- 1 Notes
- 2 Lab 2 Video Tutorials
- 3 Week 4 Lab
- 4 Three Main Techniques
- 5 Laboratory 6 Immunochemistry
Week 2 Lab
Must find copyright notice on the article. Can always site the reference. Can't directly take any of the content images or text or use it without a copyright notice.
Journal of Cell Biology : Can reuse contet BUT cannot use content until 6 months AFTER the publication date. (Permissions)
- Text & Data Mining
- All articles published in JCB, JEM and JGP dating back to volume 1, issue 1 are available for harvesting on PubMed Central, 6 months after publication, for text and data mining. For details refer to PMC's OAI-PMH site. The RUP acknowledges that text or data mining by commercial entities for their internal research purposes is allowed without further permission from RUP. Commercial entities may develop indexing or search services—available to the public for free or for a fee—based on text or data mining without further permission from RUP, but they may reproduce only snippets of text up to 156 characters in length, or thumbnails of images up to 72 pixels in the long direction, as part of such a service.
Bio Med Central : Whole journal is open access. Article will always state copyright. Need to look for this in the article.
- We also source the original copyright notice on our webpages?
Copyright notice :
- Image produced by z3459996. I, the author, hereby release this image into the public domain. Feel free to use it for whatever.
Note - This image was originally uploaded as part of a student project and may contain inaccuracies in either description or acknowledgements. Please contact the site coordinator if the uploaded content does not meet the original copyright permission or requirements, for immediate removal."
- Note : Was away for half of laboratory.
Week 3 Lab
- Micron : 10^-6m.
- One mammalian cell ~ 20 microns.
- ~50 Cells in 1 mm.
Histology Problem : Have to stain and use dead cells.
Electron Microscopy : Breakthrough. Using electron beams instead of light to look at tissue. However material still has to be dead and replace all the contents with things that can be seen which an electron. Contrasting agent like a heavy metal. (1931)
Phase Contrast Microscopy : Tool for looking at living structures. Has ability to give us a better resolution for the edges of cells and see them as they're alive. (1937)
Confocal Microscopy : Technology based on lasers. Replaced lights with lasers. Laser emits light at a specific frequency based upon crystal. More control over this technique of microscopy. (1953)
Differential interference contrast microscopy : 3D Image of structure. Can see plasma membrane. (1955)
- Many microscopes now have video cameras attached to them. Everything is now an electronic file rather than a normal 30mm film.
- 21st century all about high resolution microscopy. (Eg. nuclear pore images generated by this) - want to see single molecule within cell
- Light Microscopes mostly replaced with Virtual Slides and Digitalised images. Easier to teach.
- 60x with optics is the greatest you can get before using water/oil immersion. This is based upon the concept of how light travels.
- This was good for fixed and not moving slides.
Problem : When use focus light using a condenser on a microscope. The lens has to be EXTREMELY close to the tissue to be in focus. (Focal Length) Bigger lens = bigger diameter = greater focal length (distances you can move the lens away from tissue to still be in focus)
- Detail : Want resolution. Clarity of things that you'll be able to clearly see and distinguish from other things. (Distinguishing two things from each other)
- Based on wavelength of life
- Based on aperture
How do we look at living cells? They're usually in cell culture preserving it. Therefore preventing us getting closer to the living tissue.
- Turn it upside down. You can get closer to tissue culture. Only distance of the thickness of the slide as opposed to the thickness of the cell culture above it.
- This is called an inverted microscope
- Therefore light has to come from above instead of below.
Unless you stain the cell with a histological stain, you can't see it very well.
- Refer to : http://www.ncbi.nlm.nih.gov/books/NBK26880/figure/A1729/
- 4 Ways of Looking at a Cell
Phase Transfer Microscopy - leads to diffraction in image around the sides of cell. Called the "Halo of Health" - can see this in live cells. If it doesn't have this halo it suggests that the cell is dying as the cell membrane is NOT intact.
- Internal structures are also clearer.
- This diffraction effect is useful when it hits an intact membrane. Therefore super good for looking at live cells.
- Can watch this in real time and watch it do real life interactions.\
- Cell can move out of field of view so you can't see it any more.
- It has to be able to live in that environment. Needs nutrients, oxygen, temperature, adherent to a substrate (otherwise it'd be floating around)
- Need to do this process quickly. Get it out of an incubator. Put it in moist environment. Take pictures quickly. Then put it back into the incubator. This is ONLY okay for short. Therefore super hard if you want to do long observation for your live cells. 10 minutes, 30 minutes etc.
- Vast majority of your cells live in the dark. We usually put a layer of dead keratinized cells over them. Light harmful for these cells, you might just kill them in that exposure. Limitation of exposure to light. Use high contrast video cameras to help us determine slight changes. Can potentially just put light on when you're taking photo (like flash)
Inverted Phase Contrast Microscopy
Differential Interference Contrast Microscopy (DIC) - It's almost 3 dimensional. Shadow, idea of depth. 3rd picture on above link. Can see curve on cell. Can see nucleus, can see cell membrane. Can see distortion of cytoplasm as well as granules within it. Tool used by electro physiologists - Want to be able to measure electrical changes across membrane.
