Practical Implementation of Log-Scale Active Illumination Microscopy

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.National Institutes of Healt

Blind fluorescence structured illumination microscopy: A new reconstruction strategy

In this communication, a fast reconstruction algorithm is proposed for fluorescence \textit{blind} structured illumination microscopy (SIM) under the sample positivity constraint. This new algorithm is by far simpler and faster than existing solutions, paving the way to 3D and/or real-time 2D reconstruction.Comment: submitted to IEEE ICIP 201

Selective Plane Illumination Microscopy

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a microscopy technique that allows you to acquire high resolution fluorescence images of biological samples by illuminating the sample with a thin plane from the side, instead of along the imaging axis as in traditional transillumination or epi-illumination. The purpose of this SPIM research assignment was to combine two previously built systems, an inverted SPIM and a tunable lens system. This report includes use of optics, coupling lasers and proper technique to building optical systems. Programming in Matlab, LabVIEW, and other programming languages was used to synchronize the shutter and camera electronics and acquire and process images. The paper is concluded with expected results to ensure to detection path is optimized.https://engagedscholarship.csuohio.edu/u_poster_2015/1063/thumbnail.jp

Selective Plane Illumination Microscopy

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a microscopy technique that allows you to acquire high resolution fluorescence images of biological samples by illuminating the sample with a thin plane from the side, instead of along the imaging axis as in traditional transillumination or epi-illumination. The purpose of this SPIM research assignment was to combine two previously built systems, an inverted SPIM and a tunable lens system. This report includes use of optics, coupling lasers and proper technique to building optical systems. Programming in Matlab, LabVIEW, and other programming languages was used to synchronize the shutter and camera electronics and acquire and process images. The paper is concluded with expected results to ensure to detection path is optimized.https://engagedscholarship.csuohio.edu/u_poster_2015/1063/thumbnail.jp

Structured illumination microscopy using micro-pixellated light-emitting diodes

Structured illumination is a flexible and economical method of obtaining optical sectioning in wide-field microscopy [1]. In this technique the illumination system is modified to project a single-spatial frequency grid pattern onto the sample [2, 3]. The pattern can only be resolved in the focal plane and by recording images for different transverse grid positions (or phases) an image of the in-focus parts of the object can be calculated. Light emitting diodes (LEDs) are becoming increasingly popular for lighting and illumination systems due to their low cost, small dimensions, low coherence, uniform illumination, high efficiency and long lifetime. These properties, together with recent developments in high brightness, ultraviolet operation and microstructured emitter design offer great potential for LEDs as light sources for microscopy. In this paper we demonstrate a novel structured illumination microscope using a blue micro-structured light emitting diode as the illumination source. The system is potentially very compact and has no-moving-parts

Structured illumination microscopy with unknown patterns and a statistical prior

Structured illumination microscopy (SIM) improves resolution by down-modulating high-frequency information of an object to fit within the passband of the optical system. Generally, the reconstruction process requires prior knowledge of the illumination patterns, which implies a well-calibrated and aberration-free system. Here, we propose a new \textit{algorithmic self-calibration} strategy for SIM that does not need to know the exact patterns {\it a priori}, but only their covariance. The algorithm, termed PE-SIMS, includes a Pattern-Estimation (PE) step requiring the uniformity of the sum of the illumination patterns and a SIM reconstruction procedure using a Statistical prior (SIMS). Additionally, we perform a pixel reassignment process (SIMS-PR) to enhance the reconstruction quality. We achieve 2$\times$ better resolution than a conventional widefield microscope, while remaining insensitive to aberration-induced pattern distortion and robust against parameter tuning

Scalable-resolution structured illumination microscopy

Structured illumination microscopy suffers from the need of sophisticated instrumentation and precise calibration. This makes structured illumination microscopes costly and skill-dependent. We present a novel approach to realize super-resolution structured illumination microscopy using an alignment non-critical illumination system and a reconstruction algorithm that does not need illumination information. The optical system is designed to encode higher order frequency components of the specimen by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in structured illumination microscopy. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. This algorithm eliminates the need of clean peaks in illumination and the knowledge of illumination patterns, which makes instrumentation simple and flexible for use with a variety of microscope objective lenses. We present a variety of experimental results on beads and cell samples to demonstrate resolution enhancement by a factor of 2.6 to 3.4 times, which is better than the enhancement supported by the conventional linear structure illumination microscopy where the same objective lens is used for structured illumination as well as collection of light. We show that the same system can be used in SIM configuration with different collection objective lenses without any careful re-calibration or realignment, thereby supporting a range of resolutions with the same system