device (not shown) may be closely attached onto the objective lens 424. An emission wavelength converter (not shown) is connected to the camera port 450 that connects the microscope 460 and a camera (not shown). Operation of the Automatic Microscopy System Continuing to refer to FIG. 3, the automatic microscopy system 300, according to the method of the present invention, acquires images as seen through the optical microscope 360 and captures these images as digital data in an acquisition camera. The maximum acquisition speed of 330 or a speed close to this is recorded. At the same time, the method drives the external devices 340, 336, 316 and 320 to change the parameters related to image acquisition, ie the focal plane of the sample and / or the excitation and / or emission wavelength. The focal plane is the vertical position at which the object of interest is observed and brought to the focus of the field of view. The focal plane is
Objective lens 324 (using objective lens positioner 340) or stage 32
It can be changed by changing the vertical position of any one of 6 (using the stage mover 336). As used herein, the term Z position refers to the focal plane that is varied by either the objective locator 340 or the stage mover 336.

Accordingly, the system 300 of the present invention allows the camera 300 and the external devices 340, 336, 31 to be used during image acquisition at or near the maximum speed of the camera 300.
Operating 6 and 320 asynchronously serves the function of acquiring an image by having a programmed set of instructions to perform the method of the present invention. As mentioned above, the external devices 340, 336, 316 and 320 are
Objective lens placement device 340 for vertically moving the objective lens 324, and microscope stage 3
It includes a stage mover 336 that vertically moves 26 up and down and wavelength tunable devices 316 and 320 that change the wavelength of the light used to excite the sample and / or filter the light emitted from the sample 332.

To understand how the present invention works, keep in mind that observations of living cellular material must be made at a corresponding rate to the rate at which the observed process occurs. is important. For example, the camera 330 must capture an image of biological events at the cellular level in milliseconds real time. Interaction of cameras, automated microscopes and external devices To observe live cell and tissue events, the microscope subsystem 30
0 means that the camera 330 is essentially a two-dimensional “snapshot” of the image,
Set up, or configured, to capture at different depths of the three-dimensional live configuration of interest. Changing the vertical position of the objective lens 324 has the effect, in effect, of changing the depth of focus at which a two-dimensional "snapshot" within the cell is taken. Changing the depth of focus literally changes the perceptual state of processes at different cell configurations and different depths of cells. Each time the objective lens 324 changes position, it is focused differently, providing different visual insights into the internal cell structure. Taking a quick “snapshot” from the camera each time the vertical position of the focal point changes, a sequence of images that, when played back, allows a researcher to see, for example, the top to the bottom of a cell (or vice versa). Alternatively, it is a set of “photos” displayed as a movie. The collection of images taken by system 300 for a particular number of vertical positions (and / or selected wavelengths, as discussed below) is a stack.

For example, when a researcher wants to observe an event that occurs at or on the outer wall membrane of a cell, for example, the researcher first manually moves the mechanism 336,
The microscope 300 is visually focused by positioning the stage 326 so that the outer wall of the cell is in the vertical position of the focus of the field of view. The camera 330 then captures an image of the cell's membrane and the investigator can choose to change the focus position by moving the objective lens 324. In the system of the present invention, the software method of the present invention moves the objective lens 324 vertically to move a mechanism called a piezoelectric objective 340, hereafter referred to as piezofocuser, to focus on different depths in the cell membrane. To tie. Each position that the objective lens 324 moves is referred to in the art as the Z position. The distance between the Z positions for cell studies is nanometers, so moving the objective lens with a piezoelectric mechanism allows for fine movement and the required placement without mechanical slip, typically 2 mm. Move at a speed of seconds (ms). This is sufficient, in most cases, for the camera 330 to acquire a stack of images at different depths of focus, the stack being able to quickly display the biological events taking place on the cell membrane during playback. .

In addition to changing the Z position of objective lens 324, subsystem 300 may also be configured to tune the wavelength of light used to illuminate sample 332 during acquisition of a stack of images. it can. Cellular structures, processes and events of living cellular material are often observed using fluorescence microscopy by introducing fluorescent chemicals or protein probes. Furthermore, the ability to solve problems using fluorescence microscopy is improved by systematically changing the wavelength of the light used to excite the fluorescently colored sample or by filtering the light emitted from the sample. To be done.

Different fluorophore, fluorescent chemistries, or protein molecules have different wavelengths of light.
Absorbs the electromagnetic radiation of and is excited to emit fluorescence of a specific wavelength of light. In addition, the constituents of cells colored with different fluorescent dyes are excited by and radiate by fluorescence in different wavelength ranges of light. By limiting or filtering the wavelength of the excitation light that illuminates the fluorescently colored sample or the wavelength of the emitted light that is captured by the camera 330, researchers can identify different cell structures or cell events at different times and cells or tissues. It can be observed in depth. Furthermore, by so limiting or filtering the fluorescence excitation and / or emission wavelengths, the investigator can focus on the selected cell structure or measure conditions within the cell composition. .

For example, by staining the organelles near the outer cell membrane with fluorescent dye A and the chlorosam in the cell nucleus with fluorescent dye B, researchers have shown that different cellular events involving both organelles are involved. You can get a stack of images focused on. This is due to the nature of fluorescent dyes. That is, for example, the fluorescent dye A is
This is because it is excited at the wavelength A and the fluorescent dye B is excited at the wavelength B. Camera 33
By taking a two-dimensional "snapshot" at 0 and switching back and forth between excitation wavelengths A and B, the researchers can
Can be made into a stack of images for processing to display structures excited by only wavelength A, only wavelength A, or both wavelengths, allowing changes in only one element of interest from complex cellular events. It can be cut out. The concept is similar to a camera taking a picture of a television screen after switching each channel, and then using the remote control to quickly switch back and forth between channels.

When changing the excitation or emission wavelength, some tunable devices operate at a speed of 1.2 ms. In many cameras, a stack of images at different wavelengths can be viewed by the camera 330 at playback time so that the stack shows a rapidly occurring biological event involving one or more cellular elements or biological processes. The system of the present invention, which is fast enough to cause the image acquisition, is set up to change both the Z position and the wavelength while the camera 330 is acquiring the image. When the investigator chooses to change both the Z position and the wavelength, the method of the present invention first drives the piezofocuser 340 to move the objective lens 324 to the new Z position and then the tunable device. 316 and /
Alternatively, drive 320 to switch to a new wavelength. Only after the camera 330 has finished acquiring images at each of the selected wavelengths, in this method, the piezofocuser 340 is driven to move to the new Z position. Theoretically, this method allows the researcher to choose any number of wavelengths to switch. Embodiments of the present invention allow researchers to select four different wavelengths.

As described above, the piezofocuser 340 sets the Z position of the objective lens 324 to 3 degrees.
The wavelength tunable device 316 and / or 320 has a capacity of 1.2 m
Due to its ability to change wavelength in s, the system-specific component of subsystem 300 is most often camera 330. Ultimately, the speed at which the camera 330 acquires an image is determined by whether the external device 340, 336, 316 or 320 has sufficient time to change each parameter before the next image is acquired. Method / System Principles of Operation In essence, the method of the present invention provides a stack of images that can be displayed at the acquisition speed of the camera 330 and, when played back, with continuously changing selected parameters. It depends on the difference between the operating speeds of the piezofocuser 340, the stage mover 336, the wavelength tunable device 316 and / or 320. The principle of operation of the present invention is that the piezofocuser 340 and the wavelength tunable devices 316 and / or 320 can be used by the camera 330 when the camera 330 acquires the image at a slower speed or at a speed at which most external devices operate. The Z position and the wavelength can be changed before each starts to acquire the next image. Stated differently, the camera 330 uses the external devices 340, 336, 3 in order to change the Z position and wavelength fast enough for the next captured image to capture and display these change values.
It should be slower than 16 and / or 320 by a margin.

