Key Words Lecture 5 Flashcards
1
Q
Brightfield Microscopy
A
- magnification and resolution, specimen fixation/staining
- light is diffracted by specimen and undiffracted light focused by objective lens
- image usually captured by video camera (more sensitive to low light intensities)
2
Q
Deconvolution
A
- manipulation of digital images using various computer software programs
- designed to remove background and out-of-focus light (higher contrast and clarity
3
Q
Magnification
A
- primary purpose of microscopy to generate magnified high-quality view of specimen
- objective lens x ocular lens
- ‘empty’ = continuing to enlarge image does not provide more details/information
4
Q
Resolution
A
- most important aspect of today’s microscope
- minimum distance that can separate two points that still remain identifiable as separate points
- (0.61 * wavelength) / numerical aperture
- maximized by using shorter wavelength of illuminating light, increasing numerical aperture (ie: air -> oil)
5
Q
Fixation
A
- by formaldehyde
- cross-links amino groups on adjacent proteins/nucleic acids
- results in cell death
6
Q
Embedding
A
- in plastic or wax
- for support
- can result in structural artifacts
7
Q
Sectioning
A
- imaging thin sections
- technique for 3D visualization of large specimens
- can result in structural artifacts
- uses microtome
8
Q
Staining
A
- using molecule specific dyes
- better visualization of cells and cell parts
9
Q
Microscopy artifacts
A
An artificial structure or tissue alteration on a prepared microscopic slide as a result of an extraneous factor
10
Q
Microtome
A
tool for sectioning of a specimen
11
Q
Fluorescence Microscopy
A
- microscopy technique for visualizing fluorescent molecules in living or fixed specimens
- relies on autofluorescence in specimen, applied fluorescent dyes or dye-conjugated antibodies (immunofluorescence) / autofluorescent proteins
- provdes increased contrast and allows study of structures and dynamic processes in living cells and 3D when cell is not fixed
- out-of-focus fluorescence from thick speciment can result in blurred image
- certain atoms can absorb photon of certain wavelength
- excited electron is highly unstable, loses energy and returns to ground state by emmiting photon with lower energy / longer wavelength
12
Q
Confocal Laser-Scanning Microscopy (CLSM)
A
- method of fluorescence microscopy
- one or more lasers of certain light wavelengths excite fluorescent molecules in specimen and emitted light specifically focused to obtain detailed image
- specimen usually living
- dynamic biological / cellular process live viewing
- lasers can penetrate into thicker living specimens
13
Q
Pinhole
A
- emitted fluorescent light from only a single layer (focal plane) within the specimen is focused through this and then collected and viewed
- out-of-focus fluroescence does not pass through the pinhole and is excluded
14
Q
Focal Plane
A
- layer of specimen
- CLSM yields individual 2D z-section
15
Q
z-sections
A
individual images collected at different depths in a sample
