In the pages of the Optica journal, the scientists presented a new method of microscopy, which, in theory, has no resolution limit. Oversampling does not lead to artifacts however, it does not increase the diffraction-limited spatial resolution data to the image.The Polish-Israeli team led by Radek Łapkiewicz from the Faculty of Physics of the University of Warsaw, winner of the FIRST TEAM programme of the Foundation for Polish Science, has made another significant achievement.Undersampling an image-using too few pixels to accurately describe a small feature- can yield artifacts that masquerade as real structures, which can lead to misinterpretation of the image data.The reason for 2.5-3x oversampling is to avoid image artifacts.Scientists studying live cells in time should also consider oversampling on the temporal scale to avoid artifacts.Loss of contrast can make it difficult to determine where the edges of an object are, and it can make resolving two closely adjacent objects nearly impossible. Higher levels of sampling (hyper-sampling) can yield increased accuracy of feature measurements (particularly for larger objects) however, there is often a resulting loss of contrast.Because adequate contrast is essential to correctly resolve structures in microscopy, a 2.5-3 times oversampling is more appropriate. The Nyquist sampling theorem suggests that a point object should be oversampled at least two times in X and Y. Another important issue with sampling small objects using digital image capture is the need to correctly oversample the object.Because of this problem, measurements of the size of objects that are at, or near, the diffraction limit are very suspect. Sub-resolution objects typically appear to be the same size as objects that are at the actual diffraction-limited resolution however, this is an artifact.Resolution is defined as the ability to separate two closely adjacent objects, and is limited by the diffraction of the objects. This does not mean that the microscope can resolve the structures, since visualizing sub-resolution structures only works if the objects are well separated from other objects in the image field.Microscopes can, in several imaging modes, visualize objects smaller than the diffraction-limited resolution of the instrument.The all-too-common practice of stating the magnification of a microscope objective in the figure legend-without taking into account other instrument optics and image processing-is sloppy science, and omits important information.Resizing the image makes any magnification number provided in the figure legend incorrect, whereas a scalebar resizes along with the image. Journals may resize a figure to fit the page.Since it is often impossible to know in advance what the final magnification will be, a scale bar of known size is the best way to express the magnification. The magnification of an image is determined by the difference between the original scale of the pixel and the scale of the pixel in its final form (e.g., paper printout, projected on the wall of a large lecture hall).It is imperative that the scale of the pixels in XY and Z be maintained so that features in the image can be correctly interpreted.The Z dimension in confocal microscopy is typically twice that of the XY resolution, an issue that can lead to misinterpretation if not accounted for. In confocal microscopy and other sectioning techniques, the XY pixel also represents a volume, because the image includes a Z dimension.Ideally, the scale is the same in both the X and Y dimensions however, this is not always the case.This scale may be in meters per pixel for satellite images, or in tenths of microns per pixel for microscope images. Digital images of real world objects sample the object such that each pixel in the image has a scale.Scientific digital images are data that can be compromised by inappropriate manipulations.
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