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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Protoc. Author manuscript; available in PMC 2013 July 10.
Published in final edited form as:
PMCID: PMC3707120
NIHMSID: NIHMS486029

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals

Abstract

Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, the size of the objective lenses has limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes into minimally invasive internal organs imaging. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope for visualizing fluorescent cells and microvasculature in various murine organs. We further present longitudinal imaging of immune cells in renal allografts and the tumor development in the colon. The fabrication and integration can be completed in 5–7 hours, and a typical in vivo imaging session takes 1–2 hours.

Keywords: optical probe, microscopy techniques, intravital fluorescence microscopy, graded index lens, mouse imaging

INTRODUCTION

GRIN optical probe for intravital imaging

Intravital fluorescence microscopes have been used to study a variety of cellular-level processes in vivo, such as cell trafficking, intercellular interaction, and vascular changes. However, due to the large size of objective lenses required in these instruments, their applications have been limited mostly to superficial tissues, such as the skin, or to surgically exposed internal organs. To overcome this constraint, endomicroscopy based on miniature optical probes has been developed, providing minimally invasive access to tissues in intact organs (Flusberg et al. 2005). High quality miniature optical probes can be readily fabricated with commercially available GRIN lenses into dimensions (lengths and diameters) that are well-suited for minimally invasive high-resolution imaging in small animals (Kim et al. 2010; Fan et al. 2010). A front-view GRIN probe allows in vivo imaging of internal organs after laparotomy (Fan et al. 2010). A side-view probe is particularly suited for imaging the mucosa of tubular organs such as gastrointestinal tracts and respiratory airways (Kim et al. 2010) through natural orifices.

Comparison with other methods

The advances in micro-optic and fiber-optic technologies led to the emergence of several robust design strategies for miniature optical probes and commercial instruments from, for example, Pentax (Kiesslich et al. 2007), Karl Stortz (Becker et al. 2007), Mauna Kea Technologies (Hsiung et al. 2010), and Olympus (Dela Cruz et al. 2005). The confocal endomicroscope made by Pentax and Optiscan, Ltd. offers spatial resolution similar to a GRIN probe, but the diameter of its scanning-fiber probe is relatively large (3.5–10 mm). Microprobes objectives from Olympus have narrower diameters (1.3–3.5 mm), but the objective has a fixed focal plane which restricts its use in live imaging (Dela Cruz et al. 2010). The Coloview endoscope from Karl Stortz is optimized for tracking the tumor progression in the colon on the macroscopic size scale (Waldner et al. 2011). A handheld flexible fiber-optic probe is commercialized by Mauna Kea Technologies, but its spatial resolution is typically lower than GRIN probes and is confined to the surface of the fiber bundle. In comparison, a GRIN probe provides microscopic resolution in all three dimensions, which is suitable for cellular studies. Thus it allows optical sectioning of thick tissues while possessing the mechanical rigidity adequate for the positional stability of tissue surface during imaging. Also, the view angle can be altered by attaching a prism mirror at the tip, e.g. by 90° for high-resolution side-view imaging of luminal organs. GRIN optical probes, with small diameters (0.35–1 mm), moderate lengths (20–100 mm), and high numerical aperture (0.4–0.6), present themselves as a useful tool for minimally invasive imaging of small animal models such as mice.

Limitations of GRIN probes

As in conventional confocal fluorescence microscopy, the optical penetration depth of a GRIN probe is limited to about 100 μm in most soft tissues. The penetration depth may be improved by a factor two with two-photon imaging and fluorophores emitting fluorescence in the near-infrared region. While the confocal endomicroscopy described here is well suited for imaging the outer layer of an organ, i.e. the cortex or mucosa, deeper or inner regions, such as the medulla, are not easily accessible by an optical probe without inflicting excess tissue damage. A potential solution to reach deeper tissue is to insert the probe physically into the target site (Barretto et al. 2009). With careful insertion, the collateral damage to surrounding tissue could be minimized. Another limitation stems from the mechanical rigidity of the probe, which makes it difficult to reach deep into the intestinal and respiratory tracts, such as the small intestine. This limitation may be mitigated with carefully planned surgical methods, such as a feeding-tube-like cannula (Kim et al. 2010). In addition, the field of view (FOV) of a probe is typically proportional to the diameter of the GRIN lens and is relatively small compared to a standard objective lens. Therefore, a GRIN probe is not suitable for macroscopic imaging. This limitation can be partially overcome by sequentially scanning the probe to acquire a large-area mosaic image.

Experimental design

Design and fabrication of optical probe

In this protocol, we first present a step-by-step guide for the user who needs to fabricate an optical probe. The fabrication is relatively easy to carry out. However, for those who are less skillful in optical polishing and assembly or do not have the necessary tools for fabrication, commercial services are available, where custom designed optical probes can be purchased (e.g. GRINTECH GmbH). In this case, this protocol serves as the general guideline for custom design of GRIN optical probes.

