2.1.

Turbid Sample Preparation

Turbid samples made of silicone resin and ${\mathrm{TiO}}_2$ particles (Atlantic Equipment Engineers, Ti-602) as a scattering agent, were prepared according to Ref. 28 to which fluorescent microspheres (Invitrogen FluoSpheres yellow-green, size 1 to 15 μm) were added. These samples were polymerized in petri dishes to obtain a sample size of $35\mathrm{mm}\text{diameter}\times 10\mathrm{mm}\text{thick}$. The reduced scattering and absorption coefficients of the samples were measured at 806 nm as 10 and $0.2{\mathrm{cm}}^{-1}$, respectively, by the method described in Ref. 28. Turbid samples were also prepared by dispersion of fluorescent microspheres and ${\mathrm{TiO}}_2$ particles in gelatin and agar matrices in place of the silicone resin.²⁵ The two types of samples have the same optical properties, but while the agar/gelatin samples are easier to prepare, the silicone resin samples do not degrade and thus can be used for longer periods of time.

2.2.

Specimen Preparation

For in vivo imaging, four to six-week-old $\mathrm{Lgr}5\text{-}{\mathrm{eGFP}}^{+}$ mice were fasted for 24 h prior to being anesthetized with 0.3 cc of ketamine-xylazine given intraperitoneal (IP), and immobilized (with clear tape) on their side on a glass slide. A 1-cm vertical incision was made in the lower left quadrant of the abdomen and a loop of distal small bowel exteriorized. The loop was placed on the surface of the glass slide, with a minimal volume of phosphate buffered saline (PBS) to prevent desiccation of the serosal surface. The prepared animal was placed on the stage of the multiphoton microscope and imaged. At the end of the imaging session, the animal was euthanized.

For ex vivo tissue imaging, the mouse is euthanized. Freshly excised small intestinal tissue is rinsed with PBS and imaged. All imaging occurred within 4 h of euthanasia.

All procedures were reviewed and approved by the UC Irvine Institutional Animal Care and Use Committee (protocol 2002-2357).

2.3.

Two-Photon Microscope System

The principles of operation and characteristics of our two-photon microscope system and its ability to image inside turbid media up to a depth of a few mm have been previously described.^25,26 Here we present a modified upright version of the previous model that is better adapted to image live small animals. The experimental system diagram is shown in Fig. 1. A femtosecond Ti:Sapphire laser (Mai Tai, Spectra-Physics) supplied with a group velocity dispersion (GVD) compensation attachment (DeepSee) is used for the two-photon excitation of the sample. The excitation beam is directed to an acousto-optic modulator (AOM, AA Opto-Electronics MT 110-B50A1) to regulate the power level. Afterward, the beam is passed through an $x$-$y$-scanner (ISS, Model 6220) coupled to an Olympus BX illumination module equipped with a long working distance (7-mm) Olympus LCPlanFl $20x/0.4$ air objective. Focusing and positioning of the sample is achieved via a motorized $x$-$y$-$z$ stage. The two-photon fluorescence is induced by the IR beam focused inside the sample from its top side and collected by the detector (described below) from the opposite side of the sample. In order to protect the extremely sensitive detector from external light, the components are enclosed in a black, light-tight box.

Fig. 1

Schematic diagram of the deep-tissue imaging system. A dispersion compensated Maitai DeepSee laser is attenuated by an acoustic-optic modulator (AOM) and delivered to the sample using a galvano-scanner. The detector is placed in the transmission path of the Olympus BX71 upright microscope.

The detector is the key component of the experimental setup described above. The detector and the principle of its operation are shown schematically in Fig. 2(a). The detector is composed of a head-on photomultiplier tube (PMT) with a wide area photocathode (Hamamatsu PMT R1104, diameter 25 mm, or R7600P-300, $18\times 18\mathrm{mm}$) operating in photon-counting mode. The R1104 PMT is attached to a glass cylinder with mirror-coated walls, which in turn acts as a light-guide by directing fluorescence photons to the PMT photocathode. The R7600P-300 PMT is coupled directly to the optical shutter window without use of light-guide cylinder. The optical shutter is made of two $25\text{-}\times \text{-}3\text{-}\mathrm{mm}$ optical filters (Schott BG-39) that transmit fluorescence, but prevent the excitation light from entering the PMT. A $\sim 0.2\text{-}\mathrm{mm}$ gap between the filters allows the introduction of a thin 0.1-mm aluminum plate to provide a mechanical shutter. All the detector components, including the gap between the filters, are immersed in a refractive index matching fluid to curtail losses of fluorescence photons due to reflection at the boundaries. For the same reason, during measurements, the detector assembly is placed directly in contact with the surface of the sample.

Fig. 2

(a) Fluorescence detection method: developed in this work showing the transmission geometry. (b) The conventional reflectance geometry found in most commercial two-photon fluorescence microscopes.

