Discover Simple, Private Sharing at Drop.io
Extra awesomeness points for the use of pool noodles as mitotic spindles.
Tuesday, August 17, 2010
Maybe we'll see this at the next summer Olympics?
I'd give it a 10.
Extra awesomeness points for the use of pool noodles as mitotic spindles.
Extra awesomeness points for the use of pool noodles as mitotic spindles.
Monday, August 9, 2010
It's the small things that matter
Today's topic is presented by Dr. Angel Paredes, also of the Department of Pathology and Laboratory Medicine.
For those dental students reading along, in class yesterday, Dr. Bick discussed transmission electron microscopy (TEM). Cell structures can be imaged using this technique to obtain magnifications of up to the maximum of 25,000,000X in some microscopes. Of course with this kind of power, these magnifications would be like looking at an elephant with a light microscope and that wouldn't make very much sense. Typically magnifications in the range of 2000x to 40,000x are useful for examining things like cells (eukaryotic and prokaryotic), viruses and protein complexes.
For tissue structure, specimens are stained with heavy metals (osmium tetroxide for lipids, Uranyl Acetate for proteins and lipids) and embedded in a resin. The resin is then sectioned thinly and mounted on a metal grid which is then stained again with Uranyl acetate and lead citrate. The grid is placed in the microscope where electrons are passed through the sample where the heavy metal stains interact with the beam giving the image its characteristic contrast.
The way this happens is that electrons hit a structure and change course (because of the heavy metal), some pass through with minimal interference (no heavy metal or just a little). A charged coupled device (CCD or digital camera) camera records the image by measuring the electron density at each pixel, thus creating a black and white EM image like we see in class. Electrons do not have color so images are black and white.
Since samples need to be sectioned for this technique, this doesn't really tell us much about the 3D structure of cells. For that analysis, we need to use a scanning electron microscope. Cells are coated with a very gold palladium in a sputter coater, and the electron beam is bounced off the surface. The resulting image shows only the cell surface.
Recently, however, advances in computing have allowed scientists to further develop electron cryomicroscopy, or cryoEM. This is a technique that is used to image very small biological specimens (< 1 micron) frozen and perfectly preserved in amorphous ice. NOT resin! Why is that important? The specimen that is imaged is perfectly preserved and, in the case of virus, the virus can later be removed from the microscope, thawed from the ice, and used to infect more cells - illustrating that the images recorded were indeed of infectious virus.
The process begins by applying 3-4 microliters of specimen (let's just say we're looking at a virus in this example) in buffer onto a 3 mm copper EM grid coated with a net-like layer of carbon holes. The virus is blotted with filter paper creating a thin layer of buffer in which the specimen is briefly suspended. The EM grid is then quickly plunged into a liquid ethane cup at liquid nitrogen temperature where the virus is flash frozen so quickly ice crystals cannot form and the specimen becomes embedded in a layer of glass-like ice. The virus is then placed into an electron microscope specifically designed to image these frozen specimens. Remember, this is TEM, so electrons are passing through the sample. We get a black and white picture that looks something like this:
These are alphaherpesvirus particles!
For the next step, it is important to understand that for homogenous particles such as viruses, all the particles embedded in the ice are in all possible orientations and can be considered different views of the same object. Using a computer, the digitized images from the microscope and image analysis software, the orientation of each particle is determined relative to the electron beam. Once all the orientations are determined, the computer merges all the different views into one 3D model which then represents the specimen. Because the specimen is reproduced in 3 dimensions, it can be dissected and studied by imaging software. Behold the modeled structure of Sindbis virus (an alphaherpesvirus):
A few words about resolution, from the expert:
"Ok. You would have to ask about resolution. Resolution in the world we live in means essentially the minimum distance that two small elements in an image (dots) are distinguishable as two independent elements or dots.
(Also remember from Dr. Bick's lecture that the distance (d) between points that can be resolved increases as the wavelength of light increases, i.e. the resolving power goes down.)
In cryoEM you relate everything to your pixel size and image size. The pixel is a measurement of a grey value of the specimen you scanned. The pixel size is dependent on the mag. The higher the mag, the smaller the pixel size when you image it with the digital camera on the microscope.
