Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 248

Fig. 7.5 Lamina cribrosa image with the SHG microscope showing the cribriform structure of
lamina. The SHG contrast comes from the collagen fibrils only. The image is collage of six 512
512 pixel images taken with a 20 /0.75 microscope objective. The scale bar is 100 m

7.5 Conclusion

The nonlinear process of multiphoton imaging used in an optical microscope has
inherent out-of-focus rejection of light and hence gives superior optical sectioning
without a pinhole when compared with a confocal microscope. The lateral resolution
remains close to the diffraction limit of the imaging optics. In general, the interaction
volume in the specimen decreases with an increasing order of the nonlinearity of the
process. For multiphoton absorption processes, this effect is reduced by a relative
increase in the excitation wavelength. Therefore, the real advantage of a multiphoton
microscope is not in the improved resolution but in the noninvasive and in vivo
imaging without any staining required. The nonlinear dependence on the intensity
leads to localized excitation and is ideal for intrinsic optical sectioning in scanning
laser microscopy, for example, the intensity of the SHG signal depends on square
of the incident light intensity, while the intensity of THG signal depends on the
third power of the incident light intensity. SHG microscopy is suitable for imaging
stacked membranes and proteins with organized structures. The THG microscopy
is applicable to imaging cellular or subcellular interfaces. An advantage specific
to CARS microscopy is the ability to achieve chemical specificity without the
addition of exogenous fluorophores. This is possible by making use of resonant
energy levels in the molecules of interest to greatly enhance the desired output signal
and in vivo or in vitro study of live cells without disrupting natural processes. The
main advantage of a multiphoton microscope using coherent processes like SHG
and THG is due to the virtual nature of higher harmonic generation, in which, no
saturation or bleaching results in the signal. Long-term exposure to laser light with
near infrared wavelengths at femtosecond pulses does not compromise the sample
viability, while high-resolution morphological, structural, and functional informa-
tion of biomedical specimens can be obtained. The flexibility of combining the
various nonlinear contrast mechanisms, multiphoton microscopy is likely to become
a major imaging modality in biomedical fields. With the current development of
femtosecond pulsed laser technology, it is to be expected that multiphoton optical