As shown in Fig. 2(a), the two-photon-induced fluorescence is multiply scattered into the turbid sample and collected by the detector. The multiple scattering aids detection, since photons initially headed away from the detector now have a chance to be redirected and enter the detector. For the same reason, in the multiple-scattering media, where photons constantly change direction, light losses due to total internal reflection at the sample/detector boundary are reduced and fluorescence photons can be captured by the detector from any angle of incidence. In theory, multiple scattering could additionally improve fluorescence detection, since in multiple-scattering media the intensity of the scattered photons decays as the inverse of the distance from the excitation light focus,³ which is less than the inverse of the squared distance decay in clear media. Our detection method has proven to be very efficient in harvesting photons, as indeed no microscope objective has the ability to collect photons from such a wide area.^25,26 However, we acknowledge that our detection geometry does not capture the epi-fluorescence photons, which comprise a fraction of the total fluorescence photons under conditions of deep-tissue two-photon excitation. The diagram in Fig. 2(b) illustrates the principle of operation of a conventional two-photon microscope, where fluorescence photons can only be collected by a microscope objective from a relatively small area and at a narrow angle.

We have also used a commercial two-photon scanning microscope Zeiss LSM 710 to compare results obtained with our instrument.

3.1.

Phantom Studies

Figure 3 presents images of fluorescent beads dispersed in turbid samples (1-cm thickness) and acquired at various depths. Similar results were obtained with samples made of gelatin or agar gel and silicone. Using our fluorescence detection method we were able to image fluorescent beads in turbid samples at depths up to 3 mm [Fig. 3(a)].The same imaging experiment was conducted with a commercial Zeiss LSM 710 two-photon fluorescence microscope with similar objective and excitation light power, obtaining a maximum imaging depth of about 0.5 mm [see Fig. 3(a)]. It is important to note that 0.5 mm is indeed the maximum imaging depth achieved so far in brain tissue,^6,8,27 and the turbid samples we used for imaging had brain-like optical properties (${\mu }_s^{\prime }=10{\mathrm{cm}}^{-1}$, ${\mu }_a=0.2{\mathrm{cm}}^{-1}$).

Fig. 3

Images of fluorescent beads in turbid samples: comparison of imaging depths. (a) 1 μm beads imaged at 200 μm and 450 μm depth obtained with a Zeiss LSM710 with multiphoton optics and using our system at depths of 670, 1090, 1830, 2020, 2630, and 2980 μm, respectively. (b) Detail of an Image of 15 μm beads at 1.83 mm depth. (c) images of two 2 μm beads at 2.23 mm depth (c).

To confirm that our system maintains a high resolution of images at increased depths we prepared turbid samples containing a mixture of 2-μm and 15-μm fluorescent beads. Figure 3(b) shows the image of a 15-μm bead at a depth of 1.83 mm. Figure 3(c) shows two close 2-μm fluorescent beads also at a depth of 2.23 mm. The two beads appear well resolved at this depth and their apparent sizes correspond to the expected ones.

The images depicted in Fig. 3 were acquired using an objective with $\mathrm{NA}=0.4$ (Olympus LCPlanFl $20x/0.4$) for excitation. Because the beam diameter at the focus depends on the numerical aperture, the use of higher NA objectives results in a smaller beam size, which in turn gives a higher excitation light intensity at the focus with an increased imaging depth and resolution. The drawback is that high NA objectives usually have a short working distance. It was shown in Ref. 15 that objectives with an NA of 0.6 to 0.8 are the best choice for two-photon imaging in turbid media.

3.2.

Tissue Imaging

Our system has been successfully employed in the imaging, both in vivo and ex vivo, of adult stem cells at the crypt basis of the murine small intestine and colon. Figure 4 shows the schematic diagram of colon and small intestine along with the imaging setup. In Figs. 5 and 6 the morphology of the normal colon and small intestine tissue is imaged with our two-photon fluorescence microscope.

Fig. 4

Schematic diagrams of (a) small intestine and (b) colon mucosa. (c) Imaging scheme: images were acquired from the outside of the intact gastric lumen; villus (I), crypt (II), lamina propria (III), collagen layer (IV), muscularis mucosa (V), submucosa (VI), muscularis externa (VII, VIII), $\mathrm{Lgr}5\text{-}\mathrm{eGFP}+$ stem cell (IX), and Paneth cell (X).

Fig. 5

Colon images of intrinsic contrast and $\mathrm{Lgr}5\text{-}\mathrm{eGFP}+$ crypts. Progressively deeper images reveal submucosal collagen, then cross sections of GFP-bright crypt bases (in some crypts) that transition into the transit-amplifying compartment and become more attenuated at depth. Field of view $400\times 400\mu \mathrm{m}$. Excitation wavelength 880 nm.

Fig. 6

Small intestine images. White arrows correspond to collagen fibers at 30 μm depth and to individual $\mathrm{Lgr}5\text{-}\mathrm{eGFP}+\mathrm{stem\; cells}$ at 70 μm depth. Field of view $200\times 200\mu \mathrm{m}$. Excitation wavelength 880 nm.

The deep two-photon imaging is performed with an excitation wavelength of 880 nm through the external muscularis mucosa [Fig. 4(c)] up to a depth of 420 μm (Fig. 5). The same sample imaged with the commercial Zeiss LSM 710 microscope could only be imaged up to a depth of 160 μm. Different structures can be discerned at different depths of the colon (Fig. 5) and small intestine (Fig. 6) tissue.