Ok, now you have pixel size and image size. Say you have a pixel size that is 2 angstroms/pixel. This means that the absolute best resolution you can get is 4 angstroms resolution because you cannot achieve a resolution that is equal to your scanning step size or pixel. The best you can do is twice the pixel size.
So, what does resolution mean? In cryoEM resolution is quantifiable. You have an image. The image has noise and you refine the data to achieve the best resolution and that is when the noise and signal equal each other and the ration is 1. You cannot get better than that. With the image and pixel size you know exactly at what resolution this occurs. For my newest virus image, I get to 13 ansgtroms resolution. That does not mean that I can see two dots no closer than 13 angstroms. In fact I can see detail that is smaller than 13 angstroms. The resolution in this case is a quantifiable measure of how far out I can signal from the data in terms of resolution with the maximum being 2 times the pixel size.
I know it's complicated but YOU ASKED."
For those dental students reading along, in class yesterday, Dr. Bick discussed transmission electron microscopy (TEM). Cell structures can be imaged using this technique to obtain magnifications of up to the maximum of 25,000,000X in some microscopes. Of course with this kind of power, these magnifications would be like looking at an elephant with a light microscope and that wouldn't make very much sense. Typically magnifications in the range of 2000x to 40,000x are useful for examining things like cells (eukaryotic and prokaryotic), viruses and protein complexes.
For tissue structure, specimens are stained with heavy metals (osmium tetroxide for lipids, Uranyl Acetate for proteins and lipids) and embedded in a resin. The resin is then sectioned thinly and mounted on a metal grid which is then stained again with Uranyl acetate and lead citrate. The grid is placed in the microscope where electrons are passed through the sample where the heavy metal stains interact with the beam giving the image its characteristic contrast.
The way this happens is that electrons hit a structure and change course (because of the heavy metal), some pass through with minimal interference (no heavy metal or just a little). A charged coupled device (CCD or digital camera) camera records the image by measuring the electron density at each pixel, thus creating a black and white EM image like we see in class. Electrons do not have color so images are black and white.
Since samples need to be sectioned for this technique, this doesn't really tell us much about the 3D structure of cells. For that analysis, we need to use a scanning electron microscope. Cells are coated with a very gold palladium in a sputter coater, and the electron beam is bounced off the surface. The resulting image shows only the cell surface.
Recently, however, advances in computing have allowed scientists to further develop electron cryomicroscopy, or cryoEM. This is a technique that is used to image very small biological specimens (< 1 micron) frozen and perfectly preserved in amorphous ice. NOT resin! Why is that important? The specimen that is imaged is perfectly preserved and, in the case of virus, the virus can later be removed from the microscope, thawed from the ice, and used to infect more cells - illustrating that the images recorded were indeed of infectious virus.
The process begins by applying 3-4 microliters of specimen (let's just say we're looking at a virus in this example) in buffer onto a 3 mm copper EM grid coated with a net-like layer of carbon holes. The virus is blotted with filter paper creating a thin layer of buffer in which the specimen is briefly suspended. The EM grid is then quickly plunged into a liquid ethane cup at liquid nitrogen temperature where the virus is flash frozen so quickly ice crystals cannot form and the specimen becomes embedded in a layer of glass-like ice. The virus is then placed into an electron microscope specifically designed to image these frozen specimens. Remember, this is TEM, so electrons are passing through the sample. We get a black and white picture that looks something like this:
These are alphaherpesvirus particles!
For the next step, it is important to understand that for homogenous particles such as viruses, all the particles embedded in the ice are in all possible orientations and can be considered different views of the same object. Using a computer, the digitized images from the microscope and image analysis software, the orientation of each particle is determined relative to the electron beam. Once all the orientations are determined, the computer merges all the different views into one 3D model which then represents the specimen. Because the specimen is reproduced in 3 dimensions, it can be dissected and studied by imaging software. Behold the modeled structure of Sindbis virus (an alphaherpesvirus):
A few words about resolution, from the expert:
"Ok. You would have to ask about resolution. Resolution in the world we live in means essentially the minimum distance that two small elements in an image (dots) are distinguishable as two independent elements or dots.
(Also remember from Dr. Bick's lecture that the distance (d) between points that can be resolved increases as the wavelength of light increases, i.e. the resolving power goes down.)
In cryoEM you relate everything to your pixel size and image size. The pixel is a measurement of a grey value of the specimen you scanned. The pixel size is dependent on the mag. The higher the mag, the smaller the pixel size when you image it with the digital camera on the microscope.
