1.

Introduction

In biological and biomedical studies, most of the events or functions occur in a complex tissue environment and ultimately need to be studied in preparations as intact as possible.^1,2 It is also important to image as deep as possible in living tissues. In brain research, optical imaging technique is still the only way to study neural tissues with micrometer or submicrometer spatial resolution. New methods have been applied to image the brain with submicrometer spatial resolution, among them two-photon (2P) microscopy offers the advantages of deeper tissue penetration³ and less photodamage, such as phototoxicity and photobleaching, in comparison with conventional confocal microscopy. 2P microscopy offers micrometer scale resolution in the brain, whereas magnetic resonance imaging techniques are limited to a millimeter scale. To minimize strong scattering of visible light in tissue and to obtain high quality images of deeper layers of cortex, further investigation on 2P fluorescence technique becomes important.^4,5 Previous approaches to increase imaging depth include optimization of photodetection and implementation of regenerative amplification multiphoton microscopy.^6,7 In comparison with the traditional 2P technique, a newly developed technique is proposed in this study to achieve increased depth of imaging with both excitation and emission wavelengths falling within the “tissue optical window.⁸”

According to Beer’s law, the imaging intensity of the carrying photons (ballistic photons) is determined by the wavelength-dependent scattering length ($l_{\mathrm{s}}$) and absorption length ($l_{\mathrm{a}}$),

The objective of this study was to test the hypothesis that by exciting ${\mathrm{S}}_2$ state fluorescent agents with both pumping and emission wavelength in the “tissue optical window,” the imaging depth in tissue is increased as compared to the traditional technique using the ${\mathrm{S}}_1$ state for imaging in brain tissue.

2.

Materials and Methods

Our study utilized spinach leaves covered by fresh rat brain slices at different thicknesses, and imaged the chlorophyll $\alpha$ (Chl $\alpha$) fluorescence penetrating the brain tissue layer by using 2P fluorescence and ${\mathrm{S}}_2$ technique. All procedures and animal use were approved by the Institutional Animal Care and Use Committee of the City College of New York.

2.1.

Preparation of Spinach Leaf

Spinach leaves were purchased fresh from the local market. Each selected fresh spinach leaf was glued onto a microscope slide. The fresh leaf contains the light-absorbing molecule Chl $\alpha$ and plant organelle chloroplast, which are essential for the process of photosynthesis. It is known that the Chl $\alpha$ strongly absorbs red and blue-violet light from ${\mathrm{S}}_1$ and ${\mathrm{S}}_2$ bands to give the green color of leaves. The absorption of photons could drive the molecules of Chl $\alpha$ from the ground (${\mathrm{S}}_0$) state to the first singlet (${\mathrm{S}}_1$) or excited (${\mathrm{S}}_2$) state, converting photon energy into electronic excitation. There are three ways to obtain the emission of Chl $\alpha$ in far-red light of $\sim 680\mathrm{nm}$, (1) ${\mathrm{S}}_1$ excitation caused by red light at a wavelength of about 630 nm, (2) ${\mathrm{S}}_2$ excitation by violet light at a wavelength of 404 nm,¹² or (3) ${\mathrm{S}}_2$ excitation by 2P at a wavelength of 800 nm, which gives a nonradiative process from ${\mathrm{S}}_2$ to ${\mathrm{S}}_1$ following 2P excitation. Figure 1 illustrates the mechanism of one-photon (1P) and 2P excitation of ${\mathrm{S}}_1$ and ${\mathrm{S}}_2$ bands of Chl $\alpha$ [Jablonski energy level diagram, Fig. 1(a)], and the measured absorption and fluorescence spectra of Chl $\alpha$ [Fig. 1(b)].

Fig. 1

(a) Jablonski diagram of energy level that was pumped to ${\mathrm{S}}_1$ and ${\mathrm{S}}_2$ excited singlet state by 1P or 2P absorption; (b) Chl $\alpha$ spectra of measured absorption (solid line) and fluorescence (dotted line).

The absorbed photons excite Chl $\alpha$ from the ground state (${\mathrm{S}}_0$) to the ${\mathrm{S}}_1$ or ${\mathrm{S}}_2$ excited states, converting photon energy into electronic excitation. The decay of excited Chl $\alpha$ to the ${\mathrm{S}}_0$ state can be achieved by emitting photons from ${\mathrm{S}}_1$ directly or after the nonradiative process from ${\mathrm{S}}_2$ to ${\mathrm{S}}_1$. The latter plays a key role in 2P-excited ${\mathrm{S}}_2$ for deep imaging.

2.3.