Beginning from the surface of the colon tube we first imaged the muscularis externa, the muscularis mucosa, and the submucosa [Fig. 4(b)].³⁵ In these layers, at 122 and 133 μm depths, the collagen walls of a branching blood vessel can be observed. At 175 and 195 μm, the beginning of the epithelium structure is marked by the presence of the bases of the circular crypts that contain the $\mathrm{Lgr}{5}^{+}\mathrm{GFP}$ stem cells and are surrounded by collagen fibers. Single $\mathrm{Lgr}{5}^{+}\mathrm{GFP}$ stem cells can be seen at 217 μm depth (Fig. 5) within the circular crypt because of their distinctive alternated pattern.²⁹ Below a 217 μm depth, the granular level of the colon is visible with circular crypts and the lamina propria that contains the capillary network.³⁵

The small intestine three-dimensional (3-D) structure presents similarities to the colon, but some differences can be observed.³⁵ The surface of the small intestine exteriorized loop was the starting point from which we first imaged the smooth muscle layer, characterized by smooth muscle cells at depths of 10 and 20 μm. A layer of collagen fibers is located at the base of the Lgr5-GFP stem cells positive crypts at 30 μm depth. Again, single $\mathrm{Lgr}{5}^{+}\mathrm{GFP}$ stem cells are visible at depth 70 μm within the circular crypt with a characteristic pattern of alternating fluorescent $\mathrm{Lgr}{5}^{+}$ cells and nonfluorescent Paneth cells (Fig. 6).²⁹

Imaging at different excitation wavelengths allows one to selectively investigate different intrinsic fluorophores, providing identification of different tissue components. Figure 7 shows the intrinsic contrast of the colon tissue at two different excitation wavelengths at the depth of 80 μm. In Fig. 7(a) at the 880 nm excitation wavelength two $\mathrm{Lgr}{5}^{+}\mathrm{GFP}$ crypts are visible (white arrows) and blood capillaries and collagen are also visible from within the lamina propria. Two-photon fluorescence intensity of the same field of view excited at 740 nm [Fig. 7(b)] highlights the contrast of the metabolic coenzyme nicotinamide adenine dinucleotide (NADH) within single epithelial cells in the round crypt base. Subcellular details such as the dim nuclei and bright mitochondria are distinguishable. Since NADH is the principal electron acceptor in glycolysis and electron donor in oxidative phosphorylation it provides important information about single-cell metabolism.³⁶

Fig. 7

Two-photon fluorescence images of colon from a $\mathrm{Lgr}5\text{-}\mathrm{eGFP}+$ mouse excited at 880 nm (a) and 740 nm (b). Field of view $200\times 200\mu \mathrm{m}$, depth 80 μm.

Deep in vivo imaging of NADH and other fluorophores [Fig. 7(b)] in epithelial cells and myofibroblasts, vascular endothelia, and immune cells within the lamina propria [Fig. 7(a)] could provide important information about single-cell metabolism in the native microenvironment and about physiological processes related to inflammation and cancer in colon and small intestine.

3.3.

Deep-Tissue Fluorescence Imaging

A useful conceptual framework for considerations of fluorescence imaging deep into tissue or other highly scattering media is a separation of the excitation and fluorescence emission processes. Unquestionably, two-photon excitation achieves the highest resolution (microns) images and the deepest penetration depths. Increasing excitation laser power can achieve deeper penetration, but the cost in photodamage to tissue, especially live tissue, is unacceptable. In addition, high laser excitation fluxes induce significant spurious fluorescence signals at the sample surface. In our experience, we can obtain two-photon excitation at a depth of up to 3 mm in a scattering tissue (${\mu }_s^{\prime }=10{\mathrm{cm}}^{-1}$). The excitation efficiency degrades in more highly scattered or inhomogeneous (varying indices of refraction) tissue regions, as does the optical resolution. Significantly, in our transmission geometry the emitted fluorescence photons from the two-photon excitation focal volume can still be detected after traversing an additional $\sim 10\mathrm{mm}$ of scattering media before arriving at the detector. Our success in high resolution (1 μm) imaging at depth in tissue (ex vivo or in vivo) is achieved through the combination of several factors that vastly increase the efficiency of the detection of fluorescence photons. Namely, these factors include a simplified optical path, a large area detector and importantly an index matching fluid for the optical path that minimizes photon loss. The other systems in the literature do not use these successful strategies.^21–23 Our current microscope design relies on transmission geometry, rather than the conventional epi-fluorescence detection through the objective. While the conventional epi-configuration is versatile for whole animal or large-tissue imaging experiments, its deep imaging capabilities are generally constrained to the 100 to 500-μm range. It is noteworthy, that Combs et.al.²³ have adapted their design for an epi-configuration (epiTED) for in vivo imaging of various tissue types (e.g., brain and kidney) with emission collection gains of two or more. Our group is currently adapting our design criteria to the epi-configuration.²⁵ In our transmission geometry we used highly scattered samples of about 1 cm thick. However, the thickness limit is due to the absorption in the material rather than to the scattering properties.