A graded-index lens (GRIN) is a cylindrical glass rod in which the refractive index varies radially. The refractive index increases typically with a parabolic function of radius from the center to the periphery. Due to the particular index profile, a GRIN lens, similarly to a lens waveguide, is able to focus, collimate and relay optical beams. The narrow diameter, cylindrical shape and relatively high numerical aperture (NA) make GRIN lenses a fitting building block for high-resolution miniature probes. An optical probe may comprise of three GRIN lenses (Jung & Schnitzer 2003): a coupling lens (CL), a relay lens (RL), and an imaging lens (IL). Commercial GRIN RL lenses have NA of 0.1–0.2, and CL and IL lenses have NA of 0.4–0.6. All of GRIN lenses are available in small diameters (0.35–1 mm) and specific lengths. In Fig. 1, we illustrate exemplary light paths within two types of optical probes in the front- and side-view configurations, respectively. The length of the CL corresponds to a quarter pitch (P). The RL should be sufficiently long to reach the target tissue, with a length at a multiple of half pitch (Fig. 1c). However, the length should not be overly long because it can result in mechanical fragility and lead to degradation of optical performance due to excessive chromatic and spatial aberrations. For mice imaging, the appropriate length of the RL is 20–30 mm for organs in the abdominal cavity or 60–100 mm for gastrointestinal tract imaging. For the front-view probe, the IL has a pitch of 0.25, and in the case of side-view probe the IL should have a pitch of 0.16–0.17 to compensate for the increased optical path length due to the prism mirror attached. The prism has a base length of 0.7 mm (or a diagonal length of 1 mm) to fit onto the 1-mm GRIN lens. The slanted surface of the prism is coated with metal (aluminum or silver for high reflectivity) to reflect light by 90° after the IL. The CL for a side-view probe can be purchased at custom length from a lens manufacturer or, alternatively, prepared by reducing the length of a 0.25-pitch lens (by 0.8–0.9 mm) to accommodate a 0.7 mm prism.

Figure 1
Schematic of a GRIN optical probe. (a) A front-view optical probe. A 0.25-pitch coupling lens (CL) collects light at the input (from the left) and collimates it onto a relay lens (RL, 1.0 pitch). The collimated output is directed onto a 0.25-pitch imaging ...

The steps of assembling the lenses and prism can be performed in a laboratory equipped with the appropriate tools. For polishing the IL, it is critical to ensure the flatness and parallelism of the lens surfaces to minimize optical distortion. This can be achieved with a manual lapping tool with the help of a custom-made jig (Fig. 2a and Supplementary Fig. 1). For sufficient precision and ease of assembly, we use two stereoscopes arranged in orthogonal directions (Fig. 2a, b). In Fig. 2c, we provide a pictorial outline of the fabrication processes for the front-view probe, with photographs taken at three representative steps. Figure. 2c (i) illustrates the step to glue an IL to a RL by using UV curable epoxy. A small volume of UV epoxy (about 1 μl) is dispensed onto the surface of the RL prior to mounting the lens. The user may need to manipulate the 0.25-pitch lens onto the relay lens using a pair of tweezers to achieve good alignment (within <30 μm) of the lenses with visual feedback using a stereoscope. Once the IL is rested onto the RL, UV light is illuminated to cure the epoxy (Fig. 2c ii). The same steps are repeated on the other side of the relay lens to glue a 0.25-pitch coupling lens. After the completion of the assembly of the lenses, additional protection over the optical probe is necessary. We used a metal sleeve to reinforce the optical probe, which helps to avoid damage to the optical probe during handling and imaging. After the optical probe is inserted into the metal sleeve with care, high thermal-curing epoxy is applied and cured in order to attach the metal sleeve to the optical probe (Fig. 2c iii). The attachment of GRIN lenses for same front-view and side-view probe are the same (Fig. 2d i–ii). For the side view probe, an additional step is required for the attachment of the prism onto the IL using UV curing epoxy (Fig. 2d iii–vi). The fabrication is completed with the insertion of a metal sleeve (Fig. 2d v–vi). Finally, in Fig. 2e we show the completed optical probes.

Figure 2
Probe assembly. (a, b) Photographs for workstation and GRIN lens handling. (c) Major steps for front-view optical probe assembly. (i) UV glue is dispensed onto the relay lens prior to placing the coupling (or imaging) lens onto the relay lens. (ii) After ...

Integration to microscope

The next process is to integrate the probe into a microscope system. Here we describe the steps in detail to assemble mechanical mounts for front- and side-view probes, respectively, and to connect the mount to a custom-built laser-scanning confocal microscope. Additional approaches to adapt the probes into commercial intravital microscopes are also provided.

In Fig. 3a, we show the complete mount for the front-view probe. Most components of the mount are commercially available optomechanical parts. In Fig. 3b, we show the individual components, namely the GRIN optical probe (i), probe holder (ii), XY translation stage (iii), and Z-axis translation stage (iv). Additional components are necessary to attach the probe mount (v) to a microscope. Figure 3c illustrates this process for an exemplary custom-built confocal microscope (Kim et al. 2008; Fan et al. 2010). The objective lens (vi) of the microscope is attached to an independent z translation stage (vii). The z translation stage is attached to a cage cube (viii) with cage rods, and the probe mount (v) is attached to the cage cube (viii) with cage plates (ix) and post (x). The focal plane of the probe can be remotely adjusted with the z translation stage (vii) holding the microscope objective (vi), while the probe remains fixed in position during imaging. Figure 3d shows the complete assembly, where the optical probe (i) is fully integrated into the microscope.

