Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
"micro" (small)
"scopeo" (to watch)
THE RELATIVE SIZES OF MOLECULES,
CELLS AND ORGANISMS
THE RELATIVE SIZES OF MOLECULES,
CELLS AND ORGANISMS
MICROSCOPY
MICROSCOPY
1590 2018
PARTS OF A LIGHT MICROSCOPE
THE LIGHT
Characteristic parameters
of light waves:
Wavelength: the distance between repeating units of a
propagating wave of a given frequency. (λ, expressed in nm).
Frequency: The number of oscillations within a minute.
Amplitude: distance from the center y position to the peak
COMPONENTS OF VISIBLE LIGHT
Major Imaging Functions of the Microscope
• Magnification
• Resolution
Incident light
focus
Focal
length f
Light travels more slowly in matter
The speed ratio is the Index of Refraction, n
v = c/n
• Water 1.333
• Cytoplasm 1.35–1.38 ?
• Glycerol 1.475 (anhydrous)
• Immersion oil 1.515
• Diamond 2.417
Some examples:
10x/0.3 WD = 15.2mm
20x/0.75 WD = 1.0mm
100x/1.4 WD = 0.13mm
MAGNIFICATION
MAGNIFICATION
Total visual magnification of the microscope is derived by multiplying
the magnification values of the objective and the eyepiece.
Objective:
4-100 x magnification
Maximum
magnification:
3000X
The resolution ~0.2 to .25 mm
wavelength
() = =
2 A numeric aperture
Distance objective
Higher is not good
Lower is good object
A = n·sinα
The letter n is the refraction index of the media between the cover slip
and the objective (air n=1, distilled water n=1.33, cedar oil n=1.51).
α labels the angle closed by the main optical axis and the outermost light
beam (half angle of the objective)
Possibilities of resolution improvement
Increasing α:
The half angle of a lens can be increased only until 72°, since
at larger angle than this the light beams became totally
reflected.
ADVANCED MICROSCOPY
2. Fluorescence microscopy
Fluorescence - The process by which a suitable atom or molecule, which is transiently excited by
absorption of external radiation at the proper energy level (usually ultraviolet or visible light), releases the
absorbed energy as a photon having a wavelength longer than the absorbed energy. The fluorescence
excitation and emission processes usually occur in less than a nanosecond.
Stokes’
shift
Fluorochromes
Organelle/DNA/Ca2+ probes
Organelle probes:
44
MT
DNS
CS
Fluorescent Proteins
Green Fluorescent Protein (GFP) - A naturally occurring protein fluorescent probe derived from the
jellyfish Aequorea victoria, which is commonly employed to determine the location, concentration,
interactions, and dynamics of a target protein in living cells and tissues. The excitation and emission
spectra of enhanced GFP (a genetic derivative) have maxima at 489 nanometers and 508 nanometers,
respectively. In order to incorporate the GFP (or any of its genetic derivatives) into a cell, the DNA
sequence for the gene is ligated to the DNA encoding the protein of interest. After cultured cells have been
transfected with the modified DNA, they are able to express chimeric fluorescent proteins for observation
in the microscope. There are genetically modified variants of GFP such as blue fluorescent protein (BFP),
cyan fluorescent protein (CFP), yellow fluorescent protein (YFP)
fusion protein
emits green light
upon excitation
transfection by blue light,
intracellular
localization
determined
GFP
transcription,
translation
Light sources for fluorescence/confocal microscopy
As opposed to traditional arc-discharge lamps used with the shortest range (10-20 nanometers) bandpass
interference filters in widefield fluorescence microscopy, the laser systems used for fluorophore
excitation in scanning confocal microscopy restrict excitation to specific laser spectral lines that
encompass only a few nanometers.
Z-sectioning
Z-sectioning
55
D
PLA [IFI16 M and R PLA [IFI16 M and R
abs]+KSHV+ DAPI abs]+KSHV Enlarged
Uninfected
Untreated
KSHV (30 DNA copy/cell)
C-646
Electron Microscope
Heat
Wehnelt cap
- -
(negative potential)
- -
- -
- -
- -
- e- -
e-
Space charge e-
e-
+++++++++++ +++++++++++
Anode (positive potential)
Transmission electron microscope (TEM)
Electron ray source
Condenser lenses
Condenser aperture
Sample
OBJECTIVE
Objective apertures
Intermediate lenses
PROJECTOR
Detector
Transmission electron microscope (TEM)
Scanning Electron Microscope (SEM)
Condenser aperture
2nd condenser lens
Objective aperture
Scan coils
Amplifier/Detector
OBJECTIVE
Backscattered
electrons 2nd electrons
Sample
Scanning Electron Microscope (SEM)