Microscopy Flashcards
Who invented Microscopes?
-Robert Hook
-Antoni Van Leeuwenhoek invented a better one.
Magnification light path
-visualized image
-ocular lens
-intermediate image (inverted)
-objective lens
-condenser lens
-light source
Magnification
-the extent to when the image of an object is larger than itself
-the magnification of an image under the microscope is the product of objective and ocular lens
What is the magnification of an 10x ocular and 20x objective?
200 magnification
What is the magnification of an 10x ocular and 100x objective?
1000 magnification
How big are most bacterial cells?
~1 micron = 1 um =1X10-6 m
-at 1000x magnification they would appear to our eye as if they were 1x10-3 m = 1mm
Resolution or resolving power
-the degree to which details are retained in magnified images; the ability to distinguish between two points
-resolution = R= 0.5 λ / NA
-λ = wavelength
-NA = numerical aperture (the light gathering ability of the lens)
What is the best resolution with standard microscopes?
-1000x magnification is 200nm = 0.2 microns = 0.2um
-most bacterial cells are ~1um, so they can be easily visualized at 1000x magnification
Bright Field Microscope
-most common in general microbiology lab
-light is transmitted through the specimen
-contrast between the background and the cells is generated by absorption or scattering of light
3 things about bright field microscopes
- Bacteria are transparent
-staining of the cells is generally required - Kills the cells
- Dead cells take up stains
Preparing a slide
- Preparing a smear -> dry in air
-spread culture in thin film over slide - Heat fixing and staining -> flood slide w/ stain; rinse and dry
-pass slide through flame to heat fix - Microscopy
-place drop of oil on slide; examine with 100x objective lens
Simple Stains
-stains are generally charged molecules
-basic and acidic dyes
-bacterial cell surface carries a negative charge, therefore basic stains are most commonly used for the most simple staining methods
Basic dyes
-cationic
-positively charged
-ex: methylene blue, crystal violet, safranin
-bind to negatively charged cell components
-polysaccharides, nucleic acid, phospholipoids
Acidic dyes
-anionic
-negatively charged
-binds to positively charged cell components
-amino groups -> proteins
Differential Stains
-Purpose: to tell one type from another
-Example: ~TB
~gram stain (gram stain positive vs negative)
~Acid fast stain (mycobacteria vs other bacteria)
~Endospore stain (spore within mother cell)
Gram Stain Procedure
- flood the heat-fixed smear with crystal violet
-results: all cells purple - Add iodine solution for 1 min
-All cells remain purple - Decolorize with alcohol briefly about 20 seconds
-Gram positive cells are purple, gram negative cells are colorless - Counterstain with safranin for 1-2 mins
-Gram positive cells are purple; gram negative cells are pink to red
Why the gram stain works?
Gram Positive
-peptidoglycan is thick
1. crystal violet + iodine = largo crystal
2. Ethanol dissolves membranes
Why the gram stain works?
Gram Negative
-peptidoglycan is thin
1. CV +I
Dark Field Microscope
-The only light reaching the specimen comes from the side
-The light only enters the objective lens if it is scattered by the specimen
-cells are bright against a dark background (no need to stain)
-Can’t see colors/stains
-provides better resolution than bright field, so we can see smaller cells or structure
What organism is so skinny it can’t be seen with bright field but only seen in dark field
Borrella
Plant Contrast Microscopy
-special condenser used so that ALL light entering sample area in IN-PHASE
-The phase difference between diffracted rays through cell and direct rays (through medium) appears as a difference in light intensity
-No staining required (both unstained and stained cells are dark against light background
-can view LIVE cells!
Differential Inference Contrast Microscopy (DIC)
- Uses polarized light and a prism to make two separate light beams
- The light beams are then combined through the specimen
- Gives a 3-dimensional appearance to cells, even bacteria
How does Fluorescence Microscopy work?
- Exit
2.Emission - Blue light enters and green light comes back up is what you see
Fluorescence Microscopy
-light source at wavelength causes specimen to fluoresce at a different wavelength
-some cells exhibit autofluorescence
-Fluorescent dyes are used to stain specific cell structure
-Fluorescently tagged proteins can be specifically localized in cells
Transmission electron microscope
-The electron that pass through the sample are collected to make the image
-has a vaccum
Electron Miscroscopy
-electron wavelength is 0.004 nm (compared to 500 nm for light)
-Resolution (R) is 0.2 nm (1,000 fold better than light)
-Magnification up to 100,000 x (compared to 1000x for light)
-Transmission vs Scanning
Transmission
-2D, thin sections of fixed samples =heavy metal stains
-TEM
Scanning
-3D
-coat cells with heavy metal
-reflects
-SEM
Scanning electron microscope
-The electron that are scattered back off the sample are collected to produce the image
Atomic force
-Tiny stylus is scanned across and just above the surface of the sample
-see atoms
Atomic Force Microscope
-The weak repulsive forces between the atoms of the stylus and the sample cause the styles to deflect up and down
-The deflections are recorded as a surface contour map.
-can be challenging with live/sticky/flexible samples