Figure 3
Front-view probe mount attachment (a) Front-view probe mount. (b) Individual components: optical probe (i), probe holder (ii), XY translation stage (iii), and Z-axis translation stage (iv). (c) Assembly process: complete front-view probe mount (v), microscope ...

In Fig. 4a, we present the assembled mount for the side-view probe. A side-view mount consists of several individual parts necessary for translation and rotation control of the probe. Figure 4b shows the individual components: a timing belt (xi), rotation pulley (xii), cage plate with bearing (xiii), and rotation shaft (xiv), in addition to the optical probe (i′), probe holder (ii′), XY translation stage (iii), and Z-axis translation stage (iv). In the assembled mount, the angular position of the side-view probe is controlled with the rotation shaft (xiv). Figure 4c depicts the components used to attach the assembled side-view probe mount (v′) to the custom-built laser-scanning confocal microscope with an objective lens placed in a horizontal plane (Kim et al. 2010). The complete assembly of the side-view probe in the confocal microscope is shown in Fig. 4d (Supplementary Video 1).

Figure 4
Side-view probe mount attachment. (a) Side-view probe mount. (b) Individual components: optical probe (I′), probe holder (ii′), XY translation stage (iii), Z-axis translation stage (iv), timing belt (xi), rotation pulley (xii), cage plate ...

Once the assembly is completed, fine positioning of the probe with respect to the objective lens is necessary. These steps are described in detail in Procedures and Supplementary Video 2. In brief, this process involves centering the probe with respect to the objective lens using the XY translational stage (Figs. 3 iii, 4 iii). The z translational stage is used to bring the proximal surface of the optical probe to a position coincident with the focal plane of the microscope objective. For the initial use of any optical probe, we recommend the user to measure its optical performance. Yhe field of view (FOV), spatial resolution and chromatic aberration can all be characterized with commercially available fluorescent beads. In Box 1, we describe this procedure in detail.

Box 1

Optical characterization of the fabricated probes

The field of view (FOV) and the spatial resolution can be measured using fluorescent samples. The chromatic aberration can be quantified using a multicolor fluorescent sample. Detailed mathematical background for obtaining spatial resolution and FOV can be found in the literature (Cole et al 2011).

Measuring the field of view ● TIMING 30 min

  1. Dilute large fluorescent beads (10–20 μm diameter) in water (0.1–1% vol/vol), dispense 10 μl on a coverslip, and air dry for 20 min.
  2. Position the dried sample on the focal plane of the optical probe. (Step 24)
  3. Pick a fluorescent bead in the view, move the stage laterally to position the fluorescent bead at the edge of the view and record the current position of the stage.
  4. Move the stage in one direction, either x or y, to reposition the fluorescent bead to the edge in the opposing direction and record the position of the stage.
  5. Calculate the difference of the positions to assess the FOV.
  6. Alternatively, a fluorescent sample of known size (i.e. 20 μm fluorescent beads) that is sufficiently larger than the imaging resolution can be used to measure the FOV. (Box 1 Fig. a)
    Note: The typical FOV of a triplet probe is approximately equal to the outer diameter of the lenses divided by the ratio of NA between the relay and imaging lenses. For a front-view probe made with 1-mm-dia. SRL and ILW lenses, the FOV is about 250 μm. The FOV of a side-view probe is slightly larger (270–280 μm) due to the reduced length of imaging lens.

Measuring spatial resolution ● TIMING 1 h

  1. Dilute 0.2 μm fluorescent beads in water (0.1–1% vol/vol), dispense 10 μl on a coverslip, and air dry for 20 min.
  2. Position the dried sample on the focal plane of the optical probe (Box 1 Fig. b; scale bar, 50 μm). Step 24
  3. Move the sample away from the focus until fluorescence is not detectable.
  4. Take the z-stack images of fluorescent beads with a step of 0.5–1 μm until no fluorescence is detected. Typically, z-axis scanning of 20 μm is enough. ▲ CRITICAL Minimal laser power should be used not to photo-bleach the fluorescence dye.
  5. Plot the intensity over z-axis and measure the full-width-half-maximum of the intensity profile, which is defined as the axial resolution.
    Note: The lateral resolution can be easily measured by zooming in on a single 0.2 μm fluorescent bead and measure the full-width-half-maximum of the x or y axis intensity profile. The typical lateral and axial resolutions of a GRIN probe are 1 and 10–15 μm, respectively. If the measured resolution is lower than these values, the optical probe may need to be replaced.

Characterizing chromatic aberration ● TIMING 1 h

  1. Dilute 4 μm multicolor fluorescent beads in water (0.1–1% vol/vol), dispense 10 μl on a coverslip, and air dry for 20 min.
  2. Position the dried sample on the focal plane of the optical probe.
  3. Record the first focal position of the objective lens for the sharpest and brightest image in the “green” fluorescence channel (Box 1 Fig. c, I; wherein the image in the “red” fluorescence channel is highly blurred; scale bar, 50 μm)
  4. Record the second focal position of the objective lens for the sharpest and brightest image in the red fluorescence channel (Box 1 Fig. c, ii; wherein the image in the green channel is highly blurred).
  5. The difference between the first and second focal positions is due to the shift of focal plane due to chromatic aberration of the optical probe and represents the chromatic focal shift between the two wavelengths. Merge the green- and red-channel images obtained at the first and second focal positions, respectively, to obtain a two-color image (Box 1 Fig. c, iii).
    Note: Typically the focal plane for 491- and 532-nm excitation wavelengths differs by approximately 20–40 μm depending on the types and lengths of the GRIN lenses. To compensate chromatic aberration, the focal position of the objective lens is adjusted when changing the excitation wavelength. Separate fluorescence image (maximum fluorescent signal for each channel) are taken from each excitation and focal position and later merged into a single composite image.

