SignificanceFlexible endoscopes are essential for medical internal examinations. Digital endoscopes are connected to a video processor that can apply various operations to enhance the image. One of those operations is edge enhancement, which has a major impact on the perceived image quality by medical professionals. However, the specific methods and parameters of this operation are undisclosed and the arbitrary units to express the level of edge enhancement differ per video processor.AimObjectively quantify the level of edge enhancement from the recorded images alone, and measure the effect on sharpness and noise.ApproachEdge enhancement was studied in four types of flexible digital ear nose and throat endoscopes. Measurements were performed using slanted edges and gray patches. The level of edge enhancement was determined by subtracting the step response of an image without edge enhancement from images with selected settings of edge enhancement and measuring the resulting peak-to-peak differences. These values were then normalized by the step size. Sharpness was characterized by observing the normalized modulation transfer function (MTF) and computing the spatial frequency at 50% MTF. The noise was measured on the gray patches and computed as a weighted sum of variances from the luminance and two chrominance channels of the pixel values.ResultsThe measured levels were consistent with the level set via the user interface on the video processor and varied typically from 0 to 1.3. Both sharpness and noise increase with larger levels of edge enhancement with factors of 3 and 4 respectively.ConclusionsThe presented method overcomes the issue of vendors expressing the level of edge enhancement each differently in arbitrary units. This allows us to compare the effects, and we can start exploring the relationship with the subjectively perceived image quality by medical professionals to find substantiated optimal settings.
In Single Molecule Localization Microscopy (SMLM) emission spots are fitted with a Point Spread Function (PSF) model in order to find the position of the molecules. Recently Franke et al. [Nature Methods 2017] found that the use of a Gaussian PSF model can underestimate the photon count by up to 30%. In the presentation we elucidate the reasons for this underestimate. We show that it can be traced back to differences between the simplified Gaussian and the exact vectorial PSF, that takes all effects of high-NA, polarization, and interfaces between media into account. Especially spots captured under total internal reflection conditions show major deviations from the Gaussian spot shape. Deficiencies of other simplified PSF-models such as the low-NA scalar diffraction Airy distribution or the Gibson-Lanni model will be discussed too. Furthermore, we show a simulation study of the effects of aberrations on the photon count estimation. In particular, we will discuss the impact of spherical aberration due to refractive index mismatch. Finally, we show implementation issues and the impact on the fitting outcome of the use of the exact vectorial PSF model in combination with Maximum-Likelihood Estimation, building on the treatment of Smith et al. [Optics Express 2016].
KEYWORDS: Digital micromirror devices, Modulation transfer functions, Microscopes, Cameras, Luminescence, Diffraction, Microscopy, Confocal microscopy, Signal to noise ratio, Digital imaging
We present a versatile fluorescence microscope, built by complementing a conventional fluorescence microscope with a digital micro-mirror device (DMD) in the illumination path. Arbitrary patterns can be created on the DMD and projected onto the sample. This patterned illumination can be used to improve lateral and axial resolution over the resolution of a wide-field microscope, as well as to reduce the illumination dose. Different illumination patterns require different reconstruction strategies and result in an image quality similar to confocal or structured illumination microscopy. We focus on the optical design and characterization of a DMD-based microscope. Estimation of the optical quality of the microscope has been carried out by measuring the modulation transfer function from edge profiles. We have obtained optically sectioned images by applying multi-spot illumination patterns followed by digital pinholing. The sectioning capabilities of our DMD-based microscope were estimated from the dependence of the signal-to-background and signalto-noise ratios on the pitch of the projected multi-spot patterns and the size of the digital pinhole. In addition, we provide an outlook on the use of pseudo-random illumination patterns for achieving both sectioning and resolution enhancement.
Super-resolution microscopy often employs asynchronous localizations of many single fluorescent emitters achieving resolution below the diffraction limit. This family of techniques typically uses statistical switching of emitters between dark and bright fluorescent states. Here we investigate how imaging repeated activations cycles of the same emitter influences the achieved image resolution. Furthermore, we ask the questions how long such a typical bright emitting state should be and is there an optimal number of switching events if the measurement time is fixed. We find that longer measurement times and hereby imaging more activation cycles is always beneficial for the attained image resolution. In the case of a fixed measurement time it turns out that there is a trade-off between the number of cycles and the product of localization density and uncertainty.
We measured light scattered from gold nanoparticles with darkfield microscopy in order to perform single molecule detection based on tethered particle motion (TPM). This combination results in a signal to noise ratio of about 40 dB, which allowed us to use 80 nm diameter gold particles as reporters instead of the typically used polystyrene particles whose sizes are up to 1 µm. The particle size is crucial in TPM experiments as it can induce a volume-exclusion effect, which results in a stretching force acting on the DNA tether. This affects both the biophysical and statistical properties of the anchored DNA and hence the interpretation of the experimental data. We demonstrated that the gold nanoparticles and darkfield microscopy can be used to characterize the confined Brownian motion of dsDNA-tethered gold particles with a spatial precision of 3 nm. Physical parameters such as the spring constant of the tethered DNA fragment and the persistence length can be derived from the two dimensional (2D) (x, y) projected image data. We have applied this method to various MgCl2 and glycerol concentrations as a proof of principle.
We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence
microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light
source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS)
spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in
collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The
prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED
illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an
Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning
stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging
(FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible.
The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime
imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial
resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual
ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those
of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits
photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins
such as DRONPA.
Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular
interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal
with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been
tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We
further report on the derivation and use of several physical parameters and how these parameters change under differing
experimental conditions.
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