Multiphoton Microscope and Image Collection

Twelve-bit two-dimensional images were captured by a multiphoton microscopy system (Prairie Technologies Inc., Middleton, Wisconsin) equipped with a Ti:Sapphire femtosecond laser source ($<140\mathrm{fs}$, Coherent Inc., Santa Clara, California), as illustrated in Fig. 2. The excitation wavelength 800 nm was used to achieve the 2P pumping ${\mathrm{S}}_2$ band of 400 nm and to accomplish fluorescence imaging in Chl $\alpha$’s spectral range of around 680 nm. This is the optimal condition for studying 2P ${\mathrm{S}}_2$ excitation of Chl $\alpha$ due to the strong ${\mathrm{S}}_1$ band absorption and emission at 680 nm. Images of spinach leaves, with regions of interest (ROIs) $469.5\times 469.5{\mu \text{m}}^2$ and $512\times 512$ resolution, were obtained by the 2P microscope with a water immersion objective lens ($20\times$, $\mathrm{NA}=0.5$, Olympus, Center Valley, Pennsylvania) through two-different photomultiplier tube channels, a testing channel and a control channel outfitted with a wide band filter of $685\pm 40$ and $525\pm 35\mathrm{nm}$ (Chroma Technology, Bellows Falls, Vermont), respectively, while other imaging parameters were kept constant.

Fig. 2

Schematic diagram of the two-photon microscopy system for the second singlet (${\mathrm{S}}_2$) state excitation.

3.

Results and Discussion

Figure 3 shows 2P microscopy images of the spinach leaf under the testing and control channels without any tissue covered. Red or green dots inside the cells were likely the chloroplast organelles which contain Chl $\alpha$ and other fluorescent molecules. The red channel represented the 2P ${\mathrm{S}}_2$ state of Chl $\alpha$ and showed a much stronger peak at 685 nm. The 2P microscopy images of spinach leaves covered with 200, 400, and 450 μm freshly cut brain slices under testing and control channels are displayed in Figs. 4(a) and 4(b), 5(a) and 5(b), and 6(a) and 6(b), respectively. The 2P microscopy images of Chl $\alpha$ can be clearly observed under the testing channel with a 200 or 400-μm-thick brain tissue on top, but others cannot be clearly distinguished under the control channel. With a 450-μm brain tissue covering, the testing channel shows some vague profiles of Chl $\alpha$ but no profile visible in the control channel, indicating that brain tissue with a thickness of 450 μm is the maximum penetration depth for the Chl $\alpha$ at ${\mathrm{S}}_2$ state among the slices prepared in our study, while the actual maximum penetration depth could be slightly deeper.

Fig. 3

2P microscopy images of Chl $\alpha$ in spinach leaf without covering tissue. The excitation wavelength was 800 nm. (a) Under $685\pm 40\text{-}\mathrm{nm}$ filter; (b) under $525\pm 35\text{-}\mathrm{nm}$ filter; (c) surface plot of the light intensity under $685\pm 40\text{-}\mathrm{nm}$ filter; (d) surface plot of the light intensity under $525\pm 35\text{-}\mathrm{nm}$ filter.

Fig. 4

2P microscopy images of Chl $\alpha$ in spinach leaf covered with 200-μm brain tissue. The excitation wavelength was 800 nm. (a) Under $685\pm 40\text{-}\mathrm{nm}$ filter; (b) under $525\pm 35\text{-}\mathrm{nm}$ filter; (c) surface plot of the light intensity under $685\pm 40\text{-}\mathrm{nm}$ filter; (d) surface plot of the light intensity under $525\pm 35\text{-}\mathrm{nm}$ filter.

Fig. 5

2P microscopy images of Chl $\alpha$ in spinach leaf covered with 400-μm brain tissue. The excitation wavelength was 800 nm. (a) Under $685\pm 40\text{-}\mathrm{nm}$ filter; (b) under $525\pm 35\text{-}\mathrm{nm}$ filter; (c) surface plot of the light intensity under $685\pm 40\text{-}\mathrm{nm}$ filter; (d) surface plot of the light intensity under $525\pm 35\text{-}\mathrm{nm}$ filter.

Fig. 6

2P microscopy images of Chl $\alpha$ in spinach leaf covered with 450-μm brain tissue. The excitation wavelength was 800 nm. (a) Under $685\pm 40\text{-}\mathrm{nm}$ filter; (b) under $525\pm 35\text{-}\mathrm{nm}$ filter; (c) surface plot of the light intensity under $685\pm 40\text{-}\mathrm{nm}$ filter; (d) surface plot of the light intensity under $525\pm 35\text{-}\mathrm{nm}$ filter.

