Q1. What is the difference between Confocal and Multi-Photon Microscopy?
In 1-photon fluorescence excitation, a single photon has sufficient energy to excite the fluorescence molecule from the ground state to the excited state, (A - left). The excited molecule then relaxes to a state from which it decays back to ground state with the emission of a longer wavelength photon. In single photon confocal microscopy, excitation is present above a below the plane of focus causing labelled structures in this region to fluorescence (A - right). In this case the optical section is generated by refocusing the emitted light back through confocal apertures or pinholes. A sub-resolution green fluorescent object is depicted in the focal volume.
In multi-photon fluorescence excitation, 2 or more photons, which individually have insufficient energy to excite the fluorescent molecule, interact co-operatively to achieve excitation (B - left). The excitation process depends on 2 or more photons arriving in a very short space of time (10-16 seconds). As in 1-photon fluorescence, the excited molecule relaxes to a state from which it decays back to ground state with the same emission wavelength spectrum as in 1-photon excitation. In multi-photon microscopy ultra-short pulses of infrared excitation produce fluorophore excitation only in the focal plane where the photon density is sufficiently high (B - right). Excitation does not occur to labelled structures above or below the plane of focus. Since multi-photon excitation generates an image at a single plane within the sample all of the emitted light can be collected and it is therefore unnecessary to refocus the emitted light back through confocal apertures.
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Q2. What dyes can be used in Multi-Photon Microscopy?
Most of the dyes used in confocal microscopy can be used in multi-photon microscopy. The main exceptions are the far red dyes such as Cy5 which are not good with multi-photon.
The above figure shows examples of some dye multi-photon cross sections (efficiency of excitation) at different wavelengths. It is of interest to note that some dyes have broad wavelength ranges of excitation resulting in the possibility to image more than one dye at one wavelength of excitation. For example, at an excitation of 750nm DAPI, fluorescein and rhodamine can be excited. The emitted light from these dyes is then separated as in confocal microscopy. For details of other dyes please refer to Xu et al, (1996) Bioimaging 4, 198-207 and Xu et al, (1996) PNAS 93, 10763-10768.
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Q3. What are Non Descanned Detectors?
In confocal microscopy, optical sectioning is generated by refocusing the emitted light back through confocal apertures, which requires that the beam is descanned back through the scanning system. By its nature, multi-photon excitation, and therefore fluorescence emission, occurs at the focal plane of the exciting laser. It is therefore unnecessary to descan the emitted fluorescence and it can be collected directly.
Figure 1
All biological tissue scatters light. The degree of scattering depends on the depth of imaging and the type of tissue. Because of this scattering, a significant percentage of the emitted fluorescence cannot be imaged in a confocal system. Figure 1 shows how the fluorescent signal is attenuated due to scattering as a function of depth into the tissue. At a depth of as little as 100 mm, 70% of the fluorescence signal is lost due to scattering within the sample. In order to efficiently image from deep within tissue, non descanned detectors are optimised to collect as much of this scattered light as possible.
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 Q1. Difference between Confocal and Multi-Photon
Q2. Dyes
Q3. Non Descanned Detectors |