Ok, now you have pixel size and image size. Say you have a pixel size that is 2 angstroms/pixel. This means that the absolute best resolution you can get is 4 angstroms resolution because you cannot achieve a resolution that is equal to your scanning step size or pixel. The best you can do is twice the pixel size.
So, what does resolution mean? In cryoEM resolution is quantifiable. You have an image. The image has noise and you refine the data to achieve the best resolution and that is when the noise and signal equal each other and the ration is 1. You cannot get better than that. With the image and pixel size you know exactly at what resolution this occurs. For my newest virus image, I get to 13 ansgtroms resolution. That does not mean that I can see two dots no closer than 13 angstroms. In fact I can see detail that is smaller than 13 angstroms. The resolution in this case is a quantifiable measure of how far out I can signal from the data in terms of resolution with the maximum being 2 times the pixel size.
I know it's complicated but YOU ASKED."
Friday, July 23, 2010
When Neutrophils attack
Today in class the dental students learned about blood, and the cells contained within. I promised to post a cool video of a neutrophil "chasing" a bacteria, so here it is:
="">
Poor bacteria never stood a chance.
There are a lot of interesting processes going on here. The first, obviously, is that the neutrophil appears to "see" the bacteria. Since we all got a good look at cell structure last week, we know that cells don't have eyes! So just how does this neutrophil know to attack?
The neutrophil is most likely sensing chemical signals coming from the bacteria. So-called "quorum sensing signals" are basically messages to other bacteria to come and join the infection party. It is likely that our neutrophil has picked up on these quorum sensing molecules and is using them to sniff out the bacteria. In addition, we know that neutrophils, like other phagocytes, express what are called "Toll-like receptors",or TLRs, on their cell surface. These receptors, important for innate immunity, are designed to recognize bacteria-specific sugars and DNA sequences.
So the neutrophil has identified the target, the target is escaping - how does the neutrophil move? This involves a whole lot of specialized cell signaling. First, the neutrophil is exhibiting "chemotaxis", or movement along a chemical gradient. It wants to go where the chemical signal from the bacteria is most concentrated. So, receptors on the cell surface bind the bacterial proteins, then trigger changes in the cell.
One thing that's happening is that our neutrophil has developed polarity -now it has a front end and a back end. We're not quite sure how this happens, but it seems likely that the cell is probably sending out random pseudopodia in all directions. Whichever psuedopod gets positive reinforcement (e.g. lots of binding to bacterial protein receptors) gets to stay and grow, while the other pseudopodia which don't encounter their target are pulled back into the cell.
To move, the neutrophil needs to rearrange some cellular structures. This involves polymerization of actin and myosin, and probably some growth of microtubules as well.
Once the neutrophil catches up to the bacteria, the process of phagocytosis begins. The bacteria is "grabbed" by specific receptors and pulled into an endosome. That endosome will eventually fuse with a lysosome, and the resulting phagosome is where the bacteria ultimately meets its end.
="">
Poor bacteria never stood a chance.
There are a lot of interesting processes going on here. The first, obviously, is that the neutrophil appears to "see" the bacteria. Since we all got a good look at cell structure last week, we know that cells don't have eyes! So just how does this neutrophil know to attack?
The neutrophil is most likely sensing chemical signals coming from the bacteria. So-called "quorum sensing signals" are basically messages to other bacteria to come and join the infection party. It is likely that our neutrophil has picked up on these quorum sensing molecules and is using them to sniff out the bacteria. In addition, we know that neutrophils, like other phagocytes, express what are called "Toll-like receptors",or TLRs, on their cell surface. These receptors, important for innate immunity, are designed to recognize bacteria-specific sugars and DNA sequences.
So the neutrophil has identified the target, the target is escaping - how does the neutrophil move? This involves a whole lot of specialized cell signaling. First, the neutrophil is exhibiting "chemotaxis", or movement along a chemical gradient. It wants to go where the chemical signal from the bacteria is most concentrated. So, receptors on the cell surface bind the bacterial proteins, then trigger changes in the cell.