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Detachment of the probe from the microscope is performed in the reverse order of the integration process. Typically, the probe mount (Fig. 3a, ,4a)4a) is first removed from the microscope, the probe holder is dismounted from the probe mount, and finally the probe itself is removed. The optical probe can be cleaned and stored until required.

Integration of the optical probes into a different microscope would require a modification of the protocol. In Box 2, we illustrate alternative approaches in adapting the probe mount to a typical commercial microscope.

Box 2

Integration of the probe into a commercial microscope

The integration of a GRIN optical probe into an existing microscope system can be done with minimal modification. Here we describe two integration approaches onto a standard upright microsocpe system. Briefly, the front-view probe can be held in place using a micro-V clamp mounted on an XYZ stage. On the other hand, the side-view probe requires the redirection of the excitation beam to the side (rotate by 90°) using a simple 4f lens system.

Integration of front-view probe ● TIMING 1 h

As illustrated in Box 2 Fig. a, a micro-V clamp is mounted to a three axis motorized translation stage (Sutter Instruments, MP-285) using a standard ½ inch post (Thorlabs, TR series) and ½ inch post holder(Thorlabs, PH series). A front-view optical probe is held in place with a micro V-clamp (Thorlabs, VK 250). Alternatively, a manual stage (Thorlabs, PT3A) can also be used to hold the micro V-clamp.

Integration of side-view probe ● TIMING 4 h

An additional side-view imaging path can be built to accommodate the associated relay optics. The purpose of the side-view microscope adaptor is to flip and relay the conjugate scanning plane, located close to commercial objective mounts by 90 degrees and onto the back aperture of a microscope objective (Box 2 Fig. b). A silver mirror is used to reflect light exiting the microscope mount. The conjugate scanning plane is relayed onto the back aperture of the microscope objective with a 4-f lens system. The first lens is placed at one focal length away from the scanning plane. The second lens is placed at two focal lengths away from first lens. The back aperture of the microscope objective is placed at one focal length away from second lens. A detailed layout of the mount assembly is shown in Box 2 Fig. c. The mount comprises of 30 mm cage system cubes (Thorlabs, C4W; n=2), a rotatable kinematic cage cube platform (Thorlabs, B4C), a 1″ cage cube optic mount (Thorlabs, B5C), optical component threading adapters (SM1S10 or SM1A25), a SM1-threaded 30 mm cage plate, 0.35″ thick (Thorlabs, CP02, n=4), cage assembly rods (Thorlabs, ER1, n=4; ER2, n=2), z-axis translation mount (Thorlabs, SM1Z), a 0.5″ stainless steel optical post (Thorlabs, TR2), and an optical probe holder (Fig. 3b). Box 2 Fig. d shows the complete assembly of the side-view probe in an upright microscope.

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Operation for mouse imaging

The imaging procedure consists of handling live animals and acquiring in vivo fluorescence images/videos in the organs of interest using an optical probe. Here, we briefly describe the general outline of the steps in inserting the front- and side-view probes into anesthetized mice for in vivo imaging. An anesthetized mouse is placed on the plate and its body is kept warm with a heating pad (Fig. 3d). The mouse is immobilized on the animal stage using a flexible adhesive tape. For intra-abdominal organ imaging, the front-view optical probe is inserted through a 3 to 5 mm-long incision in the skin and peritoneum (Fan et al. 2010). While monitoring the fluorescence images, the XYZ animal stage is controlled to transverse the mouse about the probe until the tissue of interest is found. The focal plane of the optical probe is adjusted by controlling the z translation stage (Fig. 3c vii). The objective focus control allows the focal plane in the tissue to be varied without moving the optical probe.

Prior to colon imaging with a side-view probe, the mouse is starved for 24 hours to empty the colon, and immediately before imaging the belly of the mouse is gently swept toward the rectum to squeeze out any stool remaining in the colon. The anesthetized mouse is placed on its back on the plate with its anus facing the optical probe (Fig. 4d) and moved slowly toward the probe by the XYZ translation stage. By orienting the view window of the optical probe toward the ventral side, the position of the tip of the probe within the colon can be observed. It is helpful to monitor the length of the optical probe inserted into the animal for position registration. A side view optical probe is rotated and translated along the mucosa using the customized probe mount (Supplementary Video 3). Once the optical probe is in place, the user may observe the images on a computer display and record images. The acquisition of the images can take part in either a single movie file or multiple still images depending on the need.