A second principle of operation of the method of the present invention is that image acquisition occurs while the piezofocuser 340, stage mover 336, tunable device 316 and / or 320 operate asynchronously with the camera 330. It is a thing. Asynchronous operation includes piezofocuser 340, stage mover 336, wavelength tunable device 316 and / or 32.
0 means do not wait for a signal to finish reading the imaged image before changing Z position and / or wavelength. Asynchronous operation is that during camera readout, the external device either switches to the next required wavelength and / or moves the focal plane to the next required Z position. In other words, the camera 330 and the external devices 340, 336, 316 and 320 are operating more or less simultaneously,
It does not work sequentially. Contrary to this, sequentially,
That is, the operation is synchronized with the external devices 340, 336, 316 and / or 3.
The 20 in effect waits until the camera 330 completes the acquisition and readout before receiving a signal changing parameters. Synchronous operation thus slows down the overall acquisition speed of subsystem 300, and thus, is unable to acquire images at or near the maximum acquisition speed of camera 330, which is the main object of the present invention. . Basis for Camera and Image Acquisition Speed To provide context and rationale for these essential operating principles of the present invention, the following section briefly discusses image acquisition by a camera. Image acquisition by a camera is described in Inoue and Spring, Video Miroscopy, 2d. Edition, 1997, especially No. 5.
It is fully described in the chapter and is incorporated by reference. The camera converts the optical image illuminated on the photosensitive surface into a sequence of electrical signals to produce a video signal. For cameras using the North American (NTSC) format, the optical image is sampled as if it were a rectangular page containing 525 horizontal scanlines every 1/30 second, ie, from start to end, left to right. Read out. This is called a raster scan read sequence. For cameras using the European (PAL) format, the read speed is 1 /
There are 525 horizontal scan lines every 25 seconds. These numbers are converted to the following standards. The NTSC format camera reads one frame every 33 milliseconds, and the PAL format camera reads one frame every 40 milliseconds. These standard read speeds hold across a wide range of video rate cameras, whether they are vidicon tube cameras or solid state cameras. The latter camera uses a photodiode for light detection. With a raster scan video camera,
The interframe time is the time between the raster scan of the last digital scan line of one frame and the raster scan of the first digital scan line of the next frame.

As one type of solid-state photodiode camera, there is a charge coupled device (CCD) camera. Charge coupled devices for such cameras are
It consists of a huge rectangular array of photodiodes arranged on a silicon substrate. Each photodiode is an isolated sensor element that is electrically separated from its neighbors by a channel stop. During the time of exposure, photons fall on the photodiodes and the electrons in the depletion layer of each photodiode move to the junction isolated potential well. Each well collects many electrons and stores a specific charge. When all the wells in the rectangular array are detected as filled by the camera and thus signal the end of exposure signal, a read-out of the accumulated charge occurs.

The CCD solid-state camera is read out systematically by moving electrons from the wells to the output node, and the charge of the electrons in each well is the same raster scan sequence from the array of wells that make up the photodiode array. Is read out. The output nodes are read in a top-to-bottom, left-to-right sequence, with each well having its own specific charge. The output node is connected to an amplifier that converts the charge in each well into a voltage, which is proportional to the charge stored in each well or pixel.

There are three best known arrays of photodiode architecture in charge coupled devices. Full frame, frame transfer and interline. The full frame CCD architecture means that the entire photodiode frame is integrated, i.e. exposed to contain charge in all frame wells before reading occurs. Therefore, in the case of a full frame CCD camera, the interframe time is the read time of the chip.

Full-frame architecture is currently used for slow scan CCD cameras that use mechanical shutters. The shutter is opened during image acquisition and closed during charge transfer and readout. The full frame architecture shows that the image acquisition speed is limited by the detector size of the photodiode array, the speed of the mechanical shutter and the speed of reading the image data from the charge coupled device to the computer.

A camera using a frame transfer CCD architecture divides the full frame of photodiodes into two regions. An imaging unit and a masked storage unit. During integration (exposure), the image is stored as electron charges only in the wells of the imaging part. When the exposure is completed, the accumulated charge is
Moved to the masked part, two processes occur simultaneously. The imaging section is emptied to start exposure again, and the masked section reads out the charge accumulated from the first exposure. As a result, this type of interframe time of a CCD camera is the time it takes to transfer the accumulated charge from the imaging section to the masked section. The frame transfer CCD camera can perform two operations, exposure and readout, almost at the same time.

In a camera with an interline CCD architecture, a full frame of photodiodes is arranged in alternating rows, with one row containing the imaging photodiode and the next row containing the masked photodiode. To do. During integration, the columns of imaging photodiodes transfer the charge in the wells to adjacent columns containing the masked photodiode portion. In operation of a frame transfer camera, after the charge has been transferred to the masked columns, the unmasked columns of photodiodes are exposed again. Similar to the frame transfer camera, image acquisition is performed in the same manner as the interline transform camera and the frame transfer camera including overlapping masked and unmasked diodes. Interline CC operating in sequential mode
In the D camera, the interframe time is the same as the frame transfer CCD, that is, the readout time of the photodiode array. Interline CC
If the D camera operates in overlap mode, the interframe time is the shift time for transferring the charge stored under the masked portion. System Configuration and Asynchronous Operation of External Devices When the external devices 340, 336, 316 and / or 320 operate at a speed faster than or similar to the acquisition speed of the camera 330, the external devices 340, 336, 3
16 and / or 320 can operate asynchronously with the camera 330, and this is an indication of system configuration. When the external devices 340, 336, 316 and / or 320 pumped into the system can operate asynchronously with the camera 330, there is no need to directly connect the camera 330 to the external device.
Therefore, in the system of the present invention, the external devices 340, 336, 316 and / or
Alternatively, 320 is connected to system computer 211 by, for example, connections 283 and 261. These devices are not directly connected to the camera 330. Asynchronous operation is achieved by the signaling method 340, 33 according to the software method of the present invention.
6, 316 and 320 are performed by changing the parameter values in conjunction with the acquisition routine of the camera 330. Implementation of the Method of the Invention In principle, the method of the invention operates as follows. Researchers configure the system to include a camera 330 and selected external devices 340, 336, 316 and / or 320. The researcher inputs the Z position and / or the number of wavelengths at which the image is acquired. With that input, the researcher will actually be selecting the number of frames that the camera 330 will capture in a stack between experimental or capture events. For example, entering 20 Z positions and 2 wavelengths translates into a stack of 40 frames. The stack is Unversal I
With appropriate application software such as MetaMorph obtained from maging
Processed into a sequence displaying successive changes in the selected Z position and wavelength parameters.

When the researcher issues a command to start the image acquisition, the method uses the camera 330
To start exposure at the first selected Z position and the first selected wavelength. Processing is well known in the art, where an image is digitized, read into computer 211, digitized into a temporary memory location or buffer and stored as an image. When the camera 330 begins reading the exposed image, the method drives the external devices 340, 336, 316 and / or 320 to change the values. If the piezofocuser 340 and the wavelength tunable devices 316 and / or 320 are incorporated into the system, the method is prior to driving the piezofocuser 340 to move the objective lens 324 to the next Z position. , At the current Z position, drive tunable device 316 or 320 to switch to all selected wavelengths. The method continues to drive external devices 340, 336, 316 and / or 320, changing the values at the beginning of each image readout until these devices move for the entire set of selected values.

Important to the understanding of the method of the present invention is that the camera 330 uses the external device 34 as the method.
There is, so to speak, no limitation on acquiring an image at or near its acquisition rate while matching the operation of 0, 336, 316 and / or 320 with the readout from the camera 330. Important to one embodiment of the method and system of the present invention is the limitation that the operating speed of the external device 340, 336, 316 and / or 320 must be equal to or faster than the interframe time of the camera 330. is there. For example, researchers have found that fast interframe time cameras, ie, frame transfer cameras that are comparable in speed to the piezofocusers 340 and / or wavelength tuners 316 and / or 320 for 1-2 milliseconds, or
Interline transfer cameras operating in overwrapping mode can be used. In this embodiment, the camera 330 is, in effect, the device 340, 3,
Images are acquired at the same rate that 36, 316 and 320 change the Z position or wavelength.

