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Two-Photon Microscopy
Two-photon microscopy is a technique that avoids the limitations of traditional fluorescence microscopy. Typical fluorescence microscopy involves using illumination of a specific wavelength in order to excite fluorophores within a sample. However, standard widefield epifluorescence imaging also collects fluorescence from outside the focal plane, resulting in background illumination and image degradation.
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Anatomy Of A Microscope
At its core, a typical microscope is essentially a box designed to hold two lenses in precise positions so that light can be accurately magnified from the sample to the detector. The first of these two lenses is the objective lens, which is located close to the sample, moves when the focus dial is turned and has useful information such as magnification written on its side.
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Filters
The light microscope remains one of the most used tools for research, particularly in the fields of biological or biophysical sciences and offers a means to observe the dynamics of cellular processes. A critical component of these methods lies in obtaining the best possible sample representation, whilst simultaneously minimizing specimen damage, artifacts, and uncertainty.
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Phase-Contrast Microscopy
Light microscopy offers a powerful technique for label-free imaging of biological samples such as cells. Label-free imaging is particularly well-placed for understanding more about cells as they are free of any modifications that could potentially alter structure, function or behavior.
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Mizar Tilt
Conventional fluorescence microscopy uses high-intensity light to illuminate the sample but this excites all fluorophores in the light path, not just the plane of interest. The result is that light emitted from outside the focal plane contributes to the image.
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OpenSPIM
Conventional fluorescence microscopy uses high intensity light to illuminate the sample but this excites all fluorophores in the light path, not just the plane of interest. The result is that light emitted from outside the focal plane contributes to the image.
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Analysis Programs For High Content Imaging
High-content imaging (HCI) involves a powerful imaging system paired with smart analysis software, in order to parse hundreds of thousands of dense images into quantifiable data. As HCI involves maximizing the data output, HCI experiments can involve imaging millions of cells with multi-parameter analysis, resulting in the need for efficient, often automated, smart specialized analysis software.
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Resolution and Numerical Aperture
An often-asked question in imaging is whether two objects are in the same or separate places. Resolution, the ability to tell two nearby features apart, is a key parameter of microscope optics that becomes more challenging at smaller length scales.
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Introduction To High Content Imaging
High content imaging (HCI) is an area of imaging where the aim is to maximize data capture. Any kind of imaging can be high-content if the objective is to obtain as much data as feasibly possible, regardless of imaging system, sample, magnification, fluorophores, and camera used. This makes specifics in HCI difficult to define, but in general, HCI involves performing normal imaging thousands or millions of times in order to maximize data capture effectively.
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Total Internal Reflection Fluorescence (TIRF) Microscopy
Fluorescence microscopy is a fundamental set of techniques in the life sciences for visualizing structures in living systems. Typically, a fluorescent molecule, either synthetic or biological, is associated with a structure of interest in a biological sample.
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Super-Resolution Radial Fluctuations (SRRF)
Some super-resolution data cannot be visualized directly, only after images are reconstructed and processed can they be displayed, such as in PALM and STORM. This processing requires specialized algorithms and analysis software, which must be capable of dealing with the large datasets of thousands of frames from 3D, fast-moving dynamic samples with hundreds of data points of varying densities.
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Intravital NIR Imaging
The group of Prof. Gousopoulos at the University Hospital Zurich is focused on researching lymphedema, a condition where lymphatic system dysfunction results in swelling in parts of the body. The group has established a mouse model in order to investigate this disease.