MATERIALS

REAGENTS

  • UV curable epoxy (Thorlabs, cat. no. NOA81)
  • High temperature epoxy (Thorlabs, cat. no. 353 NDPK)
  • Epoxy mixing kit (Thorlabs, cat. no. EMK 100)
  • Methanol (Fisher Scientific, cat. no. AC32790-0010)
  • Eye ointment (Opticlox)
  • Distilled water
  • Mice of the preferred mouse strain (7–16 weeks old). In this protocol, we used wild-type C57BL/6 mice (Jackson laboratory, C57BL/6J), MHC class II-GFP mice (kindly gifted from Dr. Boes and Dr. Ploegh), and Tie2-GFP mice (Jackson laboratory, stock no. 003658).
    CRITICAL All animal experiments must be performed in accordance with the guidelines and regulations of the relevant authorities.
  • Ketamine (Ketavest 100 mg/ml, Pfizer)
  • Xylazine (Rompun 2% wt/vol, Bayer Healthcare)
  • Buprenorphine (Reckitt Benckiser Pharmaceuticals)
  • 10–20 μm fluorescent microspheres (Duke Scientific, cat no. G1000B)
  • 4 μm multicolor fluorescent microspheres (Invitrogen, cat. no. T7284; altenatively F36909)
  • 0.2 μm nm fluorescent microspheres (Duke Scientific, cat. no. G200, and R200)
  • Tetramethylrhodamine (TAMRA) dextran conjugate (average mol. wt 2,000,000; Invitrogen, cat. no. D7139)
  • Fluorescein isothiocyanate (FITC) dextran (average mol. wt 2,000,000; Sigma Aldrich, cat. no. FD2000S)
  • Nylon suture (Ethilon 6-0)
  • Polishing film (Thorlabs, cat. no. LFG1P, and LFG5P)

EQUIPMENT

Fabrication of optical probes

  • GRIN lens (GoFoton, formerly NSG America, IL: cat. no. ILH-100, ILW-100 and RL: SRL-100), alternatively, custom probes (GRINTECH GmbH)
  • Prism mirror (Precision Optics Co., cat. no. 8531-601; aluminum coating)
  • Stainless steel sleeve (NSG America; i.d. ~1.02 mm, o.d. 1.25 mm)
  • Stereoscope (Scienscope, cat. no. NZ-BD-T3)
  • Fiber illuminator (Amscope, cat. no. HL250-AY)
  • Polishing pad (Thorlabs, cat. no. CTG913)
  • Polishing disc (Thorlabs, cat. no. D50-SMA)
  • Ferrule (Thorlabs, cat. no. 11040A)
  • Multi-purpose V-groove clamp (Thorlabs, cat. no. HFF001)
  • Lock-down clamps (Thorlabs, cat. no. AMA010)
  • Fixed angle brackets (Thorlabs, cat. no. AMA009)
  • Cross action tweezers (Roboz, cat. no. RS5020, RS5027)
  • Caliper (Thorlabs, cat. no. CPM1)
  • Needles (BD Medical, cat. no. 328418)
  • Kimwipes (Thorlabs, cat. no. KW32)
  • Lens cleaning tissue (Thorlabs, cat. no. MC-5)
  • Dusting kit (Thorlabs, cat. no. CA1)
  • UV curing lamp (Thorlabs, cat. no. CS410-EC)
  • Heating pad (Barnstead, cat. no. HP130915)
  • Glass bead sterilizer (Dent-EQ, BS-500)

Probe holder

Probe mount

  • Single axis translation stage (Newport, cat. no. Gothic-Arch X)
  • XY translation stage (Thorlabs, cat. no. HPT1)
  • Cage mounting adapter (Thorlabs, cat. no. CP02B)
  • Cage assembly rod (Thorlabs, cat. no. ER1; 1″ Long, Ø6 mm)
  • Cage cube (Thorlabs, cat no. C4W)
  • Adapter with external SM1 threads and internal RMS threads (Thorlabs, cat. no. SM1A3)
  • Rotation pulley (SDP/SI, cat. no. A6A51-)
  • Timing belt (SDP/SI, cat. no. A6R51M-)
  • Ball bearing (SDP/SI, cat. no. A7T55-FS5031)
  • Shaft collars (SDP/SI, cat. no. A7X2-13218)
  • Rotation shaft (SDP/SI, cat. no. S4012Y-US2-29)

Probe mount attachment

  • Assembled probe mount (front-view and side-view)
  • Microscope objective (Olympus, LUCPlanF1, Magnification: 40X, NA: 0.6, dry)
  • Cage plate (Thorlabs, cat. no. CP02)
  • Cage assembly rod (Thorlabs, cat. no. ER1; 1″ Long, Ø6 mm)
  • 30 mm cage cube (Thorlabs, cat. no. C4W)
  • Post (Thorlabs, cat. no. TR series)
  • Linear translation cage mount (Thorlabs, cat. no. SM1Z)

Imaging system

  • A confocal microscope system. It is either a replica of the custom-designed confocal microscope (Fan et al. 2010; Kim et al. 2010) or one of the commercial upright confocal or two-photon microscopes (e.g. Zeiss, Axio Examiner; Thorlabs, Multiphoton microscope; Prairie technology, Ultima multiphoton microscope; LaVision BioTec, TriM scope II). ▲ CRITICAL The microscope should have a sufficient work space under its objective lens, more than 7–10 cm, to accommodate the probe mount and animal stage. If the space is insufficient, the sample stage of the microscope needs to be modified (i.e. LaVision BioTec, Intravital microscope stage).
  • Silicone rubber heating sheet (Jxsingtai Electrical)
  • XYZ-axis motorized stage and controller (Sutter Instruments, cat no. MP285)