One thing that's happening is that our neutrophil has developed polarity -now it has a front end and a back end. We're not quite sure how this happens, but it seems likely that the cell is probably sending out random pseudopodia in all directions. Whichever psuedopod gets positive reinforcement (e.g. lots of binding to bacterial protein receptors) gets to stay and grow, while the other pseudopodia which don't encounter their target are pulled back into the cell.
To move, the neutrophil needs to rearrange some cellular structures. This involves polymerization of actin and myosin, and probably some growth of microtubules as well.
Once the neutrophil catches up to the bacteria, the process of phagocytosis begins. The bacteria is "grabbed" by specific receptors and pulled into an endosome. That endosome will eventually fuse with a lysosome, and the resulting phagosome is where the bacteria ultimately meets its end.
Tuesday, October 27, 2009
Thursday, October 8, 2009
It's Auerbach's plexus
(answer for the Friday what is it)
Here is an H&E stained version of an Auerbach's, or myenteric plexus. Remember that this structure is part of the intrinsic component of the innervation of the intestines. The Auerbach's plexus contains sensory neurons that receive information from nerve endings in the smooth muscle layer regarding the composition of the intestinal content (chemoreceptors) and the degree of expansion of the intestinal wall (mechanoreceptors). Together with the Meissner's plexus found in the submucosa, the Auerbach's plexus is responsible for intestinal contractions.
This bundle of nerves was first described by Leopold Auerbach, a German anatomist, neuropathologist, and histologist. His work was some of the first to define the nervous system via histological staining.
Here is an H&E stained version of an Auerbach's, or myenteric plexus. Remember that this structure is part of the intrinsic component of the innervation of the intestines. The Auerbach's plexus contains sensory neurons that receive information from nerve endings in the smooth muscle layer regarding the composition of the intestinal content (chemoreceptors) and the degree of expansion of the intestinal wall (mechanoreceptors). Together with the Meissner's plexus found in the submucosa, the Auerbach's plexus is responsible for intestinal contractions.
This bundle of nerves was first described by Leopold Auerbach, a German anatomist, neuropathologist, and histologist. His work was some of the first to define the nervous system via histological staining.
Friday, October 2, 2009
Tissue fixation and processing
Our previous description of tissue cutting and slide preparation dealt with fresh, snap-frozen tissue. In this installment, we'll describe the more common formaldehyde tissue fixation and processing into paraffin. Dr. Rittman has kindly provided us with an excellent summary of the process:
Remember, the function of fixation is to preserve tissue in as lifelike manner as possible for histological examination.
As soon as tissue is removed from the body, pH within cells falls and lysosomal enzymes are released. Tissue rapidly starts to break down in process known as autolysis. In order to examine tissue histologically the process of autolysis need to be stopped as soon as possible after tissue removal. Failure to do so will allow autolysis to progress and will also cause diffusion of substances from their original locations.
There are a variety of methods for fixing tissue but the most common is chemical fixation by immersing pieces of tissue in a chemical solution.There are several thousand fixing solutions (due to the fact that no one fixing solution is ideal for all applications.) Fixation is most often acheived by cross linking the amino acid groups. Therefore, the most commonly used fixative is a buffered formaldehyde solution.
Fixation is generally accomplished by immersion of thin pieces (less than 5 mm thick) in the fixative for various periods of time, typically 24 hours.
At this point, the histologist has a lump of tissue in formaldehyde. That's not going to be very useful for cutting. In order to be able to cut sections thin enough for microscopic examination, tissue must be embedded in a medium (such as paraffin wax) hard enough when solid for such sections to be cut.
Processing requires:
1. The removal of water by use of a dehydrating agent such as graded ethanols from 70% up to 100% ethanol.
2. The use of an intermediary agent that is miscible with both alcohol and paraffin wax. This commonly xylene, a purified form of petroleum. This stage is also called "clearing" as the tissue becomes translucent during this step.
3. Infiltration with molten paraffin wax. This is normally at 58 to 60 degrees Centigrade and results in gradual removal of the xylene and infiltration of paraffin wax into the tissue. The paraffin penetrates into tissue components and even into the cytoplasm of individual cells.
4. Solidification of the tissue in a block of wax. The tissue is oriented for sectioning by placing in a mold and the paraffin wax allowed to solidify.
