MULTIPHOTON LASER SCANNING MICROSCOPY I
LSM 510 META NLO
ntroduction to Multiphoton Laser Scanning Microscopy
Carl Zeiss
03/06
B 45-0021 e
9-9
The lack of out-of-focus excitation greatly reduces bleaching, and therefore, photodamage, throughout
the sample. This reduces damage caused by repeated or slow scans; however, photobleaching at the
focal plane is still present. In fact, some reports indicate that bleaching may be accelerated at the plane of
focus using multiphoton excitation (Patterson and Piston, 2000). Some essential, endogenous molecules
within the cell can absorb UV or visible range photons (such as NAD, FADH etc.), which can destroy and
deplete these molecules. Thus, it can be safer for vital imaging to use an excitation source outside of the
visible range like a NIR laser. A profound example of this effect is seen in a comparative study performed
by Squirrell and colleagues (Squirrell et al. 1999), where the vitality of cleavage stage hamster embryos
was assessed after repeated exposure to visible range laser light and pulsed 1047 nm laser light. In these
experiments, confocal imaging resulted in arrested cellular division and embryo lethality, whereas imaging
using multiphoton microscopy resulted in much less embryo lethality and better data collection. In fact, at
least one embryo imaged in this way was able to develop into a completely normal adult hamster named
laser, illustrating the strength of this technique.
The use of NIR light has the additional benefit of being able to penetrate deeper into tissue than visible
wavelength light. Compared to confocal microscopy, excitation can be achieved in deeper positions of
the specimen and more data along the z-axis can be obtained. However, particularly in deep tissue
imaging, care must be taken to recover as many emission photons as possible. While the incident NIR
light has an advantage over visible range excitation sources, the photons that are emitted are at visible
wavelengths and have the potential to be scattered or absorbed by the tissue. To improve the efficiency
of collection, high numerical aperture (NA) objectives should be used, although it is often difficult to
obtain lenses that have NAs above 1 with a working distance longer than 250 µm. In addition, non-
descanned detectors (NDDs), which collect photons at a point closer to the specimen and do not require
the emission to be focused back through the scan mechanism, can be used to improve deep tissue
imaging by improving the collection efficiency of scattered photons
9.2.3
Drawbacks of using NIR light for microscopy
Although the use of NIR photons has many advantages, there is a distinct disadvantage to this mode of
excitation.
Absorption of NIR light by water and some other particular molecules (such as melanocytes or condensed
particles such as calcium carbonate crystals) can create dramatic local heating effects within the sample.
This effect increases as the overall power from the laser is increased at the sample. It is very important to
use minimized power levels to reduce the effects of local heating. For live samples, power levels above
6 mW may disturb cell replication (König et al. 1996) or even cause cells to explode, as in the case of
melanocytes in zebrafish embryos. Effects of local heating can not only damage cells, but can also
contribute to image artifacts (see Section 9.3.5. Samples your mother should have warned you about). As
you will see in the next section, optimizing the pulse length at the specimen can improve multiphoton
excitation without raising the average power.