(Optional) Probe mount for commercial microscope

  • XYZ stage (Sutter Instruments, cat. no. MP285 motorized; or Thorlabs, cat. no. PT3 manual)
  • Micro V-clamp (Thorlabs, cat. no. VK250)
  • Adapter with external SM1 threads and internal RMS threads (Thorlabs, cat. no. SM1A3)
  • Cage plate (Thorlabs, cat. no. CP02)
  • Cage assembly rod (Thorlabs, cat. no. ER1; 1″ Long, Ø6 mm)
  • 30 mm cage cube (Thorlabs, cat. no. C4W)
  • Achromat lenses (Thorlabs, Edmund optics etc.)

REAGENTS SETUP

Animal housing

Prior to imaging, mouse should be housed in a cage with free access to food and water, temperature/humidity control, ventilation, and regular cleaning. After imaging, mouse should be kept in a warm environment and observed until it recovers from anesthesia.

Ketamine/xylazine solution

Mix 1 ml of ketamine (100 mg/ml), 0.15 ml of xylazine (100 mg/ml), and 8 ml of sterile saline in aseptic condition. The mixture volume can be modified depending on the number and weight of mice. ▲ CRITICAL The ketamine/xylazine solution should be prepared and used on the same day.

Fluorescent dyes

Dissolve the FITC-dextran or TAMRA-dextran in sterile saline with 5% wt/vol and filter the solution through a 0.45 μm syringe filter. The solution can be aliquoted (100 μl) and stored in light-proof vials for future use.

High temperature epoxy

Before starting the metal sleeve procedure, mix the thixotropic epoxy (pack A) and the high temperature resistance (pack B) on an aluminum tray. ▲ CRITICAL The mixture should be prepared and used on the same day because the curing starts even in room temperature (18–20 °C).

EQUIPMENT SETUP

Metal sleeve

A stainless sleeve with an inner diameter of 1.02 mm and an outer diameter of 1.25 mm is used for metal sleeve. This is to protect the adjoining points of the optical probe during routine handling and imaging. The metal sleeve needs to be cut and trimmed to the desired length to adequately cover the adjoining points of the fabricated optical probe without exceeding its total length. Metal sleeve can be prepared with a grinding machine. Alternatively, commercial metal cutting services are also available from GRINTECH GmbH or local machine shops. ! CAUTION Care is necessary to avoid physical injury during the cutting and grinding of the metal tube.

Jig

Connect polishing disk (Thorlabs, cat. no. D50-SMA) with SMA connector which has hole diameter of 1.03 mm (Thorlabs, cat. no. 11040A) after removing plastic part of SMA connector (Supplementary Fig. 1).

Optical probe

Prior to imaging, sterilize optical probes with 100% ethanol solution.

Front-view probe holder

A custom-made stainless 25.4 mm diameter disk with a 1.26 mm diameter hole in the center is used for holding the optical probe (Supplementary Fig. 2).

Side-view probe holder

A custom-made stainless tube (4.28 mm OD, 1.2 mm ID) used for holding the optical probe is inserted into a ball bearing that is already glued to a custom-made stainless bearing collar. A rotation pulley is attached to the tube using a set screw. (see Supplementary Fig. 3).

CRITICAL Due to its small features, the optical probe must be handled with care during insertion into the holder via the thru-hole. The outer diameter of the optical probe holder is matched to the internal thread of the XY translation stage (Fig. 3b iii).

Front-view probe mount

An XY translation stage is mounted onto a single axis translation stage (Newport, Gothic-Arch X) using two assembly cage rods (Thorlabs, ER2) and a cage adaptor mount (Thorlabs, CP02B) shown in Fig. 3a.

Side-view probe mount

A probe mount is designed to provide rotation as well as translation of a side-view optical probe as shown in Fig. 4a. The detailed procedure for assembling the attachment mount is illustrated in Supplementary Fig. 4. Briefly, a probe holder comprised of a rotation pulley that is attached to an XY translation cage mount. A rotation shaft is connected with a timing belt, a cage mounting adaptor (Thorlabs, CP02B) and cage assembly rod (Thorlabs, ER1) to the probe holder and XY translation cage mount. The assembled mount is attached to a single-axis translation stage (Newport, Gothic-Arch X) using two cage rods (Thorlabs, ER2) and a cage adaptor mount (Thorlabs, CP02B) (Fig. 4b, c).

Assembly of objective lens mount

The microscope objective and the probe mount are attached to a cage cube (Thorlabs, C4W) (Fig. 3c iii, 4c iii). The microscope objective has an independent focus control, z translation stage, (Thorlabs, SM1Z) that is used to manually shift the focal plane of the objective.