During fixation and processing most lipids are extracted (this is why adipose tissue looks like chicken wire). Some non proteinaceous substances will be lost in the fixing solution or during the subsequent process. Other substances such as glycogen may be retained as they are associated with proteins. In addition, due to the solvent action and to heat the tissue undergoes a certain amount of shrinkage. With soft tissues the shrinkage is generally in the order of 25 to 30%. Shrinkage often results in separation of components from each other such as separation of muscle fibers and separation of soft from hard tissue at interfaces. By now, you should be noticing this a lot in your example slides in lab!
Here's an example of shrinkage - the keratin layer in this high-power view of skin is separating from the underlying epidermal layers and from itself. The white spaces indicated by the arrows are artifact and not there in real life.
Sectioning of a paraffin-embedded tissue is very similar to frozen tissue sectioning. The solidified block of paraffin wax is fixed in a holder on the chuck on a microtome. The microtome allows a reproducible forward advancement of the block towards the knife that will slice a section from the surface of the block. Typical advancement is in the order of 5 to 10 microns.
As sections are cut, the lower edge of the block melts slightly allowing this edge to join with the trailing edge of the previous section thus forming a ribbon of sections. Such ribbons of sections as seen here may be stored for a short period of time.
During the sectioning a certain amount of compression occurs so that sections have the same width but are smaller from their upper to lower edges than the paraffin block from which they were cut.
Section mounting.
Sections are floated onto the surface of a warm water bath (usually 45 to 48 degrees F) where the paraffin section containing the tissue expands.
Sections are then collected and oriented on a glass slide and then allowed to dry.
Staining - it's business time!
Remember, most tissue components are of similar refractive indices and cannot be distinguished from each other unless we use phase contrast microscopy. The better way to differentiate tissue components is by the use of histological stains or histochemistry.
Most stains rely on binding to different components by virtue of some type of chemical bonding. We will deal with hematoxylin and eosin although the same general principals apply to many stains.
Sections on slides must first have the paraffin wax removed by soaking in xylene, followed by graded alcohols from 100% to 70% and finally into distilled water. Shown below are slides in the process of moving through the de-paraffinization process.
Sections are then stained in a solution of hematoxylin where the hematoxylin binds to acidic components. Next, we soak the sections in eosin which binds to basic components. The sections are then dehydrated, cleared in xylene and mounted in a transparent mounting medium. If done correctly, the stained and mounted slides will last for decades (as clearly demonstrated by your lab slide sets).
In a final well-stained preparation, nuclei are blue, and the cytoplasm stains a pale pink, (the cytoplasm of some cells may also be blue due to the presence of acidic components such as RER). Muscle cells stain bright pink, red blood cells bright red. Any traces of mineral will appear dark purple. This is especially noticeable in the partially mineralized enamel of developing tissue.
Decalcification (demineralization).
Embryos (while they do contain some mineral in developing teeth and bone)may be sectioned without removal of the mineral. However, tissues with considerable amounts of mineral such as bone, dentin and cementum must first have the mineral removed before paraffin wax sections can be prepared. This process is known as decalcification or more correctly as demineralization.
The tissue is first adequately fixed and then mineral is removed most often using dilute acids. During this process the tissue may show some changes due to the effects of the acid on components other than the mineral. Further hematoxylin-eosin staining then looks like this:
Bright pink staining of a tissue with very few cells is usually bone.
Remember, the function of fixation is to preserve tissue in as lifelike manner as possible for histological examination.
As soon as tissue is removed from the body, pH within cells falls and lysosomal enzymes are released. Tissue rapidly starts to break down in process known as autolysis. In order to examine tissue histologically the process of autolysis need to be stopped as soon as possible after tissue removal. Failure to do so will allow autolysis to progress and will also cause diffusion of substances from their original locations.
There are a variety of methods for fixing tissue but the most common is chemical fixation by immersing pieces of tissue in a chemical solution.There are several thousand fixing solutions (due to the fact that no one fixing solution is ideal for all applications.) Fixation is most often acheived by cross linking the amino acid groups. Therefore, the most commonly used fixative is a buffered formaldehyde solution.
Fixation is generally accomplished by immersion of thin pieces (less than 5 mm thick) in the fixative for various periods of time, typically 24 hours.
At this point, the histologist has a lump of tissue in formaldehyde. That's not going to be very useful for cutting. In order to be able to cut sections thin enough for microscopic examination, tissue must be embedded in a medium (such as paraffin wax) hard enough when solid for such sections to be cut.
