Biology Reference
In-Depth Information
pathway, and di
ect this. However, several
factors associated with the confocal principle allow for improving also XY-
resolution, as compared to widefield epifluorescence microscopy. First, spatial
resolution may be improved by further reducing the pinhole diameter in the
confocal aperture to a size smaller than the width of the central disk of the airy
unit pattern, though this also dramatically reduces light transmission. This
principle works because the pinhole is aligned with the center of the airy unit
pattern of the illuminating beam, which means that any emission originating
from any fluorescent molecules excited by the outer airy rings of the illuminating
beam will be blocked by the confocal aperture; like all light, the illumination
beam also presents with a airy wave pattern consisting of a central bright spot
and outer ring waves that in comparison are more faint. In other words, the
resultant fluorescence emission may be experimentally manipulated to originate
from an area smaller than the airy unit, which cannot be achieved by conven-
tional widefield epifluorescence microscopy. Moreover, because the PSF of the
confocal microscope is narrower at normalized light intensities relative to that of
the conventional widefield microscope, it means the XY spatial resolution will be
V
erent media and surfaces, will also a
V
greater with a confocal microscope than a widefield microscope
(
Conchello and Lichtman, 2005
). Finally, some of the in-focus light will scatter
on its way through the specimen, due to di
1.4
raction, reflection, and refraction as
cell structures interfere with the light path. This also compromises fluorescence,
but not confocal microscopy, as the confocal aperture also blocks scattered light
from reaching the signal detector.
In addition, combining confocal microscopy with total internal reflection fluo-
rescence (TIRF) or F
¨
rster resonance energy transfer (FRET) microscopy techni-
ques has the capacity to increase resolution to only a few tens of nanometers
(see below for more detailed information). Other techniques such as narrowing
the boundaries of the PSF by suppressing (de-exciting) the fluorescence from the
edge of the center spot of the airy pattern by stimulated emission depletion
(STED), and other nonlinear optical masking techniques, have further enhanced
optical resolution of confocal microscopes (
Bullen, 2008; Willig et al., 2006
),
though these techniques are not yet compatible with fast scanning of Ca
2
þ
events
that take place over fast timescales, and will therefore not be discussed here.
Finally, secondary signal processing or deconvolution (computationally reverse
optical distortion to enhance resolution) of the recorded images also serve to
enhance spatial resolution of both confocal and epifluorescence microscopy by a
factor of 2-3.
Because light scattering increases proportionally to increasing thickness of the
specimen, this becomes more of an issue with deeper imaging of thicker speci-
mens. Therefore, appropriately setting the pinhole not only allows for imaging of
thin optical sections, but also a
V
ects the signal-to-noise (SNR) ratio. Whereas a
case made be made that reducing the pinhole diameter increases XYZ-resolution
(especially Z-, but also XY-resolution; see above), opening the pinhole to ap-
proximately match the projected image of the di
V
V
raction-limited spot (1.22
l
/NA,