NIGHTSEA Applications Pre-Screening Samples for Fluorescence


The NIGHTSEA Model SFA Stereo Microscope Fluorescence Adapter can turn your routine laboratory stereo microscope into a valuable tool for pre- screening your sample preparations for fluorescence before moving on to higher resolution systems.


The Challenge


High resolution imaging of biological samples is heavily based on fluorescence techniques. Confocal, 2-photon, and high resolution compound fluorescence microscopes are almost always a limited resource. They are often located only in imaging core facilities and accessible on a scheduled, pay-per-use basis.


The processes for introducing fluorophores to specimens are not always successful. Staining, introduction of GFP-bearing plasmids to cells, immunohistochemistry – all are fallible. It is not unusual to spend time searching for fluorescence on a high end system when there is not even any there to be found.


The Practical Solution


Rabbit psoas muscle fibers stained with Alexa Fluor 488 Phalloidin, in white light and fluorescence. Images made using NIGHTSEA’s white LED (top) and the Royal Blue excitation/ emission light+filter set. Samples courtesy of Dr. Beth Brainerd and Natividad Chen, Brown University.


The NIGHTSEA SFA enables fluorescence pre-screening of specimens on a standard stereo microscope. The detail that you see is not important – the simple presence or absence and general location of fluorescence lets you know whether it is worth taking your specimen to the imaging core. Between the direct expense of the use fee and the time wasted to look at a non-fluorescent specimen it will not take many saved trips for the NIGHTSEA system to more than pay for itself.


One researcher’s work requires staining rabbit psoas muscle fibers with Alexa Fluor 488 Phalloidin. There was some frustration with samples that did not take up the stain. After acquiring the SFA she wrote:


“The NIGHTSEA fluorescence setup is a great way to quickly check whether the stain was successful before we try to image the muscle fiber at a higher magnification on the confocal.”


Another researcher uses zebrafish as a system to look at the way different toxicants (pharmaceuticals, pesticides, food additives, etc.) alter brain development. He writes:


Confocal image of brain of transgenic zebrafish (Dania rerio). Kaede protein – green is unconverted, red is


photoconverted. Image courtesy of Robert Thorn, Creton Lab, Brown University.


23


“Before using NIGHTSEA to screen my samples, I would have to select samples to mount, go to the confocal and then hope that some of my samples were actually fluorescent. Now that I use NIGHTSEA to prescreen my samples I save both time and money by making sure the only samples I image are fluorescent.”


Arabidopsis Seeds


Arabidopsis thaliana is a small flowering plant that is widely used as a model organism for a variety of genetic studies. Dr. Scott Poethig and colleagues at the University of Pennsylvania have developed a novel transgenic strain of A. thaliana that has chromosomal segments with eGFP on one end and dsRed at the other. The segments can be followed in genetic crosses and manipulated via recombination. The transgenic strains will enable a variety of experiments, including phenotypic analyses of mutations with weak or environmentally sensitive phenotypes. They are intended for use in both research and education.


Dr. Poethig was looking for a cost-effective way to sort the genetically modified seeds in a teaching setting. He learned about the new NIGHTSEA Stereo Microscope Fluorescence Adapter and sent a set of seeds for testing. There were five varieties - strong and weak green fluorescence, strong and weak red fluorescence, and non-fluorescent control. All of the variations were easy to see, even with the room lights on.


In the example above, the image on the left was taken with white light illumination, the image in the center with the Royal Blue excitation/emission combination, and the image on the right with the Green excitation/ emission combination. Equipment - NIGHTSEA Stereo Microscope Fluorescence Adapter, Motic SMZ168 trinocular stereo microscope, Canon EOS Rebel T2i camera.


Coral Recruitment Through The Microscope


Fluorescence is a valuable tool for coral recruitment research and one of the ways to apply it is to use a stereo microscope to examine corals on settlement tiles or other surfaces. The NIGHTSEA Stereo Microscope Fluorescence Adapter is an economical system that adds fluorescence capability to existing stereo microscopes and is rugged enough for use in field laboratories in remote locations.


The images below are coral polyps viewed through a stereo microscope, with each pair, white-light (left) and fluorescence (right) showing the same area on settlement tiles. These were made by Dr. Alina Szmant (UNCW) during a research project with NIGHTSEA’s Charles Mazel to develop fluorescence tools for coral recruitment research.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24