If the operation speed of the external device 340, 336, 316 and / or 320 is slower than the frame of the camera 330, the method can be performed while the camera 330 reads the current image to the computer 211. External devices 340, 336, 316 and 320 cannot be driven to change position and / or wavelength. For example, using a slow-tuning wavelength tunable device to switch between selected wavelengths results in an image containing mixed wavelengths. In order to reduce the alteration of the image, in this embodiment, the researcher drives the camera 330 to move forward in each frame to the exposure image, but before exposing the image, a choice, such as 3, is made. Skip the specified number of frames. Then, the method of this embodiment can effectively allow the external devices 340, 336, 316 and / or 320 to change the parameters and give the camera extra time to catch up. Specific Device and Application Software In order to operate asynchronously using the method of the present invention, the specific device that constitutes the system of the present invention is as follows. Princeton Ins from Roper Scientific with a line of Coolsnap FX cameras from Roper Scientific's Photometrics division
A line of MicroMax and PentaMax cameras obtained from the trument category. Also, the present invention
The system and method can be operated with any camera that complies with the RS-170 or CCIR standards as long as it interfaces with the processor via a digitizer called a video frame grabber. An example of a video frame grabber that generates a software interrupt to allow the method of the present invention to drive a camera and simultaneously capture an image while an external device is changing is FlashBus. MV PRO PCI bus mastering digitizer card
It

Piezofocusers operating in the systems and methods of the present invention are Physik Ins
Includes Miroscope Objective Nanopositioners PIFOCR line from trumente. Suitable tunable devices operating in the system and method of the present invention include: Lambda DG5 Illuminator, La from Sutter Instruments
mbda DG4 Illuminator, Lambda 10-2 Wavelength changer and Lambda 10 Wavele
ngth Changer, Polychrome II Monochromator from TILL, Kinetic Imagi
Monochromator obtained from ng and DeltaRAM Monochromator obtained from PTI.

Application software that supports asynchronous operation of the camera and the external device includes MetaMorph (TM) and MetaFluor®. Exemplary Information Processing Subsystem With reference to FIG. 1, when the information processing device 200 comprises a computing device 211 in the form of a personal computer, the exemplary subsystem 200 of the present invention is currently 5
50MHz, 700MHz, 750MHz, 800MHz or 850MHz
Intel (R), Pentium (R) III class processors, including, can be used.

Suitable motherboards include P3BF or CUBX available from Asus. For display adapters, researchers are ATI Expert @ Play98-8 megabyte, Matrox G
400-32 megabyte dual display or ATI Tage Fury Pro-23 megabyte
be able to.

The preferred memory environment for a system configured in this way is 256 megabytes to 1 gigabyte of SDRAM memory. For a variety of equipment, see Waitern Digi
WDC WD205BA IDE (20.4 GB) hardware drive from tal
, 1.4 MB 3.5 floppy drive, CD-R58S read / write 8x24 SCSI CD-ROM drive.

Suitable cards are Adaptec 2930 Ultra SCSI2 kit for SCSI cards, SIG Cyber PCI-1serial, one parallel port for I / O cards, 3COM 3C905TX-M 10/100 PCI for network cards. is there. Method Continuing to refer to FIGS. 1 and 3, FIG. 5 is a flowchart of the method of the present invention. The method of the present invention is described herein with respect to a computer program module that acquires an image at substantially the maximum acquisition rate of the camera while an external device of the camera continuously varies the acquisition parameters, but these are , According to those skilled in the art,
It will be appreciated that it can be implemented with other program modules, routines, application programs, etc. that perform other particular operations.

The image acquisition parameters that are varied while the image is acquired are the Z position,
Includes excitation and / or emission wavelengths and vertical position of the microscope stage. The Z position can be changed by receiving the piezofocuser 340 and moving the sample objective lens 324 relatively, or by using the stage mover 336 to move the microscope stage 326. The image acquisition parameter shown in FIG. 5 is Z
Contains position and wavelength. Before the cameras 330 that make up the system begin to acquire images, the researcher decides the purpose of the study,
Preparing sample 332, configuring the specific hardware used by the system,
That is, there are preparations to identify to the system. As shown in FIG. 5, the method of the present invention begins at step 500 and a researcher configures the system of the present invention by selecting a particular image acquisition device. Configuring the system is
In fact, it is the selection of an appropriate device driver, which is a pre-stored software file in the image acquisition / processing program that contains the information the program module needs to operate the corresponding hardware. The configurable image acquisition device includes a camera 330, a piezofocuser 340, and a stage mover 336.
They are means for changing the position and wavelength changers 316 and 320.

With continued reference to FIGS. 3 and 5, FIGS. 6-9 show exemplary user dialogs that make up the automated optical microscope subsystem 300. FIG. 6 shows an exemplary dialog for installing the camera 330, shown on the left is a list of available camera drivers 610 included in an exemplary software package for image acquisition / processing 245. . By highlighting a particular driver 612 from the list of available drivers 610 and clicking the ADD 614 button, the selected driver will appear in the list of installed drivers 616. The system illustrated in the driver configuration 618 window is configured to include only one camera.

FIG. 7 illustrates an exemplary dialog that configures parameters for the installed camera of FIG. 6, specifically exposure time 620 and camera shutter 622. The exposure time 620 means the total exposure time, and in 620, it is illustrated as 100 ms. The user may also choose to leave the camera shutter open at 626 for the exposure time. By so selecting and incorporating the tunable device into the subsystem 300 occupied at the bottom of FIG.
00, the shutter can be operated as a wavelength tunable device (316, 320).

Mechanical shutters external to the camera 330, such as shown at 308 or 314 in FIG. 3, are driven by summer in terms that take time to dissipate, so must be operated at cycle times greater than 25 ms. Not becoming important is important. If these shutters are driven with a cycle length shorter than 25 ms, heat will accumulate and eventually cause puncture. For this reason, a fast tunable device with 1.2 ms switching time is convenient to drive as a shutter. This allows camera 330 to capture images at or near its maximum acquisition rate, facilitating asynchronous operation with external devices 340, 336, 316 and / or 320.

FIG. 8 shows an exemplary dialog for incorporating an external device into the system. At 632,
The device 632 is displayed on the installation device 636 by highlighting the name of the particular device and clicking the ADD 634 button. As shown in Figure 7, two external devices are installed in the exemplary system. 632 is a piezofocuser and 638 wavelength tunable device.

FIG. 9 shows an exemplary dialog for incorporating a piezofocuser into the system 300. At the bottom of the dialog, the researcher finds that the piezo focuser 340 is the objective lens 324.
Three button switches 644, 646, 648 are shown that determine the range of distances to move. By clicking on the set home 646, the user causes the piezofocuser 340 to set the objective lens 324 to the home position at the end of image acquisition. The home can be in any position and depends on the research goals. For example, a researcher determines some critical Z-positions by eye focusing or by processing previously acquired stack events. Such a Z position is set as the home. The set top 644 position is the objective lens 324.
Is the maximum limit on the home that moves. The set bottom 648 position is the maximum lower bound for the home. The top, home, and bottom positions define the total distance that the piezofocuser 340 moves the objective lens 324 during the exposure time. The researcher can initialize the starting position of the objective lens 324 by checking the current position 642. The move increment 640 option allows the user to select a distance to increment the Z position, which is illustratively set to 1.0 micrometer.

By setting the top and bottom positions at 644 and 648, along with the move increment at 640, the researcher calculates the total number of Z positions at which the image is acquired. For example, in FIG. 9, the total vertical distance that the piezofocuser moves is
22 micrometers. When the increment distance is 1 micrometer, the image has a minimum of 23 Z positions (start position = 1 + 22 micrometers =
All 23 positions are acquired. User Inputs Referring back to FIG. 5, the next step in the method after building the system is the user input of step 502. Here, the user drives the piezofocuser 340 and the wavelength tunable devices 316 and / or 320 according to the method, and inputs a range of values so as to tune a specific Z position and wavelength during image acquisition. .

User Input Stream Acquisition Embodiment FIGS. 10 to 13 are a first embodiment of a user dialog for inputting the Z position and wavelength value, and are shown as an embodiment of stream acquisition 702. 14 to 20
Is a second embodiment of such a user dialog and is shown as an embodiment of multidimensional acquisition 802.

FIG. 10 shows that an embodiment of stream acquisition 702 includes four separate user dialogs or tabs, acquisition 704, focus 706, wavelength 708, and camera parameters 710, thereby The researcher inputs the value of the acquisition condition. FIG. 10 shows the Get 704 dialog, showing the user selection within the dialog. Options 718 and 720 allow researchers to check if they are working with a camera 330 piezofocuser and a fast tunable device. By checking 718 and 720 and having a specific device built into the system, as shown in FIG. 8, the user can enable the installed device to support asynchronous operation with the camera during image capture and Confirmation is received in field 730.

