Impact of the optical depth of field on cytogenetic image quality

Abstract

Abstract. In digital pathology, clinical specimen slides are converted into digital images by microscopic image scanners. Since random vibration and mechanical drifting are unavoidable on even high-precision moving stages, the optical depth of field (DOF) of microscopic systems may affect image quality, in particular when using an objective lens with high magnification power. The DOF of a microscopic system was theoretically analyzed and experimentally validated using standard resolution targets under 60× dry and 100× oil objective lenses, respectively. Then cytogenetic samples were imaged at in-focused and off-focused states to analyze the impact of DOF on the acquired image qualities. For the investigated system equipped with the 60× dry and 100× oil objective lenses, the theoretical estimation of the DOF are 0.855 μm and 0.703 μm, and the measured DOF are 3.0 μm and 1.8 μm, respectively. The observation reveals that the chromosomal bands of metaphase cells are distinguishable when images are acquired up to approximately 1.5 μm or 1 μm out of focus using the 60× dry and 100× oil objective lenses, respectively. The results of this investigation provide important designing trade-off parameters to optimize the digital microscopic image scanning systems in the future.

Using Fourier ptychography microscopy to achieve high-resolution chromosome imaging: an initial evaluation

“…Accordingly, a high-resolution (up to ) USAF 1951 target (Newport, California, USA) was adopted, and the contrast of the imaged bar patterns was measured at a sequence of discrete frequencies from zero to the spatial resolution. 17 During the measurement, the test target was placed on the sample stage and adjusted to the in-focused position. For each pattern on the captured target, the contrast was calculated by 18 , where and are the average maximal and minimal values of the bar patterns, respectively.…”

“…The chromosome band patterns depicted on each generated image were assessed and compared between these two groups of results (i.e., FPM versus conventional system). To evaluate the impact of the DOF on the feature qualities, 17 each chromosome was imaged in a series of different positions, including the in-focused position and other positions outside the focal plane.…”

Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

“…Alternatively, one can manipulate the point-spread function to obtain information about the position of the molecule in the z axis ( i.e ., perpendicular to the imaging plane). In astigmatic imaging, a cylindrical lens is inserted into the emitted light path, distorting the symmetric point spread function (PSF) of dipole emission into an elliptical point-spread function (PSF) whose aspect ratio depends on the z position [142,143]. Another example of emission-beam engineering is the creation of a double-helix PSF.…”

“…Bacteria are small enough (typically 0.5–1 µm in diameter [ 36 ]) to fit entirely within the focal depth of even a high-NA light microscope (0.703 µm [ 141 ]). However, though bacteria are thin, they are not two-dimensional; the actual locations and motions of molecules inside a bacterium may appear distorted in two-dimensional images.…”

Impact of partial coherence on the apparent optical transfer function derived from the response to amplitude edges

“…It appears empirically that the Full Width Half Maximum (FWHM) of the through-focus transfer function Q ( q) corresponds to β ≈ √ 2. The use of a threshold on the through-focus MTF for defining a DOF metric has been proposed for incoherent imaging systems previously by Qiu et al [22]. The scaling of Eq.…”