One can use techniques of light microscopy or e- microscopy to image samples.
Organotypic slices are live tissues that can be put into cell cultures and analyzed. This is an exception.
Electric signals analyzed from this tissue
Confocal live imaging uses layers to excite fluorescent molecules within distinct optical layers of that tissue.
Three principles of microscopy
Contrast – requires light pass through a sample. Often cells are too clear.
Add dyes, or contrast, to the cell to enhance features. Works well in light and fluorescent microscopy, but often this requires the cells to be fixed and will not be able to observe movement/behavior or cells to the signals.
Objective lens, near to specimen.
Condenser lens focuses light below onto the sample of interest.
Resolution – a factor that measures how close two points can be in a sample and still be distinct.
R= (0.61*lamba)/(n * sin(angular aperture))
N = refractive index. Oil is about 1.5 and the larger it is, the better the resolution. The SMALLER the resolution, the closer two points can be a still be distinct structures.
Bestlambda is 450, oil 1.5, and angular is 70 degrees. = 194 nm. This means that two objects that are 200nm apart can be seen as distinct objects. If they are closer than this, they will not be seen as distinct structures.
Lambda – wavelength. This sets the limit on how an object can be and be seen by particular microscope. Smaller the wavelength, the smaller the object that can be seen.
E- microscopes have better resolutions.
Uses an electron gun as its source of light, or energy which pass through a condesnerser lens, onto specimen and
Has a lambda of .004nm. increase in resolution generates a maximum resolution in theory of .2nm.
Must be stained and fixed – gold is a good stained.
Use of radiation can be a drawback
Must be in a vacuum.
Magnification – how large you can make an image and still distinguish independent points. LM is 1400X in an oil. In air = 1000X
E- have 100X better of light microscope, so 100,000X
Different types of light microscopy
Cell often appears transparent when there isn’t manipulation
Insertion of nomarski optic - A prism that splits light into two separate waves through specimen. Changes wavelength of light and the interference leads to increased contrast, largest difference is the edges of cells and organelles.
Differentiation of organells and differences between them w/out fixing
Good way to see edges of cell
Vesicle transport in cells
Takes advantage of physics of light and the characteristics of it as a waveform. When two waves are in phase, they are at the same frequ and combine to create a better resolution. If out of phase, reduced intensity of light. When inphase, the unstained cell will disrupt one of the light waves creating a slowed speed for the wave form.
Thicker samples and image optical layers for 3d images.
TEM vs SEM
2d vs 3d
lots of dyes to look at parts of cells at the same times.
Only limited by number of filters one can place in microscope.
Souce of light is ultraviolet passed through an excitation filter, which removes wavelengths of light not useful in expressing the fluorescence.
Dyes are chemically derived that bind carbs, lipids, DNA.
One doesn’t need to use antibodies if the dyes bind directly.
Proteins most often identified by fluorescently linked antibodies because few dyes directly link to proteins.
Primarily, the fluorescene microscope does not have a condenser like in a light microcope. Must be thin samples.
has allowed us to use laser light to image specific layers/regions within a thick sample.
Images individual parts and put together to rebuild the picture.