In the embodiments of stream acquisition 702 and multi-dimensional acquisition 802, the user can tell the piezo focuser 340 or stage mover 336 and the wavelength tunable device 316 and / or 320 to change the Z position and the excitation and / or emission wavelength. You can choose to acquire an image while doing so. This relates to the basic operating principle of the method described above. Since the external devices 340, 336, 316 and 320 operate asynchronously with the camera 330, the external devices do not wait for the camera to read long before changing each parameter. In one embodiment of the method, as long as the external device operates faster than, or comparable to, the frame-to-frame time of the camera, several external devices can be attached together and while the camera is acquiring. , All of which make the image acquisition parameters variable. In reality, however, the camera and the external device operate asynchronously, but the external device operates in synchronization with each other. In particular, when a researcher chooses to use both a piezofocuser 340 and a tunable device 316 and / or 320, as shown in fields 718 and 720 of FIG. A signal is given to the piezofocuser 340 to move it to a new Z position. The system of the present invention then provides a signal to the wavelength tuners 316 and / or 320 to change to each selected wavelength in successive frames. Only after the frame has been exposed at each selected wavelength, the method signals the piezofocuser 340 to change the Z position. The sequential operation of the external devices relative to each other is shown in more detail in FIG.

FIG. 10 shows that the user moves the objective lens 324 by the piezofocuser 340 to select the number of autonomy, indicated in the field 712 as 23. Theoretically, the user can select any number of Z positions to acquire images, but it is limited by the biological object of interest, the research subject, etc., and the camera and the external device are operated asynchronously. Therefore, it is not limited. Figure 1
The 0 dialog works with the dialog of FIG. 9 showing the top, bottom and home Z positions.

FIG. 10 illustrates external shutter 714 selection. Shown at 716 is an exemplary user selection in which a fast tunable device, the Sutter DG4, acts as an external shutter for the camera 330. In FIG. 7, the user chose to leave the camera shutter open for the entire exposure time. The externally-chosen 714 selection operates together with the dialog of FIG. 7 to drive the high-speed wavelength tunable device to function as an external shutter.

FIG. 11 shows focus 706, in which the user is
The first and last Z positions of the objective lens 324 can be selected together with the direction in which the piezofocuser 340 is moved. The Focus 706 dialog works with FIG. 9 to enter the positions of Top 644, Home 646, Bottom 648 and Current 642. As shown in FIG. 11, at the beginning of image acquisition 732, piezofocuser 340 is at the top 644 of the selected range, 646 (FIG. 9).
During the image acquisition 734, the piezofocuser 340 moves the objective lens 324 downward, towards the bottom of the range 648 (FIG. 9). After acquiring the image, the piezo focuser 340 moves the lens 324 to the current position, and 642 (FIG. 9).
, Which corresponds to home in this example, as shown at 646 (FIG. 9).

FIG. 11 shows, as example 1, pre-instance 738, which corresponds to move increment 640 of FIG. Further, the total distance 740 is illustrated as 22, and corresponds to the total access distance calculated in the above discussion of FIG.

FIG. 12 illustrates wavelength 708 for an embodiment of user input stream acquisition 702.
Show a dialog. As illustrated here, the researcher, at 750, sees FIG.
The built tunable device, shown to be implemented at 638, has selected to switch between the two wavelengths during image acquisition. As an example of wavelength, FI of 752
There is TC and 754 RHOD.

FIG. 13 shows a camera parameter 710 dialog. In selecting the acquisition mode 760, the researcher can select different embodiments of the method of the present invention. For example, a researcher can drive the camera 330 and select that the image acquisition be done at its frame rate 762, ie, once the shutter is open, the exposure is at the maximum operating speed of the camera. In other embodiments, the researcher can also choose to drive the camera and have the image captured by an external trigger 764.

Further, in different embodiments, the researcher may find that under certain conditions, the camera 33
You can choose to drive 0 and expose the image only for specific frames, which is done by entering the number of frames to skip, 7
66. Having the camera 330 skip frames may be useful when a researcher is incorporated and uses an external device 340, 336, 316 and / or 320 that is slower than the access camera's readout throat. Such an external device 340,
336, 316 and / or 320 vary the Z position and wavelength while the camera 330 is exposing the image so that the acquired stack is displayed in an obscure state. An example of a system configuration when this embodiment of the method is effective is:
Researchers decided to use full-frame CC instead of high-speed wavelength tuner to change wavelength.
It's when I'm using a D camera and a filter wheel.

For example, at 766, selecting the camera 330 to skip 3 frames would actually drive the device to change the Z position and wavelength only every 4 frames. If the exposure and readout speed of the camera used in the system is 25 ms, the operating speed of the selected wavelength tunable device is, for example, 75 ms.
Therefore, if the device is selected to skip so that the parameters are switched every four frames, it is possible to give the wavelength tunable devices 316 and / or 320 sufficient time to switch the wavelengths. Multi-Dimensional Acquisition Embodiments Continuing to refer to FIG. 5, in step 502, a researcher may select other embodiments shown in FIGS. 14-20 to enter values for various acquisition conditions. You can This is referred to as the multidimensional acquisition 802 embodiment. A different feature of the multi-dimensional acquisition 802 embodiment and the stream acquisition 702 embodiment is that in the multi-dimensional acquisition 802 embodiment, a researcher can obtain the same sample under the same acquisition conditions at regular time intervals. You can get a set of images. Under the same conditions at different time intervals, the purpose of acquiring images is to generate time-lapse video.

FIG. 14 illustrates a main 802 user dialog of an embodiment of multidimensional acquisition 802. The researcher must check each acquisition condition, time passage 814, multiple wavelengths 816, Do Z series 816 and stream 820, and the corresponding dialog dialog, time passage 806, wavelength 808, Z series 810 and stream 812. Appears and is shown to make it accessible to researchers.

FIG. 14 also illustrates a description box 8 in which a researcher enters the title of the file into the acquired set of images to be stored specifically in permanent memory.
22 is shown. As shown, the description box 822 shows that in an exemplary experiment, the image shows that the piezo focuser 340 switches to the Z position and the tunable devices 316 and / or 320 switch between the three wavelengths. 10
Be worshiped at three different times, separated by seconds. At box 824, the user-determined name is entered into the stored file with the retrieved stack of images.

The time lapse 806 dialog of FIG. 15 allows the researcher to enter a “time interval” 832, shown as 10 seconds, as well as a “number of time points” 830, shown as 3.

FIG. 16 shows that when the Z Series 810 dialog is opened, the researcher needs a program module to drive the piezo focuser 340 to allow variable Z position during image capture. You can enter all the information. This information includes the current position 840 of the piezofocuser 340, the increment 842 that the program module drives to move the piezofocuser 340 to the next Z position, the top 846 and bottom 848 distances from the current position 840, the piezofocus. It includes the total number of steps 852 that the Casa 340 takes during image acquisition. The Z-dialog 810 allows Z-series information to be entered more directly than the embodiment of stream acquisition 702, and the piezo focuser 340 allows the objective lens 3 to move during image acquisition.
The full range 844 moving 24 is calculated directly. In particular, the multi-dimensional smallpox 802 embodiment uses the 810 dialog to display the two shown in FIGS.
In one dialog, enter the same Z-series information as you would enter in the Get Streams 702 embodiment.

17-19 show a wavelength 808 dialog, the researcher has a Waves 860 box.
In box #, the maximum of 8 wavelengths that can be switched can be identified while the camera 330 is capturing an image. As shown here, three wavelengths have been selected. The researcher can type a unique name for each numbered wavelength in the Name 864 box. The Current Wavelength 862 box shows a list of entered wavelength names, and which program module the wavelength tuners 316 and / or 320 will drive to illuminate when the camera 330 begins exposure. .

In FIG. 17, the current wavelength 862 is 1 for DAPI, 2 for FIG. 18, FITC for 3, and 3 for RHOD in FIG. The names DAPI, FITC, RHOD are abbreviations for fluorescent dyes known in the art that each have a different illumination color when excited at a particular wavelength. Thus, identifying by name the name of a fluorescent dye that becomes particularly visible when illuminated at that wavelength provides the researcher with an easy memory method to identify various wavelengths. When entering the name of the wavelength, the researcher must kick the specific name and order into the relevant diagram. That is, each input wavelength is given a number that indicates the order of irradiation during acquisition. In this way, the program module can create a wavelength table by entering and giving an order to each wavelength.

