Tuesday, July 30, 2013

Zac - Tools and tricks of the trade

My work in the Maslakova lab involves using morpholinos to suppress the products of specific genes in M. alaskensis larvae.  After the morpholinos begin taking effect, we use a number of techniques to identify the differences between morpholino treated larvae and untreated larvae. These differences can be attributed to the suppression of these genes, and thus give us clues as to the mechanistic function of these genes. However, these are microscopic larvae and oftentimes this research requires peaking into individual cells. So how do we do it?

Bright-field vs DIC imaging, pictures by Zac Swider
For one, we use extremely powerful microscopes capable of differential interference contrast imaging (DIC). This imaging technique takes advantage of the properties of polarized light to make  a high-contrast image out of an otherwise transparent object. The image to the right shows a bright-field image of one my cheek cells (top) as well as a the exact same image with the DIC prisms in place (bottom).  Although this is a handy technique for taking pictures of our otherwise transparent larvae it is still extremely limited in its ability to visualize sub-cellular components. For this purpose can utilize fluorescent protein probes to mark specific tissues or sub-cellular components.  Since we are already injecting these larvae with a solution of morpholinos, it is no big trick to add a solution of mRNA to the needle and co-inject it with the morpholino. These cells will take the injected mRNA and express the protein that it codes for in great quantities. Once the protein has been produced it will bind to its target molecule and mark its location within the cell. We can choose specific proteins that, for example, bind specifically to DNA or specifically to microtubules, or actin.  You name it and somebody has probably made a probe for it.

Nemertean Embryo Expressing Microtubule Label
Image by George von Dassow

The image to the left shows a nemertean embryo expressing a fluorescent label for microtubules (orange) - image by George von Dassow. Microtubules are significant component of the cell "skeleton" and watching the changing behavior of these macromolecules can tell us a great deal about a cell. These images of fluorescently labeled larvae are generally obtained using laser scanning confocal microscopy.  Confocal microscopes essentially just use a laser and a pin-hole to take extremely thin scans through a specimen, and then compiles these individual scans to create a crisp image with no out of focus blurring. The image series below shows the significant difference seen between confocal microscopy and compound microscopy.  The difference in image crispness is significant between the image to the left (confocal) and the image series below (compound).  This is largely due to out of focus light that the compound microscope simply cannot filter out.  

Five day old M. alaskensis larva expressing mEos - images by Zac Swider
The image series above shows a five day old M. alaskensis larva expressing a fluorescent protein called mEos. This fluorescent protein is attached to a histone, one of the components that helps package up DNA in the nucleus, which means that it will accurately label the location and shape of every single nuclei in the the subsequent larva. It also has the neat feature of being photoactivatable. This means that the protein starts out with a green emission peak but it can be irreversible converted to have a red emission peak by exposing the protein to a 390nm laser light.  The image series above shows the larva using DIC imaging (left), epifluorescence imaging before laser exposure (middle) and epifluorescence imaging after laser exposure (right). For the sake of this picture I just exposed the entire larva to UV light, but the photoconversion can be done under much more controlled conditions to alter the emission spectrum of a single nucleus and leave all others intact.  By doing so one could photoconvert, for example, just one of eight cells (in an eight cell embryo), to red and put it back in a dish to grow.  After a couple weeks the resulting larva would show a number of red nuclei - each one a direct progeny of the originally altered cell. Needless to say this has a number of implications in allowing us to study the development of these larvae.

Spionid - Image by George von Dassow
However injections of mRNA are not the only way to label specific parts of a larva, another popular technique involves permanently fixing the larvae (using formaldehyde) and staining for specific molecules using fluorescent antibodies. Antibodies are components of the vertebrate immune system that that recognize and "tag" foreign entities for destruction. The match between an antibody and its "target" it boasted to be one of the most specific chemical matches within the cell. Biologists can take advantage of this specificity to "probe" for just about any molecule imaginable. The image to the right (by George von Dassow) demonstrates the contrast and complexity achievable by staining, even with a relatively large animal.

I know I promised some fluorescence microscopy pictures of my own larvae this week, but unfortunately the last batch could not have turned out any worse. We are trying again this week (using a different buffer, and new antibodies) and will hopefully see some better results. Again, stay tuned for some fluorescence pictures and (hopefully) some tentative results next week!


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