Custom confocal microscope

The imaging system is a custom-built video-rate scanning laser confocal microscope (Kim et al. 2008). The system uses continuous-wave lasers at 491, 532, and 635 nm (Cobalt Laser) for single-photon fluorescence excitation. Silver mirrors and dichroic splitters (Edmund Optics, Chroma Technology) were used to select and direct a laser beam to a raster beam scanner comprising a silver-coated polygon scanner (Lincoln Laser) and a galvanometer (Cambridge Technology). The microscope was designed to have a field of view of 250–300 μm when a 40X objective lens (Olympus, LUCPlanF1) is used. Three photomultiplier tubes (Hamamatsu, R3896) were used to collect fluorescence signals in three fluorescence channels, respectively. An 8-bit four-channel frame grabber (Matrox Solios eA/XA) digitizes the PMT signals at 20_MS/s for 512×512 pixels/frame. Customized software written in C++ is used to display and save the acquired images at a frame rate of 15–30 Hz in real time.

PROCEDURE

Coupling lens assembly ● TIMING 30 min

  • 1|
    Mount a relay lens on a V-groove clamp using a cross-action tweezers (Fig. 2a, b).
  • 2|
    Adjust the focus of a top-viewing stereoscope and a side-viewing stereoscope to coincide at the top surface of the mounted relay lens.
  • 3|
    Partly dip the tip of a 31G syringe needle into UV curable epoxy and extract a small droplet of UV epoxy.
  • 4|
    Apply ~1 μl of UV curable epoxy over the central portion of the relay lens surface with visual inspection through stereoscopes by carefully touching only the epoxy droplet at the syringe needle tip with the relay lens surface (Fig. 2c).
    ! CAUTION Epoxy can cause irritation of the skin. Wear protective gloves when handling epoxy.
    CRITICAL STEP Be careful not to damage the relay lens surface by the syringe needle. This step can be done manually for a skilled experimenter. A XYZ micrometer stage with a mounted syringe tip can be helpful for an unskilled experimenter. In addition, excessive amount of epoxy can cause a tilt at the lens interface in the next step (Step 5). A lens cleansing tissue soaked with 100 % methanol can be used to remove the remaining epoxy and Step 4 can be repeated. Ensure that there is no remaining epoxy or dust on the relay lens surface through a top-viewing stereoscope before repeating Step 3.
  • 5|
    Place a coupling lens (IL) on top of the relay lens (RL) with a cross action tweezers and inspect through stereoscopes to ensure the alignment of the coupling lens and the relay lens (Fig. 2c). A coupling lens will be roughly aligned automatically to minimize surface free energy at the interface.
    CRITICAL STEP This step can significantly affect the imaging performance of an optical probe. If misalignment is observed, correct the displacement by gently touching the coupling lens with a tweezers. If tilt is observed, clean the surfaces of lenses using a lens cleansing tissue soaked with 100% methanol and restart the procedure from Step 3 with reduced amount of epoxy.
  • 6|
    Illuminate the ultraviolet light at the interface using a handheld UV curing lamp (90 mW/ cm2, 365 nm) for 5 min.
    ! CAUTION Wear UV protection goggles. Avoid direct exposure of UV light to the experimenter as the UV light can seriously damage the retina and skin.
    ? TROUBLESHOOTING

Polishing (for side-view optical probe only) ● TIMING 1 h

  • 7|
    Spray distilled water on the polishing pad. Two or three pumps from a spray bottle are adequate.
  • 8|
    Place the 5 μm polishing film on the wet polishing pad. The polishing film should adhere firmly to the polishing pad.
  • 9|
    Apply distilled water on the polishing film enough to soak the whole film and remove the excessive water by a clean tissue.
  • 10|
    Place a polishing disk on the wet polishing film and insert the imaging lens using a cross action tweezers into the ferrule hole in the polishing disk (Supplementary Fig. 1).
  • 11|
    Insert the pushpin into the same ferrule hole and on top of the imaging lens. This is to ensure that the GRIN lens stays in place and in contact with the polishing film during the polishing process.
  • 12|
    Polish the imaging lens by sliding the polishing disk along a path that resembles the Arabic number 8 for 20–50 times and then measure the changes of the imaging lens’ length using a caliper.
    CRITICAL STEP Avoid damage to the imaging lens surface during length measurement with a caliper.
  • 13|
    Repeat Step 10–12 until the imaging lens is about 0.1 mm longer than the desired length (e.g. 1.8 mm for the desired length of 1.7 mm).
    CRITICAL STEP Be careful not to polish beyond the desired length. Frequent length measurement is recommended for an unskilled experimenter.
  • 14|
    Repeat Steps 7–13 with a 1 μm polishing film until the desired length is achieved. Inspect the surface quality and flatness using stereoscopes.
    CRITICAL STEP This step can significantly affect the imaging quality. Ensure the flatness and surface cleanness with stereoscopes. A lens cleansing tissue soaked with 100% methanol can be used to clean the lens surface.
    ? TROUBLESHOOTING

Imaging lens assembly ● TIMING 30 min

  • 15|
    Flip the relay lens with a cross action tweezers and mount on a V-groove clamp.
  • 16|
    Repeat Steps 2–6 with the imaging lens.