A user configures a piezofocuser and a wavelength tunable device in which the program module drives these devices to operate fast enough to change the Z position and wavelength while the camera captures the image. When fields 866 and 868 are shown in FIG.
Appears to be checked, as shown at 19. In fact, 866 and 86
A check of 8 tells the user that the system 300 can use the camera 330 and the external devices 340, 336, 316 and / or 32 to carry out the method of the invention.
It provides notification and confirmation that it is configured as zero.

FIG. 20 shows a stream 812 dialog, which only appears when the user clicks on the stream 820 option of the main 804 user dialog, due to iterations. By clicking this option and configuring the system with an appropriate camera and external device, the information processing system 200 is notified that the method of the present invention is available and thus available to the user. . Importantly, the dialog 802 provides the researcher with a one-page summary of the system parameters or magnitudes the method performs after the researcher has begun image acquisition. Shown in field 870 is a list of magnitudes that have changed during image acquisition, and shown are Z position and wavelength.

In box 872, the total exposure time between acquisitions is illustrated as 50 ms. The stream illumination 874 box allows the researcher at 882 to select a list of built-in tunables used to change the wavelength during acquisition. As shown, Sutter DG4 is selected.

[0079]   At 876, the researcher temporarily captured the digitized image during acquisition.
A memory location for storage can be selected, here illustrated as RAM. Memory allocation   With continued reference to FIGS. 5 and 10, the researcher has entered the requested acquisition information.
Then, the program module proceeds to step 504, the information processing subsystem.
200 has enough temporary memory to retrieve and store the requested stack of images.
It is determined whether or not the In the embodiment of stream acquisition 702,
The Get 704 dialog of FIG. 10, at 722, for the exemplary stack
Shows the memory conditions of. An example, as shown in field 728
The total number of frames in the illustrative stack is 46 and stores 46 frames.
The amount of temporary memory 722 needed to store is 23.00 megabytes. Use
The possible memory amount 724 is, for example, 255.50 megabytes. the study
The person therefore is given the system parameters as shown in FIGS.
System, enough sys- tem memory to obtain an exemplary stack of 46 images.
System is informed.

With continued reference to FIG. 20, multi-dimensional acquisition 802 of user input.
In an embodiment of the present invention, the researcher is informed in field 878 how much temporary memory the exemplary stack requires, illustrated as 34.50 megabytes, and in field 880, the example How much memory is available in a typical information processing system is reported and is shown as 255.50 megabytes. Initialization Continuing to refer to FIGS. 1, 3 and 5, once the system has been configured, certain values have been entered, and memory allocations have been made, the program module drives the computer 211 in step 506 to drive Z Lets determine if the position changes during stream acquisition. If so, in step 508, the program module causes the piezofocuser 340 or stage mover 3 to move.
The initial Z position is initialized by moving 36 to the position designated by the user as the start position. In the stream acquisition 702 embodiment, this is the Start At 732 field of FIG. 11 and is shown in FIG.
As such, it can be in either the top 644 or bottom 648 position. For the embodiment of multi-dimensional acquisition 802, see the discussion of components 846 and 848 of FIG. 16 above. In step 509, the program module creates the table of Z positions discussed above in the description of FIG.

In step 510, the program module drives the computer 211 to change the wavelength during stream acquisition and, if so, the wavelength tunable devices 316 and / or 320 to the position 1 by the user. It is determined whether or not to move to the position selected in step 502. In the embodiment of stream acquisition 702, this is component 752, wavelength # 1 field of FIG. In the embodiment of multidimensional acquisition 802, component 86 of FIG.
See the discussion above in 2.

In step 513, the program module is back to the discussion of component 862 in FIGS.
0 creates a table of wavelength values that specifies the order in which the wavelengths are switched.

In step 514, the program module sets the frame number to “0”.
To initialize the camera, and in step 516, it is determined whether the camera 330 has an external shutter 314. In such a case, the program module opens the external shutter at 518 and drives the camera at step 520 to ensure that the shutter is open before image acquisition begins.
Wait for a predetermined delay time. The length of such delay depends on each of the shutters. In the image acquisition step 522, the system is ready to begin image acquisition. Once the researchers start the acquisition routine, the camera is
Start image acquisition while changing Z position and / or wavelength. For the embodiment of get streamline 702, FIG. 10 shows the get 732 button, which initiates the get routine. In the embodiment of multi-dimensional acquisition 802, the acquisition 826 button is shown in FIG.

The acquisition routine of step 522 is described in more detail in FIGS. 21 and 22 and is described below. A summary of step 522 is as follows. As the exposed frame is read from camera 330 to a temporary memory location on computer 211, the program module of the present invention increments the Z position and / or wavelength to the next position. The camera then exposes the next frame and thus acquires images showing different Z positions and / or wavelengths. During the reading of that frame, the program module again returns to the Z position and / or
Alternatively, the wavelength is incremented. After reading the last frame, the program module ends the acquisition of the stack.

After all the images have been read and stored in steps 524-526, the program module drives the external shutter 314 to close it (if it was done during acquisition). . In step 528, the stack of acquired images is copied from the temporary memory location and moved to a more semi-permanent system memory location, thus freeing system memory resources at 530 and initiating the next acquisition event, or If necessary, the process ends at 532.

Different embodiments of the system of the present invention store the captured image in different temporary memory locations. In one embodiment using a personal computer as the processor, the temporary memory location consists of a buffer in computer RAM. In another system embodiment, the captured image is redundan
Embodiments using t Array of Inexpensive Drives (RAID) or other computer-readable media or distributed computing environment, or embodiments in which program modules are accessed to a local or remote server via the Internet Store on a real-time hard disk including the case. Further, different embodiments of the present invention store the captured image in different semi-permanent memory locations. Those skilled in the art will appreciate that different embodiments of the optical microscope system 100, particularly those associated with using different computer configurations 211 to process the acquired images, can be used to store various images in a semi-permanent manner. It will be appreciated that memory locations are used. Asynchronous Operation of Camera and External Device During Image Acquisition Continuing to refer to FIGS. 2 and 3, FIGS.
, 316 and / or 320 more closely describe the sequence of steps in which an image is acquired by the camera 330 operating at or near maximum speed while operating asynchronously with the camera. In step 900 of FIG. 21, the user initiates a program module acquisition routine, the whole of which corresponds to step 522 of FIG. In step 902, the program module resets the system frame number to zero. In step 904, the program module initiates the image acquisition step.

In this regard, the method of the present invention branches into two processing lines that are related to each other, one line being executed by the camera 330 and steps 912-916.
And 922-928. The shaded nodes in these steps represent the processing of the camera controller. The second interrelated line is
Related to the processing performed in the main computer, 918, 932, 936
, 938 and 940 steps. Steps 932, 936, 93
8 and 940 are executed by the main processor and are indicated by the nodes with diagonal lines. Step 918 is performed by the software interrupt handler and is indicated by the crosshatching. In the image acquisition and software interrupt step 912, the program module causes the camera 330 to generate the total number of frames requested by the researcher (equal to the number of Z positions times the number of wavelengths, FIG.
(See the discussion in) and let the camera start the exposure of the first frame. At the same time, in step 932, the computer 211 starts the process of determining whether the line of processing, that is, the number of frames has been incremented.

Between the end of step 912 and the time before the beginning of reading in step 916, the computer 211 executes a software interrupt (computer 21
By receiving a command to stop 1 from consecutive processing of frame numbers), an alert that the exposure is completed is given. The step of raising an alert occurs at node 914. In step 918, the software interrupt handler, which is the description of the acquisition routine of the program module, is executed by the computer 2.
11 is notified to update the frame number. This is node 918 and node 9
It is indicated by a dotted line 919 connecting 32. Once the computer 211 increments the frame number by 1, it returns to loop 933 which spends most of the processing time waiting for the frame number to increment.

In step 916, the exposure image is read by direct memory access (DMA) to a temporary memory location, eg RAM. Referring to FIG. 1, in the DMA, the camera interface card 225 provided in the computer 211 transfers the image data from the camera 130 to the memory of the computer 211 without requesting the intermediary of the application software 245 directly. Is the way.

