Our Work Tools
The Zaidel-Bar lab is fully equipped for and proficient at molecular biology, basic biochemistry, tissue culture, C. elegans husbandry, and bioinformatics. But that’s not what makes us special. Without a doubt, our forte is in live-imaging and quantitative image analysis, and we are also experts at generating transgenic animals and cells that express fluorescently-tagged protein for us to image. In the following sections we highlight the major precision tools and methods that we use on a regular basis to advance our research projects.
Spinning disk microscopy with live super-resolution
‘Seeing is Believing’ is our motto and imaging is our ‘bread and butter’. We use a custom built spinning disk confocal imaging system with a W1 spinning disk from Yokogawa, a live-super-resolution module from GATACA systems, a Prime95 sCMOS camera from Photometrics and a fully motorized Nikon TiE2 microsocpe with perfect focus system.
Sometimes we’ll fix and immunolabel cells, but life is all about dynamics so our specialty is live-cell imaging. The room is kept at 20 degrees for worms, and we can heat the chamber around the microscope to 37C for imaging of mammalian cells. Shining laser light can harm living cells so making movies while keeping the cells or worms alive and happy is not an easy skill to acquire.
Fluorescence Recovery After Photobleaching (FRAP)
With an iLAS module for photomanipulation from GATACA installed on our imaging microscope we can photoactivate or photobleach any region of interest in our field of view. One common use for this is to study the molecular dynamics of intracellular structures, such as focal adhesions or cell-cell junctions, by following the recovery of fluorescence after photobleaching.
Long-term imaging of C. elegans with microfluidics
Some developmental processes, such as gonad morphogenesis, take place over many hours. However, imaging a worm at high magnification for long periods of time is a challenge, because if the worm can’t move and feed it will stop developing, and when it is moving it’s impossible to acquire high-resolution images. To overcome this challenge, we adopted microfluidics technology that was developed by Simone Berger from ETH Zurich (Berger, Simon, et al. Development 148.17, 2021) to allow imaging at 100X of worms up to 26 hours.
Quantitative image analysis
The images and movies of fluorescently-tagged proteins we generate on our spinning disk confocal are often very beautiful. However, while aesthetically pleasing images are nice to have we want much more from our images: we want numbers. Quantitative image analysis is the name of the game. Fluorescence intensity, size, shape, position, rate of change, co-localization, etc. are all parameters that can be extracted quantitatively from a series of images with the right image analysis tools. Since every biological experiment has its own unique features, it also requires customized image analysis tools to be tailored for it.
Laser ablation of cellular and subcellular structures
Imagine a doctor skillfully using a scalpel to cut a piece of tissue. Now imagine doing the same thing but at the subcellular scale. With the iAblate laser ablation module from GATACA we can not only observe the inner workings of the cell, but also cut, like with a micron-sized scalpel, subcellular structures, such as stress fibers or cell-cell junctions, and then observe the consequences. This method is useful, for example, to qualitatively probe cell mechanics.
Gene knockdown by RNA interference and siRNA
In order to figure out what the function of a protein is we often eliminate or reduce its function and by observing the consequences of its absence we deduce its normal function. A fast and usually effective way to deplete a gene’s product is with RNA interference (RNAi) in worms and small interfering RNA (siRNA) in mammalian cells. siRNA is transfected into cells by standard transfection procedures and to get RNAi in worms we simply feed the worms with bacteria expressing dsRNA (we can also inject dsRNA into worms if the feeding method doesn’t work).
Precision genome-editing with CRISPR-Cas9
By now, nearly everyone has heard of the Novel prize winning CRISPR revolution that allows precision editing of the genome at any location. For us, the CRISPR-Cas9 revolution is a weekly affair. Whether we want to delete a gene completely from the genome, or make a point mutation (e.g. human disease mutation) in a gene, or if we want to insert DNA (e.g. coding for a fluorescent protein) into an endogenous gene, we turn to CRISPR-Cas technology. We also developed a bioinformatic tool to help design CRISPR projects http://www.wormcoolkit.com.
We use molecular biology techniques (primarily PCR and Gibson cloning) to generate plasmids that can generate transgenic worms with new traits. However, these plasmids need to make their way into the germline of the worm in order to create a transmitted extra-chromosomal array. Also CRISPR-Cas9 only works if the mix of guide RNA, Cas9 protein and repair template finds its way into the syncytial gonad. We use very thin needles and a microinjection system to inject plasmids or CRISPR mixes into the worm gonad, where the magic can happen. Microinjection is performed under a high magnification microscope and is a skill not very different from in vitro fertilization.