Bright-field microscopy

Summary

Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes, and its simplicity makes it a popular technique. The typical appearance of a bright-field microscopy image is a dark sample on a bright background, hence the name. Light path light microscope The light path of a bright-field microscope is extremely simple, no additional components are required beyond the normal light-microscope setup. The light path therefore consists of:

a transillumination light source, commonly a halogen lamp in the microscope stand;
a condenser lens, which focuses light from the light source onto the sample;
an objective lens,

Official source

https://en.wikipedia.org/wiki/Bright-field_microscopy

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Modern histopathology workflows rely on the digitization of histology slides. The quality of the resulting digital representations, in the form of histology slide image mosaics, depends on various specific acquisition conditions and on the image processing steps that underlie the generation of the final mosaic, e.g. registration and blending of the contained image tiles. We introduce HISTOBREAST, an extensive collection of brightfield microscopy images that we collected in a principled manner under different acquisition conditions on Haematoxylin - Eosin (H&E) stained breast tissue. HISTOBREAST is comprised of neighbour image tiles and ensemble of mosaics composed from different combinations of the available image tiles, exhibiting progressively degraded quality levels. HISTOBREAST can be used to benchmark image processing and computer vision techniques with respect to their robustness to image modifications specific to brightfield microscopy of H&E stained tissues. Furthermore, HISTOBREAST can serve in the development of new image processing methods, with the purpose of ensuring robustness to typical image artefacts that raise interpretation problems for expert histopathologists and affect the results of computerized image analysis. H&E-stained fixed tissue slide specimen center dot breast carcinoma center dot histology images Technology Type(s) brightfield microscopy center dot histological assay Factor Type(s)Exposure center dot Gain center dot Gamma center dot magnification center dot image quality center dot image overlap center dot image mosaics Sample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data

2020

3D nanometrology of transparent objects by phase calibration of a basic bright-field microscope for multiple illumination apertures

Martinus Gijs, Daniel Migliozzi, Bingying Zhao

Optical retrieval of the structure of transparent objects at the nanoscale requires adapted techniques capable of probing their interaction with light. Here, we considered a method based on calibration of the defocusing with partially coherent illumination and explored its phase retrieval capability over a wide range of illumination angles. We imaged: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis,(2) custom-made nano-steps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis, and (3) human cancer cells deposited on a glass substrate to assess the results of the calibration on complex transparent 3-dimensional samples. We first verified the model prediction in the spatial frequency domain and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to0.5. Finally, we studied the dependence of the phase retrieval of a complex nanostructured object on the illumination aperture.

2020

Phase calibration of a basic bright-field microscope for 3D metrology of transparent samples at the nanoscale

Martinus Gijs, Daniel Migliozzi, Bingying Zhao

Optical retrieval of the structure of transparent objects at the nano-scale requires adapted methods capable of probing their interaction with suitable light. Scattering events, for instance, depend on the content and the arrangement of the medium encountered by the light along its propagation path. Physical models of scattering were used in the past to understand image formation and phase retrieval in transparent objects. Here, we considered one of them, which is based on the acquisition of defocused images obtained with partially coherent illumination, and explored its phase retrieval capability over a wide range of illumination angles. We used a basic transmission bright-field microscope with a bandpass filter and an adjustable illumination aperture. The bandpass filter was used to select a specific wavelength range to avoid light dispersion in the sample and maximize illumination capabilities. The adjustable illumination aperture was used to probe and assess the calibration over a wide range of illumination angles, which give access to different parts of the spatial frequency spectrum of the sample. We subsequently employed a computational algorithm to retrieve the local 3-dimensional phase-shift induced on the light field by the scattering through the sample. We imaged several types of samples to explore the calibration and the results in different experimental configurations. We employed: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis, (2) custom-made nanosteps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis. We first verified the model prediction in the spatial frequency domain for the scattering induced by our samples, and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to 0.5. This enabled us to calibrate a very simple optical system in a wide range of illumination angles to obtain quantitative 3D metrology of transparent samples at the nanoscale.

SPIE-INT SOC OPTICAL ENGINEERING2021

Phase calibration of a basic bright-field microscope for 3D metrology of transparent samples at the nanoscale

Martinus Gijs, Daniel Migliozzi, Bingying Zhao

SPIE-INT SOC OPTICAL ENGINEERING2021