In this respect, the program module can be ported via different embodiments of the method according to the integrated camera. Researcher has a frame transfer CCD camera or an interline C operating in overlap mode
When incorporating CD camera system, program modules, while being exposed in the current frame image is read out yet in the temporary memory, the process proceeds directly to step 926.

This implementation path depends on the special configuration of these cameras. As mentioned above,
The CCD device of a frame transfer camera has an architecture that allows the current frame to be captured while the previous frame is being transferred to temporary memory. In particular, the frame transfer CCD device has a photodiode in the imaging area and a masked area, and the exposed image of the previous frame is transferred from the imaging photodiode area to the masked area very quickly.
The imaging area of the camera is emptied to begin exposing the image in the current frame even before the previous image has been completely read into temporary memory.
In fact, the masked photodiode region represents a type of stop gap storage where the camera can overlap the exposure and readout functions. The overlap of these two functions allows for very fast image acquisition. The interline CCD camera operating in the overwrapping mode functions similarly to the frame transfer camera, and as a result, similarly high speed image acquisition is possible. The image acquisition speed of various models of these types of digital cameras is not standardized as with video cameras. For example, the Princeton Instru, a 1-frame transfer camera model listed in the Specific Equipment section above.
PentaMax line obtained from ments is about 1.5ms frame transfer
Have time

Alternatively, if the acquisition camera is not a frame transfer CCD or an interline CCD camera operating in overlap mode, the program module can be ported between different embodiments of the method. That is, before going to step 926, the program module waits until the exposed image of the current frame has been completely read, by step 924.
To execute. This path can be used, for example, when a full frame CCD camera constitutes the system's built-in camera.

The particular camera configuration gives an indication as to how a software interrupt is generated in step 914. The method of the present invention requires the camera controller or PCI interface card 225 to be capable of alerting the computer 211 during the transitional period after the end of exposure and the start of reading. In the case of a digital camera, the camera controller raises such an alert to the computer 211 by generating a software interrupt at the appropriate transition time. Different hair MR access makers will refer to transition times in different words. For example, Princeton Instruments and digital for scientific purposes
Roper Scientific, which manufactures Photometrics lines for cameras, calls this transition time the Beginning of Frame or BOF, and defines this as the beginning of reading.

Video camera conforming to RS-170 or CCIR monochrome or RGB specifications
In order to generate a software interrupt when the
A CI digitizer card (225 in Figure 1) or a frame grabber must be integrated into the system, and when the frame grabber has completely digitized the current frame, it cannot generate a signal in the form of a software interrupt. must not. Upon completion of the digitization of the frame, an event known in the art as a vertical blank or v-blank is generated. FlashBus MV-Pro frame grabber card
Integral Technologies, Inc. manufactures software when v-blanks occur.
Implements interrupts. Other manufacturers of frame grabber cards could also make software interrupts for this event. The method of the present invention can be used in a BOF of a digital camera or in a v of a video frame grabber.
-In the blank, using a software interrupt, which is generated as a signal to inform the computer 211 how the camera 330 completed one frame of exposure.

After the program module proceeds to step 926, the camera determines if the last frame was exposed. If so, the camera proceeds to step 928.
At, the acquisition ends. If not, the camera continues and exposes the next frame. Variable Z Position and / or Wavelength and Software Interrupts With continued reference to FIG. 21, an important point to note in the method of the present invention is that step 916 and step 936 occur simultaneously. That is, as the camera controller transfers the exposed and digitized image to the memory buffer of computer 211 in step 916, the program module executes the routine shown in FIG. In step 918, the software interrupt handler increments the frame number, which is a description of the program module. When a software interrupt is received from the camera controller or the video frame grabber in step 914, the software interrupt handler interrupts the processing of the subroutine 933 of the system computer 211 through the path 919 and causes the computer to increment the frame number by 1. Notice. Step 93
At 2, in updating the frame number, the program module proceeds to step 936, which is the execution of the routine of FIG.

In summary, step 936 shown in FIG. 21 varies the Z position and / or wavelength performed by computer 211 while camera 330 is performing steps 916-924 of FIG. It consists of the routine shown in FIG. That step 936 is performed concurrently with steps 916-924 constitutes an asynchronous operation of the camera with an external device.

In FIG. 22, in step 952, the program module inquires whether the system changes wavelength during acquisition. If not, the program module proceeds to step 964 to determine if the Z position changes during acquisition.

If the system is configured to tunable wavelengths, under the embodiment of stream acquisition 702 shown in FIG. 12, or performing multidimensional acquisition 802 shown in FIGS. 17-19. Under morphology, in step 954, the program module inquires whether the illumination wavelength of the last frame was the last one listed in the wavelength table. Otherwise, in steps 956 and 960, the program module drives the wavelength tunable devices 316 and / or 320 to switch the wavelength to the next one listed in the wavelength table. At this point, the method returns to step 938 of FIG. 21, at which point the program module should perform a new exposure at the changed wavelength, or
Determine if the acquisition should end.

Alternatively, if the wavelength of the last frame is the last one in the wavelength table, in steps 958 and 962, the program module causes the wavelength tunable device 3 to operate.
Drive 16 and / or 320 to switch to the wavelength listed first in the table. This means that at a particular Z position, each selected wavelength illuminates the image in a different frame.

At this point, the method proceeds to step 964, where the program module
Queries if the position changes during the acquisition of this stack of images. If not, then in step 968 the method returns to 938 of FIG. 21 and the program module determines if the last frame was acquired.

If the Z position changes during this acquisition event, in step 966,
The program module determines whether to change the Z position value. If the objective 324 or stage 326 is already at the last value in the list of Z position tables, then in step 972 the program module causes the next Z
Reset the position to the first value in the table. Alternatively, if the last value in the Z position table has not been reached, the program module increments the Z position value by 1 in step 970. In step 974, the program module drives the piezofocuser 340 or the stage mover 336 to move it to the next Z position. In step 976, the method returns to step 938 of FIG.

Here, the program module determines whether this is the last frame.
Otherwise, the program module increments the frame number by 1 in step 932 and reenters the routine of FIG. 22 in step 936 to change the wavelength and / or Z position again. The routine of FIG. 22 continues until the program module determines in step 938 that the last frame has been exposed.

As described above with respect to the user input multi-dimensional 802 embodiment, the researcher can choose to acquire a collection of images at different times. If a different time sequence is selected, as indicated by 3 in field 830 of FIG. 15, the program module will generate a number of frames equal to the number of Z positions times the number of wavelengths times the number of time points. calculate. And continuing the above example, 2
In the case of 3 Z positions, 3 wavelengths, and 3 time points, the total number of frames is equal to 23 × 3 × 3 or 207 frames.

In step 940, the program module ends the acquisition routine,
The method proceeds to step 524 in FIG. In the description of FIG. 5, as discussed above, the method proceeds to steps 524-530, closes the external shutter if necessary, copies the stack of images from temporary to semi-permanent memory, and stores temporary memory. Empty and complete the method. Post-Acquisition Processing After all images have been acquired and stored in semi-permanent memory, the method ends. However, after the end of the method, the investigator will process the acquired stack in a variety of known ways with appropriate application software to gain insights previously unavailable. For example, a very important and valuable way of processing the acquired stack is to play back an uninterrupted sequence of images, i.e. display as a "movie" showing changes in the focal plane and illuminating light as a continuum. It is to be.

FIG. 23 shows the review multidimensional data 1100 of MetaMorph (TM).
FIG. 6 is an example of a user dialog, a software application that processes a stack of images that allows the user to choose to display the acquired stack of images as a continuous stream, or movie. By clicking the Select Base File 1102 button in FIG. 23, the researcher can select the file containing the required stack of images to be processed. 14
Recall that, in field 824 of, a researcher can enter an identifiable file name for an acquisition event by using the method of the present invention. Upon selecting a file, the researcher can request in the wavelength 1104 box to display the selected file of images as illuminated at a wavelength. Here, as indicated at 1104, the researcher selects the DAPI, FITC and RHO selected in FIGS.
Any or all of these illumination wavelengths, labeled D, can be checked. Each checked wavelength, shown at 1104, appears in that window.

