Laser scanning microscopy as a versatile platform

Laser Scanning Microscopy as a Versatile Platform: Imaging based on Nonlinear and Ultrafast Effects

Fu-Jen Kao* Institute of Biophotonics Engineering, National Yang Ming University, Taipei 112, Taiwan Institute of Electro-Optical Engineering, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

ABSTRACT The nature of scanning image acquisition greatly facilitates incorporation of various contrast signals for imaging and integration with techniques in signal processing. In this study, we are reporting imaging modalities and techniques based on nonlinear optical and ultrafast effects that are excited by ultrafast laser. Additionally, the use of signal processing electronics allows signal conditioning techniques, such as dithering, lock-in detection,Ö etc, to be employed so that better signal to noise ratio can be resulted and special features in imaging can be emphasized. Keywords: Optical parametric oscillator, second harmonic generation, third harmonic generation, radio frequency, optical beam induced current, dithering, laser scanning microscopy

1. INTRODUCTION Scanning optical microscopy has become a versatile and powerful tool in many disciplines. The optical sectioning capability is increasingly applied in the study of three-dimensional structures commonly encountered in biology, medicine and material sciences. The employment of ultrafast lasers as the excitation light sources has enabled imaging contrasts based on multi-photon excitations, such as 2-p fluorescence, second- and third harmonic generation, coherent anti-Stoke Raman scattering[1]. In this study, the output from a sync-pumped OPO at 1240 nm is used to demonstrate imaging that is based on second- and third harmonic generation. It is well known that all materials possess non-vanishing third-order susceptibilities. However, if the medium at the focal point of the scanning laser beam is homogeneous, the third harmonic generated before and after the focal point interferes destructively and results in no net signal. Non-vanishing THG will result if there is inhomogenieties at the focal point[2]. This characteristic makes THG an ideal probe for interfaces and boundaries. On the other hand, intense SHG has long been observed on biological samples with the so-called bio-photonic structures[3]. We are presenting here a case study of dental sections with laser scanning THG and SHG microscopy[4-10]. Teeth are the hardest and most indestructible part in human body[11]. Their ordered structures and mineral-like properties make them an ideal subject to demonstrate harmonic microscopy on. Scanning image acquisition is effectively a sampling process, in which a high definition point spread function is used to sample an object so that a clear 2D, 3D, or even multi-dimensional image can be reconstructed from the accumulated data set. In this view, it is natural to employ techniques in signal processing to condition and to improve the signals for imaging. For instance, the very short temporal width of ultrafast laser pulses enables generation of very high frequency current on fast photo-detectors[12]. Through these unique capabilities we introduces the novel method of optical beam induced current (OBIC)[13-17] that is used to map the frequency transfer properties of optically active electronic devices. This technique is based on and is hence termed radio frequency (RF) OBIC[12]. We have demonstrated this novel RF OBIC contrast on a PIN photodiode. These results were then compared to DC OBIC generation. The DC response of the particular photodiode revealed a uniform spatial profile, whereas the spatial distribution of the response at RF excitation showed radial symmetry with center located at the wire-bonded electrode. In addition, we are demonstrating the use of lock-in techniques in detecting differential signals[18-20]. * Tel: (02) 2820-4624, Fax: (02) 2823-5460, Email: [email protected]

Information Optics and Photonics Technology, edited by Guoguang Mu, Francis T. S. Yu, Suganda Jutamulia, Proceedings of SPIE Vol. 5642 (SPIE, Bellingham, WA, 2005) · 0277-786X/05/$15 · doi: 10.1117/12.569454

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2. HARMONICS AND UVB EQUIVALENT 2-P IMAGING 2.1 OPO and laser scanning microscope setup In harmonic generation imaging, a KTP based sync-pumped OPO (Mira-OPO, Coherent) is used to generate the available spectral range in 1.05~1.3 µm for harmonic excitation and 525~650 nm (the signal waveís frequency double) for 2-p UV fluorescence excitation as shown in Fig. 1. The added spectral range enables multiphoton processes that were beyond the reach of a Ti:sapphire laser, which is commonly employed in 2-p confocal microscopy. The pulse width is approximately 150 femtoseconds and the repetition rate is 76 MHz. Output power of 200 mW or less from the above laser system is sufficient for various nonlinear optical excitations. Sets of IR cut-off and bandpass filters are used to discriminate SH and TH against background and other signals. THG and SHG are collected in transmission mode to maximize signal strength due to their forward scattering nature. A set of objectives with NA ranging from 0.4 to 1.2 is used for both microscopy and micro-spectroscopy. The harmonic signalsí authenticity is verified by tuning the pumping wavelength and observing the corresponding spectral shift. SHG in the dental sections is produced with pulses in both 700-920nm and 1050-1300nm while THG is only observed with pulses in 1050-1300nm. No THG is found with 700-920nm excitation in the samples used.

