Widefield Microscopy

A widefield microscope is one type of Fluorescence Microscope. With the Widefield & Brightfield optical option in the Huygens software, widefield images can be deconvolved, and corrected for spherical aberration and blaeching issues. This leads to significantly sharper 2D-3D images in which objects are more clearly visible - even deep within very thick samples.

In a conventional widefield microscope (WFM), the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto an image capture device or photographic film. When fluorescent specimens are imaged using such a microscope, secondary fluorescence emitted by the specimen that appears away from the region of interest often interferes with the resolution of those features that are in focus. This situation is especially problematic for specimens having a thickness greater than about 2 micrometers. Thus, such a microscope is not an intrinsic 3D Microscope. (source)

Because of this lack of resolution, in widefield microscopes the Nyquist Rate is about double of that in confocal ones. See Confocal Microscope.

Huygens widefield deconvolution

Below you see two examples of widefield microscope images that are deconvolved with the Huygens software. The image on the right is a single XY (2D) slice of an original Multi Channel widefield stack. The image is restored with deconvolving the whole stack using a QMLE algorithm as detailed in this deconvolution procedure.

Raw Widefield
Huygens Deconvolved
Data courtesy of Dr. Livio Kleij, Medical Oncology, UMC Utrecht, The Netherlands.
Raw Widefield
Huygens Deconvolved
Data courtesy of Dr. Rita Reis, Lab. Molecular Genetics, IBMC, Porto, Portugal.

Brightfield microscope

Brightfield microscopy 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. The most common use of the microscope involves the use of a sample mounted to a glass microscope slide. (Source)

Huygens Professional also includes the non-iterative Tikhonov-Miller algorithm which proves to give excellent results combined with brightfield microscopes tuned and controlled using using a Shack-Hartmann sensor.
See also the article of Dr. Oberlaender a.o. for more explanation.
See Huygens References.

Restoring a 2D Widefield Image

A widefield Point Spread Function (PSF) has the property that its plane-integrated intensity is constant. As a result the blur cone extending from objects is always truncated.

Truncated images can be handled by allowing the measured and object data space to be different, while computing the quality measure only over the measured region. In this way an object can be restored with larger extent than the measured region.

The ultimate consequence of this is the possibility to restore single-plane images.