Dark Field Illumination - Majority of image is now black. Good for identifying vesicles. Not very good for looking at dynamics of cell or plasma membrane. 4th picture on above link.
Total Internal Reflection Fluorescence Microscope (TIRFM) : Video - Light coming from three different lasers. Red, green and blue. Laser light reflected back on the glass water interface and the fluorescence is collected through the very sensitive camera
Robert Y. Tsien won 2008 Nobel Prize in Chemistry. Discovered Green Fluorescent Protein (GFP). Found from a jellyfish. He isolated the protein that makes the fluorescence. Breakthrough : Took coding sequence of GFP and attach it to any other proteins and now the new protein will glow green in response to the correct frequency of light. You can now see a lot of things in microscopy. Now commercially - can buy GFP tagging kits. Developed into other fluorescent proteins. Really helpful in microscopy. Illuminates at 405 nm. Jellyfish illuminates when stimulated/disrupted. Aequorin fluoresces blue. (Chemical Energy --> Light Energy) GFP turns it from blue to green when stimulated. GFP self sufficient. Can generate fluorescence by itself as long as it's sequence is intact (Can develop it's own chromophores)
Each chromophore illuminates at one particular wavelength
Fluorescent microscopes generally in a dark room. White light disrupts this fluorescence. Going from a non visible wavelength to a visible wavelength. This is where the green/different colours come in. Living cells have proteins that naturally fluoresce. NADH and NADPH fluoresce normally - part of normal energy process. Vitamin B also auto fluoresces.
Fluorescent microscopes has all the limitations of all the light microscopes as it still deals with light. Hasn't got the high resolution that we need. This is solved by the introduction of lasers ~ 20 years ago. Lasers have the unique property because lasers have a known wavelength that it emits. Therefore you can do a lot of manipulation of the material you're looking at by knowing the wavelength of the light. Can focus it on a very small structure and move the laser over the object (over that small spot). Illuminating just a small region of the cell and detecting the fluorescence that is emitting from that spot - this gives you a very high resolution image of these spots.
- Laser Scanning Technique - scans rapidly over small parts. (Micron scale)
- Spinning Disc Confocal Technique - at any one time only illuminating only one part of the cell. Uses a spinning disc with small holes. This is better for living cells because it gives less exposure of light to the cell. Like using a shutter for small parts of cells. Can combine images to form an entire high resolution image.
Two Photon Confocal Technique - If one laser is good, why not use two? Two lasers which are scanning over our fixed living tissue. You can focus them in a particular 3D point of space (because you know the wavelength that the light emits) Use two suboptimal wavelengths where they combine in one point in space to excite the molecules which fluoresce. Not illuminating through the whole tissue then, just one particular spot which is illuminated by that one wavelength.
Total Internal Reflection Fluorescence Microscope (TIRFM) - Light hitting it at such an angle that instead of going through glass it's being reflected. It doesn't even pass through the slide, it reflects off glass slide.... how is this useful? The energy of this laser beam can stimulate structures which are close to that point of reflection. This stimulation in the near field area (nanometres range) means that it will excite cell tissue which is in close contact to the glass slide. Useful for studying Cell Adhesion or Cell Migration.
Laser Capture Microscopy - Use laser beams to cut out just where the tumor is from the tissue. This piece of tissue is then sucked up and you can do molecular analysis on the cell to work out the genetic changes in that section.
Scanning Tunneling Microscopy - Shows 3D images of the sample
Super Resolution - Can bypass the wavelength of light. Highest resolution that we can currently achieve. Various tricks :
- Changing wavelength of light over time = slightly different images of region = combine together and get higher resolution in comparison to without the combination
We've got these images now so... what are we going to do with them? Problems :
- HUGE Images. Single image = 1.8 gb. Then if you're doing a constant analyzing ie video. Then HUGE space usage.
- High resolution cameras that are stimulated by very low amounts of light (remember light damages DNA and kills our cells) High resolution CCTV cameras
- What's to stop us as a scientist and then deciding exactly what I want it to show, so how about I just photoshop it? Suspicion. Ethical Issue - See : http://jcb.rupress.org/cgi/content/full/166/1/11
- What can we do to stop it? Journals now require scientists to submit raw data and final figure. Raw data HAS to be the figure that they were working with originally.
- See : http://www.nytimes.com/2005/12/25/science/clone-scientist-relied-on-peers-and-korean-pride.html
- Types of manipulation can be very subtle and hard to detect. Have to rely on the moral of the creator. Causes many issues and problems.
- Different image formats. (Eg. JPEG, PNG) These forms bad for science because they compress the image down and this means you lose some information.
- Have to use something like TIF (Tagged Image Formations) - uncompressed image format. Every original pixel is still there. (Large format images) Is our scientific standard image format.
