Overview # There are four steps to this process. First is to create a dataset file that allows BigSticher to interpret the data. Second is to run a macro that will flip the opposing views, so they are roughly aligned. Third is to find the best parameters that register the views together. Forth is to register and export the reconstructed data. Inital Setup # First we must install ImageJ and the BigStitcher plugin. ...
Starting in August 2019, the Advanced Imaging Center (AIC) introduced its first pre-commercial electron microscope. This system is based on work done in Janelia’s Hess lab and makes use of a powerful technique: Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). This is the first in a series of blog posts that introduces this unique methodology, and the various considerations that should be made, to prospective AIC users.
In a previous blog post, we discussed probe selection for single molecule localization microscopy (SMLM) techniques such as PALM and STORM. This blog post continues earlier discussions to give some practical guidance toward both probe selection and imaging considerations when performing SMLM across multiple channels.
The last 10 years has seen an explosion of techniques aimed at creating biological fluorescence images with unsurpassed clarity via super-resolution microscopy. Among these various techniques that circumvent the diffraction barrier, there is a group of methods that can be termed single-molecule localization microscopy (SMLM). This blog introduces the conceptual basis, and gives pointers for selecting these fluorescent tags for use in SMLM.
Needless to say that, just like in any type of microscopy, low signal samples in general do not work well for SIM. Besides that, there are other factors unrelated to the general signal level that will determine if a sample, either fixed or living, is suitable for SIM. The following is by no means a complete list of those factors.
As mentioned in an earlier post, the lattice light-sheet microscope (LLSM) requires special consideration when preparing and mounting samples for imaging. Even after properly mounting a sample for imaging, the raw data produced by the microscope can be hard to intuitively understand due to the angle between the detection objective lens and the sample stage.
The lattice light-sheet microscope (LLSM) is a powerful optical tool for observing rapidly-occurring processes in live samples. This technique offers multiple advantages over more common optical microscopy methods, like spinning-disk confocal, with improved optical sectioning, reduced photobleaching, and minimized phototoxicity. This blog post will cover the most common model systems and the steps we take to mount and adapt the sample for lattice light-sheet microscopy.
For many researchers, the prospects afforded by super-resolution (SR) imaging are both vast and exciting. Unfortunately, the dizzying array of acronyms reported in the literature, including PALM, STED, SIM, GSDIM, RESOLOFT, iPALM, dSTORM, among many others, can leave researchers unsure as to the optimal technique for their specific application(s). This article will provide a brief overview of the major classes of SR techniques, with suggested reading for further details. The second part of the article will outline a practical comparison of the two SR systems currently available at the Advanced Imaging Center (AIC).