Figure 1: Spectral distribution of commonly used lasers for scanning microscopy. Note the available spectral range of the syncpumped OPO and mode-locked Ti:sapphire laser.

A customized microscope setup capable of both transmission and reflection detection is shown in Fig. 2. Specialized projection lenses are employed so that power loss in the chain of coupling optics can be minimized. A fiber coupling based miniature spectrometer (USB2000, Ocean Optics) is also integrated into the system to facilitate spectroscopic analysis. 2.2 Harmonic generation microscopy of dental sections Human teeth consist of three primary components: enamel, dentin and pulp. The outer surface of a tooth is covered by a thin and transparent layer of enamel that is made of calcium salts in the form of hydroxyapatite crystals.

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Figure 2: Multiphoton microscopy and micro-spectroscopy setup.

The microcrystals form enamel prisms or rods with 4-6 µm transverse dimensions. The interprismatic space is filled with proteins that form an organic matrix[11]. The microscopic SHG and THG images near the dentinoenamel junction of a dental section are shown in Fig. 3(a) and (b), respectively. Strong SHG and THG are produced in dentin. Microtubule structures there can be clearly seen in the THG image. The SHG image, however, does not exhibit specific features and may simply reflect the distribution of collagen. For comparison, much weaker THG and no SHG are found in the enamel, which forms the dark part in the upper-left corner of the image. The prismatic structures in enamel can be clearly seen in the THG image of enamel, shown in Fig. 3(c) with adjusted contrast. Enamel, however, does generate fluorescence covering from 450 nm to 550 nm under 2-photon excitation of Ti:sapphire laser or single photon excitation from UV with wavelength less than 365 nm, as reported earlier[21,22]. Enamel

Enamel

Enamel

Dentin

Dentin (a)

(b)

(c)

Figure 3: (a) SHG image acquired near the dentinoenamel junction (b) corresponding THG image (c) THG image of enamel. Note that a much higher electronic gain was used since THG is a lot weaker in enamel than in dentin


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The spectra of harmonics from dentin and enamel are shown in Fig. 4 (a) and (b), respectively. Note that THG is much stronger (~7X) in dentin than in enamel. SHG is only found in dentin. Strong SH has been found on various biological specimens, such as collagen, potato starch, and skeletal muscles[3]. These materials all possess periodical nano-structures that are often referred as (nonlinear) bio-photonic structures. In particular, collagen is an extracellular structural protein and is a major component of bone, cartilage, skin, and other tissues. Collagen fibrils have a triple-helical structure and it has been shown that this structure enables collagen to generate SH from a wide range of wavelengths in the infrared region. The SH responses from dentin and collagen are compared in Fig. 4(c) and 4(d). The sharp rise in SH signal during 820-828 nm for dentin and 796-806nm for collagen indicates the existence of resonance effects, as shown in the inset of Fig. 4 (c) and (d). The 2-p auto-fluorescence, on the other hand, stays the same both in spectral profile and signal strength. For incident wavelength longer than 830 nm, the SH signal greatly surpasses that of 2-p auto-fluorescence and becomes the dominant signal.

Dentin

Enamel

(a)

(b)

(c)

(d)

Figure 4: THG and SHG spectra measured at (a) dentin (b) enamel near the dentinoenamel junction. (c) SHG and 2-p fluorescence from dentin (d) SHG and 2-p fluorescence from collagen (Type I). The corresponding excitation spectra of SHG are shown in the inset in (c) and (d).

3. USE OF SIGNAL PROCESSING IN SCANNING IMAGING 3.1 Radio frequency optical beam induced current (RFOBIC) In addition to nonlinear optical excitation, ultrafast laser pulses will generate current at radio frequencies when a photo-diode is irradiated. The principle of OBIC is simple: a tightly focused pulsed laser light is shone on the surface of a photosensitive semiconductor device. The relative position of the illumination and the specimen is changed in a raster scanning fashion. The signal, corresponding to a given spatial location, is obtained via the electrical contact

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and a bias voltage applied on the device. As shown in the power spectra in Fig. 5, the spacing between adjacent spectral peaks is equal to the pulse repetition rate (~76 MHz) of the laser. Overall, the RF spectra trail off at 3.0 GHz, reflecting the diodeís sub-nanosecond response. At low bias voltage, Fig.5(c) 4.0V, the high frequency response (1.5GHz~3.0GHz) of the photodiode is suppressed. As shown in Fig. 5(d)-(f), the high frequency response is substantially improved with increasing bias voltage, while the low frequency (0. It is straightforward to carry out the calculation and obtain

I sig (t ) =

where

R0 i1

α2 +ω2

cos(ωt − φ ) ,

(2)

ω φ = tan −1 ( ) is the phase shift and contains the information of response time. The response time, τ α

, can

then be easily derived by the following expression,

τ=

1

α

=

1

ω

tan(φ ) .