Processing requires:
1. The removal of water by use of a dehydrating agent such as graded ethanols from 70% up to 100% ethanol.
2. The use of an intermediary agent that is miscible with both alcohol and paraffin wax. This commonly xylene, a purified form of petroleum. This stage is also called "clearing" as the tissue becomes translucent during this step.
3. Infiltration with molten paraffin wax. This is normally at 58 to 60 degrees Centigrade and results in gradual removal of the xylene and infiltration of paraffin wax into the tissue. The paraffin penetrates into tissue components and even into the cytoplasm of individual cells.
4. Solidification of the tissue in a block of wax. The tissue is oriented for sectioning by placing in a mold and the paraffin wax allowed to solidify.
During fixation and processing most lipids are extracted (this is why adipose tissue looks like chicken wire). Some non proteinaceous substances will be lost in the fixing solution or during the subsequent process. Other substances such as glycogen may be retained as they are associated with proteins. In addition, due to the solvent action and to heat the tissue undergoes a certain amount of shrinkage. With soft tissues the shrinkage is generally in the order of 25 to 30%. Shrinkage often results in separation of components from each other such as separation of muscle fibers and separation of soft from hard tissue at interfaces. By now, you should be noticing this a lot in your example slides in lab!
Here's an example of shrinkage - the keratin layer in this high-power view of skin is separating from the underlying epidermal layers and from itself. The white spaces indicated by the arrows are artifact and not there in real life.
Sectioning of a paraffin-embedded tissue is very similar to frozen tissue sectioning. The solidified block of paraffin wax is fixed in a holder on the chuck on a microtome. The microtome allows a reproducible forward advancement of the block towards the knife that will slice a section from the surface of the block. Typical advancement is in the order of 5 to 10 microns.
As sections are cut, the lower edge of the block melts slightly allowing this edge to join with the trailing edge of the previous section thus forming a ribbon of sections. Such ribbons of sections as seen here may be stored for a short period of time.
During the sectioning a certain amount of compression occurs so that sections have the same width but are smaller from their upper to lower edges than the paraffin block from which they were cut.
Section mounting.
Sections are floated onto the surface of a warm water bath (usually 45 to 48 degrees F) where the paraffin section containing the tissue expands.
Sections are then collected and oriented on a glass slide and then allowed to dry.
Staining - it's business time!
Remember, most tissue components are of similar refractive indices and cannot be distinguished from each other unless we use phase contrast microscopy. The better way to differentiate tissue components is by the use of histological stains or histochemistry.
Most stains rely on binding to different components by virtue of some type of chemical bonding. We will deal with hematoxylin and eosin although the same general principals apply to many stains.
Sections on slides must first have the paraffin wax removed by soaking in xylene, followed by graded alcohols from 100% to 70% and finally into distilled water. Shown below are slides in the process of moving through the de-paraffinization process.
Sections are then stained in a solution of hematoxylin where the hematoxylin binds to acidic components. Next, we soak the sections in eosin which binds to basic components. The sections are then dehydrated, cleared in xylene and mounted in a transparent mounting medium. If done correctly, the stained and mounted slides will last for decades (as clearly demonstrated by your lab slide sets).
In a final well-stained preparation, nuclei are blue, and the cytoplasm stains a pale pink, (the cytoplasm of some cells may also be blue due to the presence of acidic components such as RER). Muscle cells stain bright pink, red blood cells bright red. Any traces of mineral will appear dark purple. This is especially noticeable in the partially mineralized enamel of developing tissue.
Decalcification (demineralization).
Embryos (while they do contain some mineral in developing teeth and bone)may be sectioned without removal of the mineral. However, tissues with considerable amounts of mineral such as bone, dentin and cementum must first have the mineral removed before paraffin wax sections can be prepared. This process is known as decalcification or more correctly as demineralization.
The tissue is first adequately fixed and then mineral is removed most often using dilute acids. During this process the tissue may show some changes due to the effects of the acid on components other than the mineral. Further hematoxylin-eosin staining then looks like this:
Bright pink staining of a tissue with very few cells is usually bone.
Friday "What is it?"
This one might be obvious to the dental students who paid attention during Dr. Rittman's lecture on Thursday. Any guesses?
Subscribe to:
Posts (Atom)