**3.2 Liquid crystal-based photonic crystals for pulse compression and signal enhancement in fluorescence applications**

Multiphoton fluorescence microscopy, devised in 1990, has become an important technology for bio-applications. The improved axial depth and image penetration depth can reduce the bio-sample damage. This approach demonstrates potential applications in bio-imaging *in vivo* [35]. However, the high intensity of the excitation pulsed laser used in multiphoton fluorescence microscopy inducing the photo-damaging in specimens [36]. In the past, the method to reduce the photo damage in the bio-sample with strengthened multiphoton signal employs the excitation laser with narrower pulse widths. Recently, researchers found that the multiphoton fluorescence can be strengthened with the fluorescence signal, which

**63**

**Figure 13.**

*(adapted from [38]).*

*Hybrid Liquid-Crystal/Photonic-Crystal Devices: Current Research and Applications*

is being proportional to the laser pulse widths. In 2001, both scientists McConnell and Riis [37] observed a seven-fold enhancement in two-photon fluorescence by the excitation-compressed laser pulses (~35 fs) [36]. However, by using this laser pulse compression techniques, a dispersion compensator is needed. Thus, the different microscope objectives contribute to different degrees of laser pulse broadening. Recently, Hsiao et al. propose the first PC device enabling on-specimen compression of excited laser pulse. The compression effect occurs after the laser light passing through the objective and photonic components. This will be significant to enhance the multiphoton fluorescence signal. In addition, the PC devices combining with LC materials as defect layer can make the device with the tunable property. From now on, the LC-based PCs for the pulse compression and signal enhancement

Moreover, in order to measure the pulse widths through the PC device onspecimen, an optical autocorrelator was employed in the multiphoton fluorescence microscopy. This new approach allows us to detect the autocorrelation signal at the focal plane of the objective, which are shown in **Figure 13a**. In addition, the Ti:sapphire laser is sent through a 50% beam splitter, and one of the optical beams passes through a variable delay line system. Moreover, the multiphoton fluorescence signals can be detected by a photomultiplier tube (PMT). The autocorrelation signal traces with a peak-to-background ratio of the interferences are 8:1 and shown in **Figure 13b** and **c**. We can observe that the envelope of the interferences is fitted to the function of Gaussian. The original pulse width of the commercial Ti-Sapphire laser is about 100 fs. However, the laser pulse width was broadened to be 270 fs after passing through the optical components and objective, (**Figure 13b**). In addition, the laser pulse width decreases in a nonlinear fashion when we increase the power. The most important is the shortest pulse duration is 30 fs (**Figure 13d**). **Figure 14a** shows the images of the red channel at different exposure time under the operating power 40 mW with device and 150 mW without device. If the PC/LC device was not used, the photo damage becomes apparent when the exposure time beyond 1.5 h. If the proposed PC device was

*(a) The system design of optical autocorrector in multiphoton fluorescence microscopy. HW is half-wave plates, LP stands for linear polarizers, QW is quarter-wave plates, M means mirrors, B is a beam splitter, L is lenses, O stands for the objective, D means dichroic mirrors, and F stands for filters; (b) and (c) are the autocorrection traces without and with the PC devices; (d) pulse duration time versus the applied laser powers* 

*DOI: http://dx.doi.org/10.5772/intechopen.82833*

in multiphoton fluorescence have been proposed.

#### *Hybrid Liquid-Crystal/Photonic-Crystal Devices: Current Research and Applications DOI: http://dx.doi.org/10.5772/intechopen.82833*

is being proportional to the laser pulse widths. In 2001, both scientists McConnell and Riis [37] observed a seven-fold enhancement in two-photon fluorescence by the excitation-compressed laser pulses (~35 fs) [36]. However, by using this laser pulse compression techniques, a dispersion compensator is needed. Thus, the different microscope objectives contribute to different degrees of laser pulse broadening. Recently, Hsiao et al. propose the first PC device enabling on-specimen compression of excited laser pulse. The compression effect occurs after the laser light passing through the objective and photonic components. This will be significant to enhance the multiphoton fluorescence signal. In addition, the PC devices combining with LC materials as defect layer can make the device with the tunable property. From now on, the LC-based PCs for the pulse compression and signal enhancement in multiphoton fluorescence have been proposed.