(3)

Note that the response of the specimen is represented by R(t), with a single primary time constant so that the analysis can be simplified. Experimentally the pertinent phase information, φ , can be easily obtained by employing a duel phase lock-in amplifier, in this case a SR830 made by Stanford Research. The duel channels within the lock-in allow simultaneous acquisition of the detected signal at two different phases, i.e. V ∝ R0i1 cos(θ + δ ) and X

α2 +ω2

sin(θ + δ ) , where δ represents the total phase delay introduced by the optical and electronic system as a α2 +ω2 whole. In this way, signals from the two channels can be used to calculate the phase and thus the response time directly. VY ∝

R0i1

A high-speed 1550nm traveling-wave electroabsorption modulator (TWEAM) is used as the specimen and the photocurrent from it is used as the contrast signal. The photo-current is fed into the lock-in amplifier that used the signal from the function generator as the reference. Two of the input channels in the confocal microscope (with a maximum number of 5 channels) are used to take the duel outputs from the lock-in amplifier, VX and VY. In this way, mapping at two different phases can be accomplished simultaneously. The two correlated images thus obtained can then be used to calculate the corresponding response time according to Eqs. (2) and (3). As shown in Fig. 8, the contrast of the photocurrent images is greatly improved with the employment of lock-in detection.

320


(a)

(b)

Figure 8: Improvement of scanning OBIC images with the use of lock-in detection. (a) A conventional OBIC image obtained from the TWEAM device. Note the large background noise. (b) OBIC image obtained with modulation and lock-in detection.

4. CONCLUSIONS In conclusion, scanning optical microscopy presents to be a versatile platform to build images on various contrast mechanisms. This unique capability has made it a powerful tool in numerous disciplines. Through the employment of OPO, we have demonstrated the use of harmonic generation in imaging the enamel and dentin sections. These novel imaging modes reveal unique contrast and do not suffer from photobleaching. The non-invasive nature also enables live sample observation. The deep penetration depth due to the long wavelength used allows high quality and optically sectioned SH and TH image acquisition without denaturing sample preparation procedures, such as polishing, decalcifying, and dye labeling. As expected, THG is sensitive to interfaces and boundaries while SHG is attributed to collagen, one of the many interesting bio-photonic structures. In addition, we have found that the intensity of SHG from collagen and dentin depends strongly on the incident wavelengths. Though the exact causes are to be further identified. The RF OBIC method is expected to find further applications in non-contact radio frequency injection, near-field radio frequency generation, and high-speed device characterization. In addition, the high peak power of the ultrafast laser pulse also allows generation of photocurrent based on multi-photon excitation, which is the foundation of multiphoton confocal microscopy. This new application of ultrafast lasers in optical microscopy might provide insight currently not available through conventional methods.[26] Dithering is different from image processing, which is performed after image acquisition and does not increase the dynamic range of the original image. Contrast enhancement through dithering and lock-in detection is particularly evident at high spatial frequency due to the differential nature of the signal processing. However, the absolute spatial resolution remains the same as a result such signal processing, i.e. cut-off frequency is not extended. It is expected that the principles of dithering can be further applied to other scanning microscopy. We have also shown that through modulation and lock-in detection, the S/N ratio of the acquired images is greatly enhanced and the corresponding temporal response can be obtained from the phase information. This method permits rapid measurement of single or primary lifetime components and presents an effective approach toward lifetime or response time imaging. It is simple, robust and easy to be implemented when compared with many other methods. The nature of scanning laser microscopy allows easy integration with techniques in signal processing and presents numerous opportunities worth looking into. Many tasks remain, however, in fully developing these signals and techniques into reliable and useful tools for basic research and/or diagnosis. For instance, much more work is required to fully quantify and relate the conditions of samples to the signals so obtained. Nonetheless these developments have demonstrated to be new, noninvasive, and effective methods in probing a wide variety of subjects. Proc. of SPIE Vol. 5642

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ACKNOWLEDGMENTS Support of this research by the National Science Council of Taiwan under grant No. NSC91-2112-M-110-009 and NSC91-2736-L-110-001, and by the Ministry of Education of Taiwan under grant No. 89-B-FA08-1-4 is gratefully acknowledged.

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