Open main menu
Home
Random
Recent changes
Special pages
Community portal
Preferences
About Wikipedia
Disclaimers
Incubator escapee wiki
Search
User menu
Talk
Dark mode
Contributions
Create account
Log in
Editing
Confocal microscopy
(section)
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
== Basic concept == {{refimprove section|date = October 2024}} [[File:Minsky Confocal Reflection Microscope.png|thumb|Confocal point sensor principle from Minsky's patent]] [[Image:MP-30-GFP.jpg|thumb|[[Green Fluorescent Protein|GFP]] fusion protein being expressed in ''Nicotiana benthamiana''. The fluorescence is visible by confocal microscopy.]] The principle of confocal imaging was patented in 1957 by [[Marvin Minsky]]<ref name="Minsky-1961">{{cite patent|country=US|number=3013467|pubdate=1961-12-19|title=Microscopy apparatus|inventor1-last=Minsky|inventor1-first=Marvin}}</ref> and aims to overcome some limitations of traditional wide-field [[fluorescence microscope]]s.<ref>[http://web.media.mit.edu/~minsky/papers/ConfocalMemoir.html Memoir on Inventing the Confocal Scanning Microscope], ''Scanning'' '''10''' (1988), pp128β138.</ref> In a conventional (i.e., wide-field) [[fluorescence microscope]], the entire [[Laboratory specimen|specimen]] is flooded evenly in light from a light source. All parts of the sample can be excited at the same time and the resulting [[fluorescence]] is detected by the microscope's [[photodetector]] or [[camera]] including a large unfocused background part. In contrast, a confocal microscope uses point illumination (see [[Point Spread Function]]) and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal β the name "confocal" stems from this configuration. As only light produced by fluorescence very close to the [[focal plane]] can be detected, the image's [[optical resolution]], particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity β so long [[exposure (photography)|exposure]]s are often required. To offset this drop in signal after the ''pinhole'', the light intensity is detected by a sensitive detector, usually a [[photomultiplier]] tube (PMT) or [[avalanche photodiode]], transforming the light signal into an electrical one.<ref name="Fellers-2007">{{cite web |vauthors=Fellers TJ, Davidson MW | title = Introduction to Confocal Microscopy | work = Olympus Fluoview Resource Center | publisher = National High Magnetic Field Laboratory | year = 2007 | url = http://www.olympusconfocal.com/theory/confocalintro.html | access-date = 2007-07-25}}</ref> As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The beam is scanned across the sample in the horizontal plane by using one or more ([[servomechanism|servo]] controlled) oscillating mirrors. This scanning method usually has a low reaction [[Latency (engineering)|latency]] and the scan speed can be varied. Slower scans provide a better [[signal-to-noise ratio]], resulting in better [[Contrast (vision)|contrast]]. The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the [[numerical aperture]] of the [[objective lens]], but also by the optical properties of the specimen. The thin [[optical sectioning]] possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples. Successive slices make up a 'z-stack', which can either be processed to create a 3D image, or it is merged into a 2D stack (predominately the maximum pixel intensity is taken, other common methods include using the standard deviation or summing the pixels).<ref name="Pawley-2006" /> Confocal microscopy provides the capacity for direct, noninvasive, serial [[optical sectioning]] of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution compared to wide-field microscopy.<ref name="Fellers-2007" /> Biological samples are often treated with [[fluorophore|fluorescent dyes]] to make selected objects visible. However, the actual dye concentration can be low to minimize the disturbance of biological systems: some instruments can track single fluorescent molecules. Also, [[transgenic]] techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, [[green fluorescent protein]] with the protein of interest). Confocal microscopes work on the principle of point excitation in the specimen (diffraction limited spot) and point detection of the resulting fluorescent signal. A pinhole at the detector provides a physical barrier that blocks out-of-focus fluorescence. Only the in-focus, or central spot of the [[Airy disk]], is recorded.
Edit summary
(Briefly describe your changes)
By publishing changes, you agree to the
Terms of Use
, and you irrevocably agree to release your contribution under the
CC BY-SA 4.0 License
and the
GFDL
. You agree that a hyperlink or URL is sufficient attribution under the Creative Commons license.
Cancel
Editing help
(opens in new window)