Abstract
HiLo microscopy is a recently developed technique that provides both optical sectioning and fast imaging with a simple implementation and at a very low cost. The methodology combines widefield and speckled illumination images to obtain one optically sectioned image. Hence, the characteristics of such speckle illumination ultimately determine the quality of HiLo images and the overall performance of the method. In this work, we study how speckle contrast influence local variations of fluorescence intensity and brightness profiles of thick samples. We present this article as a guide to adjust the parameters of the system for optimizing the capabilities of this novel technology.
Highlights
HiLo is a novel fluorescence microscopy modality that provides both optical sectioning and fast imaging at a very low cost
Speckle contrast measurement and processing is key for obtaining optimum results with HiLo microscopy
In this work we have studied two aspects that determine the characteristics of HiLo images
Summary
HiLo is a novel fluorescence microscopy modality that provides both optical sectioning and fast imaging at a very low cost. As opposed to laser scanning microscopes, optical sectioning is obtained combining two images, one obtained with wide-field illumination and a second one using speckle pattern illumination. This required combination of two images makes the sampling frequency only two times slower than the camera acquisition rate, what is typically one order of magnitude faster than a standard confocal microscope. Since only a coherent light source must be added to a standard fluorescence microscope, the cost of HiLo microscopes is approximately ten times lower than a laser-scanning configuration. We present this work as a guide to profit from this novel microscopy, and to understand how certain features of the illumination profile and the sample characteristics render HiLo images that can be difficult to comprehend
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