The Z-table 1106 is a two-dimensional array of all the frames acquired in different time series at the selected Z position. The number of columns shown in table 1106 is equal to the number of time series entered by the user. Field 83 of FIG.
As shown in 0, the number of time series is 3, which is table 1106.
Corresponds to the number of columns in. The number of rows in the table 1106 corresponds to the number of Z positions entered in the field 852 in FIG. 16, where there was 23 as an example. Therefore, column 1 of table 1106 contains 23 Z's acquired during the first time series.
Position, column 2 represents the 23 Z positions acquired during the second time series, and so on.

To view each frame acquired by illuminating with one of the wavelengths checked in box 1104 at a particular Z position and in a particular time series, the researcher should
Click on a particular cell at 106. The image corresponding to that Z position of the time series for each wavelength checked at 1104 is displayed in box 1122. For example, highlighted cell 1120 corresponds to all checked wavelengths in the fifth Z position of the second time series.

To view a movie at all Z positions in a time series, the researcher would
For example, highlight the cell in column 1 of the array and click the appropriate arrow button 1108 to display the 23 Z positions of the first time series in the forward or backward direction. To see an image of a Z position over time, the researcher would highlight a cell, for example cell 5120 at the fifth Z position, and click on the appropriate arrow button at 1112. Three images at the Z position # 5 in three time series are displayed in the front and rear directions.

When the load image 1114 button is clicked, all selected frames are related as a subset of the stack originally acquired. in this way,
The subset stack is played back as a movie to see the change over time of its parameters. More importantly, Select Best Focus 111
By clicking the 6 button, the researcher autofocuses all Z-position images in a time series to determine which position, or which focal plane, contains the most focused image. The algorithm can be activated. When the algorithm finds the most in-focus location, an "X" is placed in that location, as shown at 1120. Automatic focusing is 1120
, 1124, and 1126, until the table of best focus positions for each time series is generated. The researcher can use the appropriate buttons at 1112 to view these frames, or click 1114 to assemble the subset stacks at these frames and position the most focused position over time. Play back the movie.

This discussion of post-acquisition processing of stacks of frames acquired using the present invention does not describe the claimed components of the present invention, but at the cellular level that researchers could not previously do. It was included to demonstrate how the present invention provides a previously unknown class of image sets that can be processed in known ways to generate knowledge of biological events in.

Although the present invention has been particularly shown and described with reference to particular embodiments, various modifications in form and detail are departing from the spirit and scope of the invention by those skilled in the art. It will be understood what can be done.

[Brief description of drawings]

FIG. 1 is a block diagram of an automated optical microscopy system that provides an operating environment for an exemplary embodiment of the invention.

FIG. 2 is a schematic representation of an exemplary hardware connection configuration between an exemplary embodiment of an information processing subsystem and a microscope subsystem.

FIG. 3 is a schematic diagram of an exemplary microscope subsystem used to implement the method of the present invention.

FIG. 4 is a schematic representation of an exemplary microscope subsystem used to implement the method of the present invention.

[Figure 5]   3 is a flow chart illustrating an exemplary embodiment of the overall method of the present invention.

FIG. 6 illustrates an exemplary user interface dialog for installing a camera in the system of the present invention.

FIG. 7 illustrates an exemplary user interface dialog for building parameters associated with the installed camera of FIG.

FIG. 8 illustrates an exemplary user interface dialog for installing a microscope external device with varying image acquisition parameters in the system of the present invention.

FIG. 9 illustrates an exemplary user interface dialog that configures an objective lens placer installed in the system of the present invention.

FIG. 10 illustrates an Acquire user interface dialog of a stream acquisition embodiment of the method of the present invention.

FIG. 11 illustrates a focus user interface dialog of a stream acquisition embodiment of the present invention for a user to enter start and end focus positions for an installed objective lens dispositioner.

FIG. 12 shows a wavelength user interface dialog of a stream acquisition embodiment of the present invention in which the user inputs the wavelength used to illuminate the sample during image acquisition.

FIG. 13 is a diagram showing a camera parameter user interface dialog of the stream acquisition embodiment of the present invention.

FIG. 14 is a diagram illustrating a main user interface dialog of a multidimensional acquisition embodiment of the present invention.

FIG. 15 illustrates a Time Lapse User Interface dialog of a multi-dimensional acquisition embodiment of the present invention in which the user enters the number of times to acquire an image with the method of the present invention at any acquisition event.

FIG. 16 illustrates a Z-series user interface dialog of a multidimensional acquisition embodiment of the present invention in which a user inputs a value to change the Z position while performing the method of the present invention.

FIG. 17 shows a wavelength user interface dialog of a multidimensional acquisition embodiment of the present invention in which a user inputs a value for changing the wavelength while performing the method of the present invention.

FIG. 18 shows a wavelength user interface dialog of a multi-dimensional acquisition embodiment of the present invention in which a user inputs a value for changing the wavelength while performing the method of the present invention.

FIG. 19 shows a wavelength user interface dialog of a multi-dimensional acquisition embodiment of the present invention in which a user inputs a value for changing the wavelength while performing the method of the present invention.

FIG. 20 is a diagram illustrating a stream user interface dialog of a multidimensional acquisition embodiment of the present invention.

FIG. 21 is a flow chart illustrating an exemplary embodiment of the steps of an Acquire routine of the present invention in which a camera operates at or near the maximum rate of image acquisition while an external device is operating asynchronously with the camera. Is.

FIG. 22 is a flow chart illustrating an exemplary embodiment of the method steps of the present invention for moving an external device to change image acquisition parameters while the external device is operating asynchronously with the camera.

FIG. 23 shows a review multi-dimensional data user interface dialog of an exemplary image processing software application that allows a user to play back a sequence of images created by the method of the present invention as a continuous stream or movie. is there.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Petterson, William             Pennsylvania, United States             19380, West Chester, Shaw             Dead Drive 1329 (72) Inventor Cohen, Abram             Pennsylvania, United States             19355, Malvern, Charlestong             Linee 812 (72) Inventor Sujastad, Michael, Dee.             California, United States             94301, Palo Alto, Guindustri             525 (72) Inventor Stucky, Jeffrey, A ..             Pennsylvania, United States             19403, Jeffersonville, East             Indian lane 28 F term (reference) 2H052 AC04 AD03 AD16 AF00 AF14                       AF25                 5C022 AA01 AA08 AB15 AC42 AC52                       AC69                 5C053 FA09 FA11 FA27 GB06 GB10                       KA04 KA24 LA02 LA11                 5C054 AA01 CH02 EA01 EA05 EA07                       EB05 ED07 FC15 FF02 GA04                       GB11 HA05

Claims (26)

