1.

Introduction

Both two-photon microscopy (TPM) and optical coherence tomography (OCT) are three-dimensional (3-D) tissue imaging techniques with high imaging depths. TPM is a 3-D microscopic technique based on two-photon excitation and other nonlinear processes, and provides molecular and cellular information of tissues at subcellular resolutions down to a few hundred micrometers deep from the surface.¹ OCT is another 3-D technique based on light back scattering, and provides structural information at $<10$ micrometer resolutions down to a few millimeters deep from the surface.² Since TPM and OCT are based on different contrast mechanisms, their combination is beneficial by providing complementary information. Various methods to combine TPM and OCT or optical coherence microscopy (OCM) have been developed.^3–5 One method was to use single Ti-sapphire lasers with a very short pulse width in the range of 10 fs, for a combined TPM and OCM.³ This method was applied to miniaturized imaging probes with double clad fibers.^3,4 A method of combined TPM and OCT was developed by using two objective lenses of different magnifications for imaging at different length scales.⁵ We recently developed a combined TPM and OCT by using separate light sources for optimal imaging conditions of individual modalities: a wavelength tunable Ti-sapphire laser and a wavelength-swept light source with its center wavelength of $\sim 1310\mathrm{nm}$ for TPM and OCT, respectively.⁶ Both TPM and OCT images were obtained with a single objective lens by controlling illumination or excitation beam sizes. Local cellular distribution within tissues and tissue structure in the surrounding regions were visualized in 3-D. Additional important information of in vivo tissues is vasculature. In tumor microenvironment, abnormal tumor vasculature is one of the hallmarks of cancer. Angiogenesis is required for the growth of tumor and provides route for cancer cell metastasis.⁷ There are various OCT methods for vasculature visualization.

In this article, we developed a combined TPM and angiographic OCT in order to provide vascular information of tissues together with molecular, cellular, and structural information in the same tissue sections. Angiographic OCT was implemented by adapting one of OCT vasculature visualization methods. Combined TPM and angiographic OCT was applied to in vivo imaging of mouse models including a cancer model as demonstration.

2.

Materials and Methods

3.

Results

In vivo mouse brain images with the combined system were shown in Fig. 1. A projected angiographic OCT image in the $x$ to $y$ plane was in (a), and it showed dense vasculature in the brain. FOV of TPM image was marked as a rectangle in (a). A TPM image at 144 μm depth from the surface and its overlaid image with an angiographic OCT image were shown in (b) and (c), respectively. This overlaid angiographic OCT image was not a projection image but the one at a single depth range. TPM image showed neural cells and extracellular matrix (ECM) expressing GFP in the brain. Blood vessels appeared dark in the image due to absence of GFP. TPM image was coregistered well with the angiographic OCT image based on vascular structure. A mosaic image of $3\times 3$ projected angiographic OCT images was shown in (d). Its size was $2\times 2{\mathrm{mm}}^2$ with some overlap between images. Large-sectional vasculature distribution could be visualized.

Fig. 1

(a) Two-photon microscopy (TPM) and angiographic optical coherence tomography (OCT) images of a mouse cranial window model. Angiographic OCT image, (b) TPM image (Video 1, MOV, 1.51 MB) [URL: http://dx.doi.org/10.1117/1.JBO.18.8.080502.1], (c) overlaid angiographic and TPM image in TPM field of view (FOV), and (d) large sectional angiographic OCT image. Black circular shapes in the images are air bubbles.

The images of the mouse ear cancer model were shown in Fig. 2. Both the normal and cancer sections were shown in (a to c) and (d to f), respectively. In each section, an OCT cross-sectional intensity image in the $x$ to $z$ plane, an OCT angiographic image in the $x$ to $y$ plane, and a TPM image in the $x$ to $y$ plane were shown. TPM images were acquired based on intrinsic signal. OCT cross-sectional image of the normal section showed typical layered structures of mouse ear: superficial epidermis and dermis on both sides of the ear and cartilage in the middle as two lines in the image (a). The entire cross section of the mouse ear was shown, and its thickness was $\sim 440\mu \text{m}$. Below the mouse ear, the top surface of a mouse ear holder was shown as an artifact. OCT angiographic image, which was generated by maximum intensity projection, showed vasculature of the mouse ear (b). OCT angiographic image shows regular smooth vascular distribution. A box in the angiographic OCT image indicates TPM FOV. Zoomed TPM image showed dermal layer at 68 μm depth (c). Mostly fibrous structures of ECM were shown, and there were some epidermis left in the image due to surface irregularity.

Fig. 2

Combined TPM and angiographic OCT image of a mouse ear cancer model. Normal and tumor sections are shown in (a–c) and (d–f), respectively. In each section, cross-sectional OCT intensity image (a, d), OCT angiographic image (b, e), and TPM images (c, f) are shown. (Video 2, MOV, 2.15 MB [URL: http://dx.doi.org/10.1117/1.JBO.18.8.080502.2]; Video 3, MOV, 3.18 MB [URL: http://dx.doi.org/10.1117/1.JBO.18.8.080502.3]). Brackets, stars, and arrow heads in (a) and (d) indicate whole thickness of the mouse ear, cartilage inside the mouse ear, and top surface of the mouse holder respectively. $E$ and $D$ in (c) indicated the epithelium and dermis respectively.

In the tumor section, the ear appeared thicker due to cancer cell mass in the cross-sectional image (d). Since the cartilage layer appeared close to the bottom surface, the cancer cells seemed to be located in the upper side of ear. OCT angiographic image showed that large blood vessels were mainly distributed in the periphery of the image, and this might be due to the cancer cell injection (e). TPM image in the tumor section shows some cells in the dermis, which might be injected cancer cells (f).

4.

Discussion

To our knowledge, this is the first demonstration of visualizing both cancer cells distribution and surrounding vasculature within tissues in vivo based on different contrast mechanisms. Combined TPM and angiographic OCT could be useful for visualization of tumor microenvironment. OCT could be used to screen tumor sections rapidly and to identify regions of interest, and then TPM could be used to do high-resolution cellular, molecular imaging at those regions. Various in vivo studies can be performed by using the combined TPM and angiographic OCT, and examples are in vivo studies of tumor or immune response.