KEYWORDS: Deconvolution, Point spread functions, Image resolution, Super resolution, Signal to noise ratio, Resolution enhancement technologies, Visibility, Confocal microscopy, Image enhancement, Optical transfer functions
Significance: It is commonly assumed that using the objective lens to create a tightly focused light spot for illumination provides a twofold resolution improvement over the Rayleigh resolution limit and that resolution improvement is independent of object properties. Nevertheless, such an assumption has not been carefully examined. We examine this assumption by analyzing the performance of two super-resolution methods, known as image scanning microscopy (ISM) and illumination-enhanced sparsity (IES).
Aim: We aim to identify the fundamental differences between the two methods, and to provide examples that help researchers determine which method to utilize for different imaging conditions.
Approach: We input the same image datasets into the two methods and analyze their restorations. In numerical simulations, we design objects of distinct brightness and sparsity levels for imaging. We use biological imaging experiments to verify the simulation results.
Results: The resolution of IES often exceeds twice the Rayleigh resolution limit when imaging sparse objects. A decrease in object sparsity negatively affects the resolution improvement in both methods.
Conclusions: The IES method is superior for imaging sparse objects with its main features being bright and small against a dark, large background. For objects that are largely bright with small dark features, the ISM method is favorable.
Although light microscopes play a pivotal role in biological discoveries, their inability to observe the workings of the tiny structures within cells – whose size fall below the classical resolution barrier – continues to frustrate biologists and optical engineers alike. It is generally assumed that the only approach to breaking the classical resolution barrier is through manipulating the fluorophores by their excitation processes, which limits their implementation to a few specific biological applications. Here, we present a new path to achieving super-resolution imaging of three-dimensional objects without the requirements of complex optics or special fluorophores; it can provide more than 3 times resolution improvement in all three dimensions. We demonstrate it on various biological samples: Drosophila brain neurons and mouse brain dendrites.
KEYWORDS: Super resolution, Point spread functions, Microscopes, Performance modeling, Super resolution microscopy, Image processing, Microscopy, Signal to noise ratio, Image acquisition, Biomedical optics and medical imaging, Image processing algorithms and systems, Digital image processing, Optimization (mathematics)
We report progress in algorithm development for a computation-based super-resolution microscopy technique. Building upon previous results, we examine our recently implemented microscope system and construct alter- native processing algorithms. Based on numerical simulations results, we evaluate the performance of each algorithm and determine the one most suitable for our super-resolution microscope.
We demonstrate a new method for two-dimensional super-resolution fluorescence imaging. Our method achieves more than 2-fold resolution enhancement over the Rayleigh resolution limit simply with a focused illumination spot and non-negative least-squares inversions, and with practical photon budgets that can be supported by common fluorophores used for biological fluorescence microscopy.
We report a novel microscopy technique, utilizing our previously reported expanded point information content (EPIC) concept [1], to extend the technique into the coherent regime. Preliminary data shows coherent EPIC (CoEPIC) can image reflective samples successfully, and can recover the 3D structure without the need to acquire an image stack at multiple depths. Numerical simulation demonstrates the potential super-resolution capability of CoEPIC.
Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.
The most concerned factors for cancer prognosis are tumor invasion and metastasis. The patterns of tumor invasion can
be characterized as random infiltration to surrounding extracellular matrix (ECM) or formation of long-range path for
collective migration. Recent studies indicate that mechanical force plays an important role in tumor infiltration and
collective migration. However, how tumor colonies develop mechanical interactions with each other to initiate various
invasion patterns is unclear. Using a micro-patterning technique, we partition cells into clusters to mimic tumor colonies
and quantitatively induce colony-ECM interactions. We find that pre-malignant epithelial cells, in response to
concentrations of type I collagen in ECM ([COL]), develop various branching patterns resembling those observed in
tumor invasion. In contrast with conventional thought, these patterns require long-range (~ 600 μm) transmission of
traction force, but not biochemical factors. At low [COL], cell colonies synergistically develop pairwise and directed
branching mimicking the formation of long-range path. By contrast, at high [COL] or high colony density, cell colonies
develop random branching and scattering patterns independent of each other. Our results suggest that tumor colonies
might select different invasive patterns depending on their interactions with each other and with the ECM.
Collagen denaturation is of fundamental importance for clinical treatment. Conventionally, the denaturation process
is quantified by the shrinkage of collagen fibers, but the underlying molecular origin has not been fully understood.
Since second harmonic generation (SHG) is related to the molecular packing of the triple helix in collagen fibers,
this nonlinear signal provides an insight of molecular dynamics during thermal denaturation. With the aid of SHG
microscopy, we found a new step in collagen thermal denaturation process, de-crimp. During the de-crimp step, the
characteristic crimp pattern of collagen fascicles disappeared due to the breakage of interconnecting bonds between
collagen fibrils, while SHG intensity remained unchanged, suggesting the intactness of the triple helical molecules.
At higher temperature, shrinkage is observed with strongly reduced SHG intensity, indicating denaturation at the
molecular level.
Confocal scanning laser ophthalmoscope (CSLO) has been established to be an important diagnostic tool for
retinopathies like age-related macular degeneration, glaucoma and diabetes. Compared to a confocal laser
scanning microscope, CSLO is also capable of providing optical sectioning on retina with the aid of a pinhole,
but the microscope objective is replaced by the optics of eye. Since optical spectrum is the fingerprint of local
chemical composition, it is attractive to incorporate spectral acquisition into CSLO. However, due to the
limitation of laser bandwidth and chromatic/geometric aberration, the scanning systems in current CSLO are
not compatible with spectral imaging. Here we demonstrate a spectral CSLO by combining a
diffraction-limited broadband scanning system and a supercontinuum laser source. Both optical sectioning
capability and sub-cellular resolution are demonstrated on zebrafish's retina. To our knowledge, it is also the
first time that CSLO is applied onto the study of fish vision. The versatile spectral CSLO system will be
useful to retinopathy diagnosis and neuroscience research.
To obtain specific biochemical information in optical scanning microscopy, labeling technique is routinely required. Instead of the complex and invasive sample preparation procedures, incorporating spectral acquisition, which commonly requires a broadband light source, provides another mechanism to enhance molecular contrast. But most current optical scanning system is lens-based and thus the spectral bandwidth is limited to several hundred nanometers due to anti-reflection coating and chromatic aberration. The spectral range of interest in biological research covers ultraviolet to infrared. For example, the absorption peak of water falls around 3 μm, while most proteins exhibit absorption in the UV-visible regime. For imaging purpose, the transmission window of skin and cerebral tissues fall around 1300 and 1800 nm, respectively. Therefore, to extend the spectral bandwidth of an optical scanning system from visible to mid-infrared, we propose a system composed of metallic coated mirrors. A common issue in such a mirror-based system is aberrations induced by oblique incidence. We propose to compensate astigmatism by exchanging the sagittal and tangential planes of the converging spherical mirrors in the scanning system. With the aid of an optical design software, we build a diffraction-limited broadband scanning system with wavefront flatness better than λ/4 at focal plane. Combined with a mirror-based objective this microscopic system will exhibit full spectral capability and will be useful in microscopic imaging and therapeutic applications.
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