Figures 3 through 6 show the surface plots of emission intensity in the testing channel (panel c) and control channel (panel d). The vertical axis ($z$-axis) in these plots represents the fluorescent intensity. More regions show higher emission intensity in Figs. 3(c) through 6(c) than those in Figs. 3(d) through 6(d), indicating a much stronger emission intensity of the Chl $\alpha$ under the testing channel (685 nm) over the control channel (525 nm). These surface plot results demonstrate that the optimized 2P microscopy imaging of Chl $\alpha$ at 685 nm was exactly the strong fluorescence peak of Chl $\alpha$ under the 2P ${\mathrm{S}}_2$ state, which leads to an excellent tissue penetration depth of more than 450 μm and with a much better image quality than that at 525 nm (control channel). Although the scattering properties of the brain tissue were likely changed shortly after it was cut into slices, experiments were conducted in a sufficiently oxygenated environment and in an acute way to keep the maximum penetration depth with limited variation and to avoid a reduction in the maximum penetration depth. The technique of combining 2P and ${\mathrm{S}}_2$ to achieve deep tissue imaging can be further optimized and tested with in vivo experiments of the brain vasculature¹³ and neural structures.⁹

In order to quantify the emission through the tissue, in each image, five different ROIs with peak intensity were selected and another five ROIs were also selected from the background. The integrated light intensity of each region was calculated and then averaged for each image as $I_{\text{peak}}$ and $I_{\text{background}}$. Figure 7 shows the normalized intensity $I_{\text{normalized}}=(I_{\text{peak}}-I_{\text{background}})/I_{\text{background}}$ for each test group. As the thickness of the covering tissue increased from 0 to 400 μm, the intensity in the control channel dropped tremendously from 119 to 7, whereas that in the testing channel dropped from 183 to 140.

Fig. 7

Comparison of normalized peak intensity for the testing and control channels covered with 0, 200, and 400-μm-thick brain tissues.

In contrast to diffusion optical tomography that studies the multiple scattering optical imaging,¹⁴ 2P microscopy technique explores the optical imaging using the ballistic and snake light within a number of single scattering events governed by Eq. (1). It is well-known that the depth resolution for light transporting in tissue depends on the scattering coefficient (${\mu }_{\mathrm{s}}$) and absorption coefficient (${\mu }_{\mathrm{a}}$). Within the range of far-red to near infrared, ${\mu }_{\mathrm{s}}\gg {\mu }_{\mathrm{a}}$ in tissue and their inverses lead to the penetration lengths in tissue, where $l_{\mathrm{s}}(=1/{\mu }_{\mathrm{s}})$ is the mean free scattering length and $l_{\mathrm{a}}(=1/{\mu }_{\mathrm{a}})$ is the absorption length. In tissue, the ${\mu }_{\mathrm{s}}$ at 525 nm is larger than that at 685 nm, and therefore a higher depth resolution is expected for 685 nm over 525 nm in 2P microscopes. This led to the objective of the present 2P imaging depth study. In another study,¹⁵ the scattering coefficients were measured for fresh rat brain tissue at different wavelengths, the corresponding $l_{\mathrm{a}}$ and $l_{\mathrm{s}}$ can be calculated as 2 and 0.06 mm, respectively, at a wavelength of 525 nm, and 2.2 and 0.09 mm, respectively, at a wavelength of 685 nm. van der Zee et al.¹⁶ measured ${\mu }_{\mathrm{a}}$ and ${\mu }_{\mathrm{s}}$ of 40-week-old human brain gray matter; the corresponding $l_{\mathrm{a}}$ and $l_{\mathrm{s}}$ at a wavelength of 525 nm are calculated to be 5.88 and 0.02 mm, respectively, at a wavelength of 525 nm, and 20 and 0.025 mm, respectively, at a wavelength of 685 nm. Both studies evidence higher depth resolutions at a wavelength of 685 nm.

In our previous study,⁸ the intensity was almost zero under 525 nm as demonstrated in Fig. 4(b) in Ref. 8, since the beads were passively coated with Chl $\alpha$ by soaking in the Chl $\alpha$-ethyl solution, which could lead to nonuniform distribution of Chl $\alpha$ on the surface of the beads, and did not present enough detectable signal from the shoulder emission at 525 nm. There are no accessory pigments in Chl $\alpha$ soaked beads. The present study used spinach leaves that contain cells and chloroplasts organelles with a high concentration of Chl $\alpha$ and other accessory fluorophores, such as flavins (the emission peak of ${\mathrm{S}}_1$ is close to 525 nm), such that detectable signals could be observed at 525 nm [Fig. 3(b)]. For the first time, the present study imaged Chl $\alpha$ through fresh rat brain tissue layers with 2P ${\mathrm{S}}_2$ excitation where both the emission and excitation were in the “tissue optical window,” and can be applied for further studies.

4.

Conclusions

The 2P ${\mathrm{S}}_2$ excitation of Chl $\alpha$ in chloroplast of spinach leaf under brain tissue provides positive results for a deeper tissue imaging technique in comparison with a traditional 2P microscopy technique. Both the excitation and emission wavelengths fall within the “tissue optical window” and penetrate rat brain tissue up to 450 μm. This is the first study that applies a 2P ${\mathrm{S}}_2$ state technique to investigate the imaging depth of Chl $\alpha$ inside rat brain tissue. This ${\mathrm{S}}_2$ pumping technique can be potentially used as a reference for deeper and better quality images in future studies of brain blood vessels and neural tissue in vivo.