Moreover, in order to measure the pulse widths through the PC device onspecimen, an optical autocorrelator was employed in the multiphoton fluorescence microscopy. This new approach allows us to detect the autocorrelation signal at the focal plane of the objective, which are shown in **Figure 13a**. In addition, the Ti:sapphire laser is sent through a 50% beam splitter, and one of the optical beams passes through a variable delay line system. Moreover, the multiphoton fluorescence signals can be detected by a photomultiplier tube (PMT). The autocorrelation signal traces with a peak-to-background ratio of the interferences are 8:1 and shown in **Figure 13b** and **c**. We can observe that the envelope of the interferences is fitted to the function of Gaussian. The original pulse width of the commercial Ti-Sapphire laser is about 100 fs. However, the laser pulse width was broadened to be 270 fs after passing through the optical components and objective, (**Figure 13b**). In addition, the laser pulse width decreases in a nonlinear fashion when we increase the power. The most important is the shortest pulse duration is 30 fs (**Figure 13d**). **Figure 14a** shows the images of the red channel at different exposure time under the operating power 40 mW with device and 150 mW without device. If the PC/LC device was not used, the photo damage becomes apparent when the exposure time beyond 1.5 h. If the proposed PC device was

#### **Figure 13.**

*Photonic Crystals - A Glimpse of the Current Research Trends*

pulse in this PC lasing device.

**Figure 12.**

feasible and potential.

white light laser and depicted in **Figure 12c**. One can tell that the novel PC/DDCLC structure can be really lasing in white light. **Figure 12d** shows the relation between lasing intensity of PC device with the pumping energy. The threshold is about 7.4 μJ/

*Spectra of PC/DDCLC. (b) The white-light lasing spectrum and the spectra of PC and the CLC in the planar state. (c) Photograph of a tricolor laser device and the color space coordinates of the PC laser on the CIE 1931 chromaticity diagram. (d) the pumping energy-dependent the lasing emitted from PC/DDCLC device.*

This is the first demonstration of a discrete white light source (three-colors: red, green, and blue) lasing. The organo-inorganic PC/DDCLC cannot only generate three colors in lasers with a single pump, but also be electrically switched among the three modes lasing. With such properties, lasing wavelength can be altered back and forth in a wavelength range and in a very short response time. In addition, PC/DDCLC lasing device is also cost effective, color tunable, and can be fabricated easily. Moreover, it has been shown that the PC device can be pumped using a simple CW laser. The ability to generate a single-color, two-color, three-color or white-light laser makes a new way to full color display, lighting, and other optics applications. By employing PC/DDCLC lasing device, a small size laser system can be achieved to make the proposed PC/DDCLC applications more

**3.2 Liquid crystal-based photonic crystals for pulse compression and signal** 

Multiphoton fluorescence microscopy, devised in 1990, has become an important technology for bio-applications. The improved axial depth and image penetration depth can reduce the bio-sample damage. This approach demonstrates potential applications in bio-imaging *in vivo* [35]. However, the high intensity of the excitation pulsed laser used in multiphoton fluorescence microscopy inducing the photo-damaging in specimens [36]. In the past, the method to reduce the photo

damage in the bio-sample with strengthened multiphoton signal employs

the excitation laser with narrower pulse widths. Recently, researchers found that the multiphoton fluorescence can be strengthened with the fluorescence signal, which

**enhancement in fluorescence applications**

**62**

*(a) The system design of optical autocorrector in multiphoton fluorescence microscopy. HW is half-wave plates, LP stands for linear polarizers, QW is quarter-wave plates, M means mirrors, B is a beam splitter, L is lenses, O stands for the objective, D means dichroic mirrors, and F stands for filters; (b) and (c) are the autocorrection traces without and with the PC devices; (d) pulse duration time versus the applied laser powers (adapted from [38]).*

#### **Figure 14.**

*The photo images of (a) the red and (b) the green fluorescent balls at different illumination time with and without the PC/LC device (adapted from [38]).*

used, the photo damage effect can be easily reduce and lower the excitation power, which is applied to achieve the same signal intensity. We can observe that PC/ LC device can efficiently reduce the both operation power and photo damage. In addition, **Figure 14b** shows the same effect of photo damage reducing in the green fluorescent balls under applied voltage 10 V. Thus, this novel PC/LC device is much more powerful for bio-imaging in photo damage reducing. In addition, this PC/LC device for laser pulse compression does not need any dispersion correction, making the biologists easy to use the PC/LC device.

In conclusion, Dr. Hsiao used a PC/LC to compress the laser pulse, exhibiting a 15-fold enhancement of the fluorescence. Without any dispersion compensator, the PC/LC device can be more convenient for nonphotonic researchers. By using the both pulse compression effect of PCs and the tunability of LCs, the PC/LC device shows a new way to enhance the multiphoton fluorescence microscopy with lower photo damage.
