An autofocus circuit, based on measurement of the high spatial frequency image components, was designed for automated microscopic scanning of biological specimens. By careful consideration of the system transfer function, elimination of video end-of-line filter artifacts, correction for illumination instability, and incorporation of autogain, the focus measurement circuit attained the sensitivity and dynamic range necessary for robust operation even at the extremes of biological specimen detail encountered in exhaustive raster scans of thousands of fields. This new circuit exhibited a 25-fold improvement in dynamic range over a previous analog implementation, matched real-time digital performance at an order of magnitude lower cost, resulted in autofocus precision of 56 nm (or 10-fold better than the depth of field of the objective) in scanning experiments comprising over 10 000 microscope fields, and tracked focus at scanning speeds of up to 3.45 fields/s. Focus was correctly maintained in these scanning experiments without additional compensation for low-detail images. This circuit makes possible the widespread inclusion of high-performance autofocus as a low cost option in video microscopy systems.
Autofocus functions based on measurement of image resolution appear to be precise and robust for biological microscopy. However, the through-focus response of these functions previously exhibited unwanted local maxima, or side peaks. Here we report theoretical and experimental studies showing that side peaks are mainly a result of contrast reversals inherent in optical systems at mid-range frequencies. These contrast reversals are not present in frequencies near optical cutoff. Contrast reversals thus limit the lower cutoff for resolution measurement filters, whereas signal-to-noise limits the upper cutoff. These improved bandpass design criteria led to sharp, unimodal autofocus responses for all tested microscopy specimens.
Efficient image cytometry of a conventional microscope slide means rapid acquisition and analysis of 20 gigapixels of image data (at 0.3-microm sampling). The voluminous data motivate increased acquisition speed to enable many biomedical applications. Continuous-motion time-delay-and-integrate (TDI) scanning has the potential to speed image acquisition while retaining sensitivity, but the challenge of implementing high-resolution autofocus operating simultaneously with acquisition has limited its adoption. We develop a dynamic autofocus system for this need using: 1. a "volume camera," consisting of nine fiber optic imaging conduits to charge-coupled device (CCD) sensors, that acquires images in parallel from different focal planes, 2. an array of mixed analog-digital processing circuits that measure the high spatial frequencies of the multiple image streams to create focus indices, and 3. a software system that reads and analyzes the focus data streams and calculates best focus for closed feedback loop control. Our system updates autofocus at 56 Hz (or once every 21 microm of stage travel) to collect sharply focused images sampled at 0.3x0.3 microm(2)/pixel at a stage speed of 2.3 mms. The system, tested by focusing in phase contrast and imaging long fluorescence strips, achieves high-performance closed-loop image-content-based autofocus in continuous scanning for the first time.
Relevant to mobile health, the design of a portable electrocardiograph (ECG) device using AD823X microchips as the analog front-end is presented. Starting with the evaluation board of the chip, open-source hardware and software components were integrated into a breadboard prototype. This required modifying the microchip with the breadboard-friendly Arduino Nano board in addition to a data logger and a Bluetooth breakout board. The digitized ECG signal can be transmitted by serial cable, via Bluetooth to a PC, or to an Android smartphone system for visualization. The data logging shield provides gigabytes of storage, as the signal is recorded to a microSD card adapter. A menu incorporates the device’s several operating modes. Simulation and testing assessed the system stability and performance parameters in terms of not losing any sample data throughout the length of the recording and finding the maximum sampling frequency; and validation determined and resolved problems that arose in open-source development. Ultimately, a custom printed circuit board was produced requiring advanced manufacturing options of 2.5 mils trace widths for the small package components. The fabricated device did not degrade the AD823X noise performance, and an ECG waveform with negligible distortion was obtained. The maximum number of samples/second was 2380 Hz in serial cable transmission, whereas in microSD recording mode, a continuous ECG signal for up to 36 h at 500 Hz was verified. A low-cost, high-quality portable ECG for long-term monitoring prototype that reasonably complies with electrical safety regulations and medical equipment design was realized.
With the typically narrow depth-of-field microscope optics, biological specimens do not lie in a single focal plane across the slide and this complicates automated scanning for image cytometry. An on-the-fly autofocus system for high-resolution image cytometry is presented which keeps the image sharply focused during continuous stage travel. To track possible foci, an image volume is acquired by concurrent optical sectioning of the specimen with a dedicated imaging array. This volume scanning camera was designed for adjustment of the optical path lengths to allow simple adaptation to objectives with different depths-of-field and magnifications. The computational demand of calculating and adjusting focus dynamically is absorbed by an array of parallel autofocus circuits that measure the 3D image in real time. In conventional optical sectioning microscopy, where the image data is acquired by sequential sectioning, a priori knowledge of the specimen and its boundaries exists. In continuous volume scanning, this is usually not the case and variations in specimen thickness and information content are routine. This makes implementation of fully automatic, high-speed, high-resolution image cytometry a challenge. A model of the combined specimen and optical system was developed to evaluate strategies for tracking focus. Data from this model is described along with the volume scanning camera array design.
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