High-illumination numerical aperture-based large field-of-view high-resolution microimaging device and a method for iterative reconstruction

A high-illumination numerical aperture-based large field-of-view high-resolution microimaging iterative reconstruction method by using a high-illumination numerical aperture-based large field-of-view high-resolution microimaging device comprising:

• an LED array;
• a stage;
• a condenser;
• a microscopic objective;
• a tube lens;
• and
• a camera,
• wherein the LED array is arranged on a forward focal plane of the condenser, and a center of the LED array is on an optical axis of the microscope objective,
• wherein a back focal plane of the microscope objective coincides with a forward focal plane of the tube lens, an imaging plane of the camera is placed on a back focal plane of the tube lens, and during imaging, a specimen to be examined on the stage is adjusted to a front focal plane position of the microscope objective to form an infinity-corrected imaging system, and
• wherein light emitted by an i-th lit LED unit of the LED array passes through the condenser and converges to become parallel light illuminating the specimen to be examined, which is placed on the stage, and part of diffracted light passing through the specimen is collected by the microscopic objective, converged by the tube lens, and reaches the imaging plane of the camera, forming an intensity image recorded by the camera,
• said method comprising the steps of:
• calibrating a brightness of the LED unit, by using the LED array as an illumination source of the high-illumination numerical aperture-based large field-of-view high-resolution microimaging device, sequentially lighting up each LED unit of the LED array, and after illuminating a blank specimen, using high magnification objective lens to collect corresponding image and calculating a normalized brightness correction coefficient corresponding to each LED unit in three channels of red, green and blue;
• calibrating a position of the LED array, by using a resolution board as the specimen to be examined, using the LED array as the illumination source of the high-illumination numerical aperture-based large field-of-view high-resolution microimaging device, sequentially lighting up each LED unit of the LED array, after illuminating the specimen to be examined, in-focus images and defocus images with defocus distance h are respectively collected, then calculating an illumination angle corresponding to each of the three channels of red, green and blue for each LED unit by using a sub-pixel registration algorithm, and then determining the position of the LED array by using nonlinear regression;
• capturing raw images, by using the LED array as the illumination source of the high-illumination numerical aperture-based large field-of-view high-resolution micro-imaging device, sequentially lighting up each LED unit of the LED array, and after illuminating the specimen to be examined, collecting corresponding low-resolution raw images;
• pre-processing raw images, including threshold denoising and brightness correction, by firstly using an average value of a dark current noise of the camera as a threshold, performing threshold denoising on the captured low-resolution raw images to obtain denoised low-resolution images, and then dividing each denoised low-resolution image by the normalized brightness correction coefficient obtained in the step of calibrating the brightness of the LED unit, thereby obtaining brightness-corrected low-resolution images;
• initializing high-resolution images, by adding and averaging all the low-resolution bright-field images among the brightness-corrected low-resolution images, and then initializing amplitude and phase of the high-resolution images by up-sampling;
• performing an iterative reconstruction, wherein each of the brightness corrected low-resolution images is subjected to synthetic aperture calculation one by one in a frequency domain by using a pixel binning based Fourier ptychographic recovery method and an updating coefficient is gradually reduced, a cost function value is used as a criterion, and when the cost function value is less than a given threshold, the iteration is stopped, and the amplitude and phase of the high-resolution images are determined to be a final reconstructed large field-of-view high-resolution microscopic images;
• and
• performing a color fusion by repeating the step of initializing the high-resolution images and the step of performing the iterative reconstruction, respectively reconstructing high-resolution images of red, green and blue channels, and then synthesizing three reconstructed high-resolution images as the red, green and blue channels respectively of a final true color image.