P. Lupetti
Dept. Life Sciences
Siena University
The intensity of a small object (diameter much smaller than λ) as viewed through an optical
microscope, also called the point spread function (PSF). (a) The intensity of a small object as
observed in the plane (solid line). To distinguish two small objects from one another, the distance
between them should be approximately as plotted (see the distance between the solid and dashed blue lines). The total intensity is the sum (red line) that has been artificially raised a little. It is common to use the Rayleigh criteria for the resolution. With this criterion, the sum intensity of two close objects (red line) should have an intensity minimum that is 20–27% lower than the peak intensity. (b) The
same graphs shown along the optical axis z. Note that the resolution along the optical (z) axis is
worse than the resolution in the lateral (x,y) plane.
Two axes are shown for each graph. The upper one shows the distance for a numerical aperture (NA) of 1.0 and a wavelength of 500 nm. The lower axis is drawn for a general case and the resolution can be calculated with it for any given value of NA and λ.
The PSF as observed along the y axis for (a) a conventional
microscope and (b) a confocal microscope. The intensity is
shown along the z axis and along any axis in the xy plane (it has a circular symmetry in the plane).
This function is calculated for NA = 1.0 and λ = 500 nm. Note that in both methods the main spot is much larger along the z axis (optical axis) than in the lateral plane.
Marvin Minsky
Two-photon microscopy
Two-photon microscopy is a technique whereby two beams of lower intensity are directed to intersect at the focal point.
Two photons can excite a fluorophore if
they hit it at the same time, but alone they
do not have enough energy to excite any
molecules. The probability of two photons
hitting a fluorophore at nearly the exact
same time (less than 10
-16) is very low,
but more likely at the focal point. This
creates a bright point of light in the
sample without the usual cone of light
above and below the focal plane, since
there are almost no excitations away from
the focal point.
Strengths
Less haze, better contrast than ordinary microscopes.
3-D capability.
Illuminates a small volume.
Excludes most of the light from the sample not in the focal plane.
Depth of field may be adjusted with pinhole size.
Has both reflected light and fluorescence modes.
Can image living cells and tissues.
Fluorescence microscopy can identify several different structures simultaneously.
Accommodates samples with thickness up to 100 µm.
Can use with two-photon microscopy.
Allows for optical sectioning (no artifacts from physical
sectioning) 0.5 - 1.5 µm.
Weakness
Images are scanned slowly (one complete image every 0.1-1 second).
Must raster scan sample, no complete image exists at any given time.
There is an inherent resolution limit because of
diffraction (based on numerical aperture, ~200 nm).
Sample should be relatively transparent for good signal.
High fluorescence concentrations can quench the fluorescent signal.
Fluorophores irreversibly photobleach.
Lasers are expensive.
Angle of incident light changes slightly, introducing
slight distortion.
Bibliography
P. Davidovits and M. D. Egger, Nature, 1973, 244, 366.
A. Hibbs, Confocal Microscopy for Biologists, Twayne Publishers, Boston (2004).
M. Minsky, Scanning, 1988, 10, 128.
J. Pawley, Handbook of Biological Confocal
Microscopy, Twayne Publishers, Boston (2006).
V. Prasad, D. Semwogerere, and E.R. Weeks, J.
Phys.: Condens. Matter, 2007, 19, 113102.
D. Semwogerere and E. R. Weeks, Encyclopedia of Biomaterials and Biomedical Engineering Confocal Microscopy, Taylor Francis (2005).
