Hello everyone, this is my first attempt at writing a blog. I would like to dedicate this mostly to scientific discussions and focus on scientific imaging, flow Cytometry and clean room usage for biological applications. Emphasis will be laid upon discussing various options for imaging and image analysis; also various imaging platforms will be compared. The stem cell instrumentation foundry (SCIF) at University of California (UC), Merced will feature in most of the posts as it is the place of my work and most of my imaging and clean room experiences that I share will be using the instruments at SCIF. Feedback is welcome, spam not. Images and experiences from my previous work place; Dr. Alejandro Calderon Urrea’s laboratory at California State University, Fresno will also be featured in the blog. Without further ado let us delve into the first topic of interest which includes fluorescence microscopy, confocal imaging and why it is important for most of the bright field imaging applications.
Microscopy has evolved in leaps and bounds since the reconfirmation of Anton Von Leeuwenhoek’s observations using a modified version of the then existing model by Robert Hooke. Light microscopy in particular has advanced to a stage where microscopes are put into use to image nanoscale particles. Bright-field microscopes are widely employed for fluorescence studies but the major drawback of a conventional microscope is the excessive background that makes the specimen of interest look blurry. Confocal microscope gives sharper images as it eliminates most of the background as light passes through a pin-hole which blocks excess light and allows for the light to pass only through the pin-hole. Galvanized mirrors help focus light on the sample and in turn eliminate any unwanted exposure. Lasers are used for fluorescence applications as they provide a more stable source of light and also are very specific of the wavelength of light being focused on the specimen. Solid state lasers are used for Confocal applications nowadays and this provides a marked advantage as the intensity of laser remains stable for extended periods of time which makes Confocal imaging suitable even for extended time lapse applications. The beauty of a Confocal system is that it can be built around a simple microscope and customized based on the application of the user. Lasers, objectives, software modules and filters can be added based on the user’s needs. The resolution of a confocal microscope is greater by a factor of 1.4 and a confocal microscope is optimal for high speed scanning and ruggedness which makes it an ideal source for 3D imaging.
Most of the software used for confocal imaging renders 3D volume view and analysis possible and this has led for the increased usage of Confocal in developmental and neurobiological studies. Addition of temperature and growth controlled chambers to Confocal microscopes has improved live-cell imaging; in addition, advanced shutter systems and software help control laser exposure and intensity making it easy to monitor developmental processes for an extended period of time. Personally, I have worked with various live and fixed specimens for confocal imaging. A few examples include Algae, Nematodes, Zebra fish, Fungi, Drosophila, mouse and human stem cells. I will try to blog regularly and will start posting on the various techniques/modifications used for imaging different kinds of samples. Please post your views on using microscopy as a tool for your research needs and also any useful information that might be helpful for others in their research.