typical cell size
20 micrometers
resolution
- determined by the wavelength of light and the numerical aperture of the objective lens
- resolution improves if d gets smaller
- directly proportional to the lends numerical aperature
- inversely proportional to the wavelength used for imaging
- visible light wavelength are 360-780 nm
- resolution limit of light microscope about 0.2um
long wavelength light
- less damaging and less easily scattered than short wavelength light and illuminate deep structures
- Cy5 dyes emit near infrared light
resolution vs. deteection
objects smaller than the resolution limit of a microscope can be detected but are not resolved
magnification
the minimal adequate magnification is one that allows the smallest object you want to resolve to fall on 3 discrete elements of the imaging device
properties of interaction
Four properties of the interaction between light and matter influence the design of microscopes used to produce contrast.
- absorbtion
- reflection
- refraction
- fluorescence emmission
absorption methods: stained tissue sections
- stains are compounds that absorb light or electrons
- black stain absorbs all colors of light
- colored stains absorb some colors of light, others to pass
refraction techniques in light microscopy
- very little incident light is absorbed, reflected or refracted by a living biological specimen
Living cells are seen clearly using - to image the difference between refracted and non-refracted light.
phase contrast microscopy
fluorescence microscopy
Fluorescent molecules absorb high energy light and then emit less energetic,
longer wavelength light. The shift in wavelength between absorbed and
emitted light is called the Stokes shift.
Fluorescence Microscopes
use excitation, emission and dichroic
filters to take advantage of the Stokes shift
fluorescent stains
Chemical compounds of some fluorescent stains bind preferentially to specific cellular components
- Fluorescent phalloidin (red) stains actin
- DAPI (blue) stains nucleic acids
Fluorophores
Antibody linked fluorophores detect specific cellular antigens
Fluorescence in situ hybridization (FISH)
uses synthetic fluorescent RNA
probes to detect compatible mRNA in cells and tissue
Fluorescent Proteins
Green fluorescent proteins can be expressed in living organisms
Gene Promoters
Cell-type specific gene promoters can be used to express GFP in specific cells or tissues
Fluorescent Fusion Protiens
Green fluorescent fusion proteins can be used to label proteins in living cells
Fluorescent Probes
- Dynamic studies of fluorescent probes can be conducted using Fluorescence Recovery After Photobleaching (FRAP)
- Dynamic studies of fluorescent probes can be conducted using photo-activatable probes
electron microscope
- uses electrons to resolve fine structure of the cell
- wavelength of an electron can be 0.004 nm so the theoretical limit of resolution of an electron microscope is 1/20 angstoms or 1/20 the diameter of a H atom
EM stains
- Stains used for electron microscopy (EM) are very dense (metals) so they
absorb electrons - EM stains are generally soluble, reactive forms of metals such as lead,
uranium, gold, silver and tungsten
Metal Shadowing
Metal shadowing can be applies to surface structures or interior structures using freeze fracture and freeze etching methods
Super resolution microscope-STED (stimulated emission depletion) microscopy
An extremely strong superimposed laser beam, at a different wavelength and in the shape of a torus, or doughnut, depletes emitted fluorescence everywhere in the specimen except right in the center of the beam, reducing the effective width of the point spread function
expansion microscopy
- sample is crosslinked and embedded in a polymer gel
- proteinase digestion of the gel
- expansion by immersion of aqueous solution
Total internal reflection fluorescence (TIRF) microscopy
- incident light is reflected at the interface
- but evanescent wave or light energy penetrate through around 50nm
- only image surface molecules on glass slide
