Biology Reference
In-Depth Information
ideally want to distinguish Ca
2
þ
in distinct areas that may be within a nanometer
distance from each other, and to record localized Ca
2
þ
events that may last only a
millisecond. Although such requirements tax any given microscopy system, confo-
cal and multiphoton microscopy systems o
V
er a range of imaging capabilities that
fulfill these criteria.
II. Confocal Microscopy
Optical sectioning by confocal microscopy adds several benefits to Ca
2
þ
imaging.
Since its early development, confocal microscopy has fundamentally transformed
optical imaging to now provide a valuable addition that allows unprecedented
imaging of minute optical sections within live specimens in close to real-time speeds.
In terms of Ca
2
þ
imaging, this has opened up new fields of study, given that Ca
2
þ
signaling in many, if not all, biological systems is compartmentalized within small
sections of the cell and occurs often at very high velocities. Confocal microscopy
allows the study of these events within discrete depths of cell or tissue by blocking
light originating outside the plane of focus. This is achieved by the addition of
confocal apertures in front of the illumination source and in the image plane
directly in front of the signal detection system (see
Fig. 1
); usually, a photomulti-
plier tube (PMT) that rejects out-of-focus light originating from fluorescence
outwith the area of interest (the focal plane), and only allowing in-focus light
through to the PMT (
Webb, 1999
)(
Fig. 1
). This is in contrast to regular epifluor-
escence microscopy, in which the majority of the fluorescence is out-of-focus light
that generally reduces the contrast of the in-focus light, and also dramatically
compromises in-focus detail. This occurs since the emitted fluorescence cannot
be discriminated along the Z-axis (top to bottom), and also less along the X-and
Y-axes (although this has also to do with excitation light sources; see later) in
conventional epifluorescence microscopy (
Lichtman and Conchello, 2005
)
(See also later). Thus, although confocal microscopy also excites the specimen
along the entire Z-axis in line with conventional epifluorescence microscopy, only
in-focus light is allowed to pass the pinhole of the confocal aperture to enter the
signal detector.
Importantly, confocal imaging may be performed on live specimens residing
under physiologic conditions and that are electrically, chemically, mechanically,
and otherwise active and healthy. Specimens may also be electrically and mechani-
cally stimulated and superfused by any given solutions that would not interfere
with the confocal imaging.
The di
erence between regular epifluorescence and confocal microscopy light
capture abilities can be illustrated by the following examples. Considering that the
depth of focus of a high numerical aperture (NA
V
>
1.3) objective is restricted to
0.3
m
m, whereas the depth of a fluorescent cell may be
5-25
m
m, it becomes clear
that the depth of focus will only constitute
1-5% of the full depth of the cell. Since
epifluorescence microscopy captures light along the entire Z-axis, 95-99% of the