Lab 2 Video Tutorials
|Video Tutorial - Fluorescence Microscopy|
Published on 17 Nov 2013 - Fluorescence is a process in which matter absorbs light and re-emits at a different wavelength. Fluorescence is widely used in biological microscopy. This lecture describes the principles of fluorescence and fluorescence microscopy.
|Video Tutorial - Fluorescent Proteins|
Published on 17 Nov 2013 - Live cell imaging has been revolutionized by the discovery of the green fluorescent protein (GFP). This lecture covers the history of GFP, how it folds and becomes fluorescent, how it has been mutated to produce additional colors (blue, cyan, yellow), and the discovery of red fluorescent proteins from corals. It also covers novel photoswitchable and photoactivatible fluorescent proteins, whose color can be changed by light, and new infrared fluorescent proteins.
- Links: Fluorescence Microscope
Week 4 Lab
3) Fixed cell = extremely hardy. Live cell = extremely labile and easy to damage. Membrane very soft and easily damaged by any sort of event you carry out on it. 4) Permeability.
Biological Fixitive = not only will it fix your cells in culture. If you're exposed to those chemicals, it will fix YOU as well. xD --> Gotta be careful how you work with it.
Safety Guidelines in lab
Every chemical in laboraty has Safety Data Sheet (SDS)
- Has standard set of safety information on it. We should know what we're working with.
- Substances Name
- Alternate/Generic/Commercial Name
- Chemical and Physical Property Sheet
- Health Hazard Information
- Safe Use and Handling
- How do you handle it? What protection do you need?
- The Manufacturer's or Importer's Name.
- So you can contact them if things go wrong.
- Global Harmonization (GHS) - See bottom of page
- Set of pictograms which are clearly described by the image the precautions of working with that chemical.
- Make sure labeling is same wherever you go in world.
- Constant symbols throughout the world which mean the same thing.
- Some of it's called using old nomenclature. Material Safety Data Sheet (MSDS)
- Be careful of this. Shouldn't be referred to. Outdated information.
Protect yourself. Take precautions to prevent injuries. (Majority of injuries : Needlestick Injuries)
Find a safety sheet from a chemical supplier for Glutaraldehyde. (https://proscitech.com/msds/c002.pdf)
Three Main Techniques
Techniques used for tissue and cell fixation.
- 1. Fresh Frozen.
- 2. Precipitation.
- 3. Aldehyde Cross-Linked.
- Cross link thinks together. Physically and Chemically cross linked so structure is preserved.
3 Dimensional Tissues Cryosectioning
- Rapid process
- Often will retain the functionality of enzymes. (Eg. crosslinking proteins = gg enzymes)
- Retains fat
- Retains epitopes
- Retains 3D Organization
- Requires a cyotome (Freezing Microtome) for sectioning
- Thicker sections (8+ mm)
- Cause tissue distortion
- Thawing can degrade tissue
Organic solvents such as methanol, acetone or acids.
- Dehydrate biological material. Remove water. Therefore preservation of material.
- Retains epitomes
- Can combine with crosslinking
- Is fast process
- Dehydrating biological material leads to shrinking up to ~50%.
- Not be good for studies that look for some specific proteins since you can change that shape of protein during this process.
- Removes lipid (permeabilises cells)
- Will also permeabilize.
- Fresh acetone is better, as open acetone absorbs water and increases background. You leave it around too long it absorbs water and isn't 100% acetone.
Aldehydes cross link proteins together (chemical reaction) Uses Formalin, Paraformaldehyde, Gluteraldehyde (all have different advantages/disadvantages)
Good for EM imaging.
- Step that's carried out AFTER fixation
- Osmolality kept so to prevent heaps of shrinkage
Group Project Stuff
Don't reference Review articles as Research Articles. eg. "As reviewed in." "For future information see review."
Research topics in depth. Deep understanding of current research. What's been released in last 2 years, 6 months? Current direction for this? Current unknowns. <-- Indicates that we know what the unknown aspects are and where the research is going. Identify key research laboratories that are researching on this topic. Links to the research facilities which have further information etc.
Reflect back to learning aims of cell biology - structure + function. How is function reflected by its structure.
When giving feedback : positive and negative feedback. Needs to have a solid balance to actually help group and giving appropriate feedback.
Ask that at least one image on the project page is a STUDENT DRAWN IMAGE.
- Flow diagram? PowerPoint slide? Cartoon structure? Made something self as opposed to just copying other stuff. Make powerpoint slide --> jpeg --> upload. Make it look neat and tidy. Can use photoshop.
Email people who aren't here.
Not assessing project until end of semester. End of one of the lab --> he'll give verbal feedback on what it's like.
Look at old projects to see how they look.
Laboratory 6 Immunochemistry
|This year's main topic is Blood Cell Biology. Each group should discuss with group members the specific sub-topic that will be covered by their project.
Here is a list of some of the cell types (Structure and Function)
Cell Type (PuMed citations)
|Group Assessment Criteria|
Group Assessment Criteria
|Individual Lab Assessments|