[Claims] 1. A method of acquiring an image using an optical microscope system, comprising: a) a camera, a microscope, an information processing system and at least one external to the microscope operable to vary at least one acquisition parameter. Equipped with one device,
Assembling an optical microscope system; b) acquiring an image at a speed substantially close to the maximum image acquisition speed of the camera; and c) substantially acquiring a maximum image acquisition speed of the camera during image acquisition. Driving the at least one system unit at a near speed, modifying at least one image acquisition parameter, the acquired image displaying a change in at least one image acquisition parameter, and d) the acquired image. Is stored as at least one stack of digitized data images, and the assembly of the optical microscope system comprises: (i) initializing the system, inputting the image acquisition rate of the camera, the external device Enter the speed at which you want to work, changing the image acquisition parameters And (ii) initializing a range of values in which the image acquisition parameter changes during image acquisition, (iii) the operating speed of the external device in the change of the image acquisition parameter, and the camera Comparing speeds, and whether the assembled system can acquire images at or near the acquisition speed of the camera while the external device varies the image acquisition wave and other meters. And (iv) the system is a stack of digitized images acquired while the system unit is operating to vary the image acquisition parameters over the range of values. Determining whether or not it has sufficient memory to store. 2. A method for image acquisition using an optical microscope system, comprising: a) a camera, a microscope, an information processing system and at least one external to the microscope operative to vary at least one acquisition parameter. With one device,
Assembling an optical microscope system; b) acquiring an image at a speed substantially close to the maximum image acquisition speed of the camera; and c) substantially acquiring a maximum image acquisition speed of the camera during image acquisition. Driving the at least one system unit at a near speed, modifying at least one image acquisition parameter, the acquired image displaying a change in at least one image acquisition parameter, and d) the acquired image. Is stored as at least one stack of digitized data images, and the assembly of the optical microscope system comprises: (i) initializing the system, inputting the image acquisition rate of the camera, the external device Enter the speed at which you want to work, changing the image acquisition parameters And (ii) initializing a range of values for which the image acquisition parameter changes during image acquisition, (iii) initializing the time at which the image is acquired, and (iv) image The operating speed of the external device in the change of the acquisition parameter,
Compare the speeds of the cameras and the assembled system acquires images at or near the acquisition speed of the camera while the external device is varying the image acquisition wave and other meters. (V) the system is digitized acquired while the system unit is operating to vary the image acquisition parameters over the range of values. Determining whether or not it has sufficient memory to store a stack of images, the method comprising: 3. A computer program product in a computer-readable medium having instructions for image acquisition using an automated optical microscope system, the camera, microscope, information processing system and at least one acquisition parameter being variable. And an instruction in the computer-readable medium for assembling an optical microscope system, comprising at least one device external to the microscope, and acquiring images at a speed substantially close to the maximum image acquisition speed of the camera. Driving the at least one system unit and changing at least one image acquisition parameter at a speed substantially close to the maximum image acquisition speed of the camera during the acquisition of the instructions in the computer readable medium and the image. And the captured image contains at least one image. The instructions in the computer-readable medium for displaying a change in a capture parameter and the instructions in the computer-readable medium for storing the captured image as at least one stack of digitized data images. The instruction for assembling the optical microscope system is to initialize the system, input the image acquisition speed of the camera, and input the speed at which the external device operates to change the image acquisition parameter. During the acquisition, a command for initializing a range of values for which the image acquisition parameter changes, the operating speed of the external device in the change of the image acquisition parameter, and the speed of the camera are compared, and the assembled system is , While the external device is changing the image acquisition wave and other meters, An instruction to determine whether an image can be acquired at or near the acquisition rate, and the system operates such that the system unit varies the image acquisition parameters over the range of values. A computer program product, comprising: instructions for determining whether or not it has sufficient memory to store a stack of digitized images acquired during the operation. 4. A computer program product in a computer-readable medium having instructions for image acquisition using an automated optical microscope system, the camera, microscope, information processing system and at least one acquisition parameter being variable. And an instruction in the computer-readable medium for assembling an optical microscope system, comprising at least one device external to the microscope, and acquiring images at a speed substantially close to the maximum image acquisition speed of the camera. Driving the at least one system unit and changing at least one image acquisition parameter at a speed substantially close to the maximum image acquisition speed of the camera during the acquisition of the instructions in the computer readable medium and the image. And the captured image contains at least one image. The instructions in the computer-readable medium for displaying a change in a capture parameter and the instructions in the computer-readable medium for storing the captured image as at least one stack of digitized data images. The instruction for assembling the optical microscope system is to initialize the system, input the image acquisition speed of the camera, and input the speed at which the external device operates to change the image acquisition parameter. An instruction for initializing a range of values for which the image acquisition parameter changes during acquisition, an instruction for initializing a time at which an image is acquired, an operation speed of the external device when the image acquisition parameter changes, and Comparing camera speeds, the assembled system is An instruction to determine whether an image can be acquired at or near the acquisition speed of the camera while the acquisition wave and other meters are being varied; An instruction to determine whether it has sufficient memory to store a stack of digitized images acquired while operating to vary the image acquisition parameters over a range of values; A computer program product characterized by: 5. An automated optical microscope system programmed to include a computer program product for performing the steps of claim 1, comprising: a microscope, a camera, an information processing system comprising a computer and memory, and at least one of: At least one means external to the microscope for varying the image acquisition parameters, and a system comprising: 6. The system of claim 5, wherein the acquired image is stored in random access memory. 7. The system of claim 5, wherein the captured image is stored on a real-time hard disk. 8. The system of claim 5, wherein the captured image is stored on a computer-readable medium. 9. The system according to claim 5, wherein the acquired image is stored in a server to which the information processing system is connected. 10. Processing of the digitized stack is performed by a computerized image processing method for acquiring and processing images of fluorescently colored cellular elements, the computerized method comprising: And a) a camera, a microscope, an information processing system, and at least one device external to the microscope operative to vary at least one acquisition parameter, the method comprising:
Assembling an optical microscope system; b) acquiring an image at a speed substantially close to the maximum image acquisition speed of the camera; and c) substantially acquiring a maximum image acquisition speed of the camera during image acquisition. Driving the at least one system unit at a near speed, modifying at least one image acquisition parameter, the acquired image displaying a change in at least one image acquisition parameter, and d) the acquired image. Is stored as at least one stack of digitized data images, and the assembly of the optical microscope system comprises: (i) initializing the system, inputting the image acquisition rate of the camera, the external device Enter the speed at which you want to work, changing the image acquisition parameters And (ii) initializing a range of values in which the image acquisition parameter changes during image acquisition, (iii) the operating speed of the external device in the change of the image acquisition parameter, and the camera Comparing speeds, and whether the assembled system can acquire images at or near the acquisition speed of the camera while the external device varies the image acquisition wave and other meters. And (iv) the system is a stack of digitized images acquired while the system unit is operating to vary the image acquisition parameters over the range of values. 6. The system of claim 5, comprising: determining if it has sufficient memory to store. 11. The system according to claim 5, wherein the device external to the microscope means comprises means for varying the vertical position of the objective lens of the microscope. 12. The system of claim 5, wherein a device external to the microscope comprises means for varying the emission wavelength at which the image is acquired. 13. The system of claim 5, wherein a device external to the microscope comprises means for varying the excitation wavelength at which the image is acquired. 14. The system of claim 5, wherein a device external to the microscope comprises means for varying the vertical position of the microscope stage on which an image is acquired. 15. The system of claim 5, wherein all devices external to the microscope vary the value of their respective acquisition parameters while the camera is acquiring an image. 16. An automated optical microscope system programmed to include a computer program product for performing the steps of claim 2, comprising: a microscope, a camera, an information processing system comprising a computer and memory, and at least one of: At least one means external to the microscope for varying the image acquisition parameters, and a system comprising: 17. The system of claim 16, wherein the captured image is stored in random access memory. 18. The system of claim 16, wherein the captured image is stored on a real-time hard disk. 19. The system of claim 16, wherein the captured image is stored on a computer-readable medium. 20. The system according to claim 16, wherein the acquired image is stored in a server to which the information processing system is connected. 21. The processing of the digitized stack is performed by a computerized image processing method of acquiring and processing images of fluorescently colored cell elements, the computerized method comprising: And a) a camera, a microscope, an information processing system, and at least one device external to the microscope operative to vary at least one acquisition parameter, the method comprising:
Assembling an optical microscope system; b) acquiring an image at a speed substantially close to the maximum image acquisition speed of the camera; and c) substantially acquiring a maximum image acquisition speed of the camera during image acquisition. Driving the at least one system unit at a near speed, modifying at least one image acquisition parameter, the acquired image displaying a change in at least one image acquisition parameter, and d) the acquired image. Is stored as at least one stack of digitized data images, and the assembly of the optical microscope system comprises: (i) initializing the system, inputting the image acquisition rate of the camera, the external device Enter the speed at which you want to work, changing the image acquisition parameters And (ii) initializing a range of values for which the image acquisition parameter changes during image acquisition, (iii) initializing the time at which the image is acquired, and (iv) image The operating speed of the external device in the change of the acquisition parameter,
Compare the speeds of the cameras and the assembled system acquires images at or near the acquisition speed of the camera while the external device is varying the image acquisition wave and other meters. (V) the system is digitized acquired while the system unit is operating to vary the image acquisition parameters over the range of values. 17. The system of claim 16, comprising: determining if it has sufficient memory to store a stack of images. 22. The system of claim 16, wherein a device external to the microscope comprises means for varying the vertical position of the objective lens from which the image is acquired. 23. The system of claim 16, wherein a device external to the microscope comprises means for varying the emission wavelength at which the image is acquired. 24. The system of claim 16, wherein a device external to the microscope comprises means for varying the excitation wavelength at which the image is acquired. 25. The system of claim 16, wherein a device external to the microscope comprises means for varying the vertical position of the microscope stage on which an image is acquired. 26. The system of claim 17, wherein all devices external to the microscope vary the values of their respective acquisition parameters while the camera is acquiring an image.