Prism mirror assembly (for side-view optical probe only) ● TIMING 30 min

  • 17|
    Repeat Step 3 and apply ~1 μl of UV curable epoxy over the central portion of the imaging lens surface with visual inspection through stereoscopes by carefully touching only the epoxy droplet at the syringe needle tip with the relay lens surface (Fig. 2d).
  • 18|
    Hold the triangular sides of prism with a cross action tweezers and place it on top of the imaging lens.
  • 19|
    Align the prism to fit inside the circular face of the imaging lens by gently touching the prism mirror using a tweezer with visual feedback through a front-viewing stereoscope and then repeat Step 6.
    CRITICAL STEP Use caution not to damage the rectangular sides of the prism. Excessive glue should be removed with a lens cleaning tissue, as it can prevent insertion into the metal sleeve.

Metal sleeve ● TIMING 3 h

  • 20|
    Insert the assembled optical probe into the prepared metal sleeve (see the Equipment setup section for metal sleeve preparation). The assembled optical probe should smoothly slide into the metal sleeve without resistance.
    ? TROUBLESHOOTING
  • 21|
    Apply high temperature epoxy dipped on a syringe needle tip to fill the gap between the inserted optical probe and the metal sleeve. For a side-view optical probe, seal the prism edge with the epoxy to form smooth surface (Fig. 2b).
    CRITICAL STEP Be careful not to cover the optical path with the epoxy or outside the metal sleeve. High-temperature epoxy at the focus of the laser beam can produce strong auto-fluorescence, producing unwanted background noise during imaging.
  • 22|
    Keep the sleeved optical probe on the heating pad at 120 °C for 2 hours. This is to cure the high temperature epoxy.
    ! CAUTION Heating pad can cause burn injury. Use tweezers when handling the sleeved optical probe and avoid direct contact with skin.

Integration of the probe into a microscope (front- and side-view) ● TIMING 1 h

  • 23|
    Secure the optical probe on a custom-designed mount with 3-axis translation control or an additional rotation control for a side-view optical probe (see Figs. 3, ,4;4; Equipment Setup for probe mounting and assembly; see Supplementary Video 1).
    CRITICAL STEP Insertion and fixing of the probe in the holder should be made with great care, because bending or stress can cause damage to the epoxy glue between the GRIN lenses or cracking of the lens itself.
  • 24|
    Adjust the proximal surface of a coupling lens of the optical probe to a position coincident with the focal plane of a microscope’s objective lens. See Supplementary Video 2.
    CRITICAL STEP This step requires precision positioning of the optical probe relative to the objective by adjusting the probe mount and Z-axis translational stage. We recommend the use of fluorescence signal as a feedback for alignment. Drop a small solution of large fluorescent beads (≈ 10–20 μm diameter) onto the distal surface. Adjust the optical probe mount to maximize the fluorescent signal. Once this position is established wash the beads off of the probe surface with physiological saline. Detailed characterization of probe performance can be found in Box 1. In our custom confocal microscope, we can also remove an emission filter temporarily and image the optical probe surfaces using the reflected excitation light as a feedback for probe alignment.
    ! CAUTION Laser can cause serious damage to the retina. Wear a protective goggles and avoid direct eye exposure to laser.
    ? TROUBLESHOOTING
  • 25|
    Adjust the focus control knob such that the objectives to be around 30–50 μm closer to the optical probe. This step ensures probe’s working distance lies within the sample.

Animal preparation ● TIMING 30 min

  • 26|
    Anesthetize the mouse through an intraperitoneal injection of ketamine/xylazine solution (75 mg/kg for ketamine and 15 mg/kg for xylazine) and wait for 2–3 min until the mouse is sufficiently anesthetized.
    CRITICAL All animal experiments must be performed in accordance with the guidelines and regulations of the relevant authorities.
  • 27|
    Apply an eye ointment onto the mouse to avoid cornea damage.
  • 28|
    Remove the hair around target area using a clipper and a hair removal cream.
  • 29|
    Make a 3–5 mm incision on the skin and peritoneum. This step is not required if the optical probe is inserted through a natural opening, such as colon.
  • 30|
    Irrigate a target organ with a 37°C physiological saline.
    CRITICAL STEP This step is important to wash the natural waste or surgical artifact, such as blood and also to lubricate the target tissue. For colon, irrigate several times by injecting ~ 0.5 ml of physiologic saline with a rubber tipped syringe, taking care not to induce bleeding.
  • 31|
    Inject 100 μl of TAMRA dextran or FITC dextran (5% wt/vol in sterile saline) intravenously, if needed. This step is to fluorescently label blood vessel.

Animal imaging ● TIMING 1–2 h

  • 32|
    Place the mouse on a heated translation stage with desired position (Figs. 3d, ,4d4d).
  • 33|
    Move the mouse on the translation stage so that optical probe can be inserted through the incision or natural opening and reach the target tissue (Supplementary Video 3).
    CRITICAL STEP Do not press the target tissue with optical probe to avoid tissue damage, such as bleeding. In addition, care is necessary not to bend the optical probe by pushing against the tissue, because it can cause crack in the individual GRIN lenses in the probe or damage the epoxy glue between lenses.
  • 34|
    Adjust laser power and PMT gain until sufficient signal-to-noise ratio is achieved. Keep laser power as low as possible to prevent photobleaching and tissue damage.
  • 35|
    Acquire images or movies with changing focus, position of mouse stage, and rotation (side-view optical probe only; see Supplementary Video 3).
    ? TROUBLESHOOTING
    CRITICAL STEP Monitor the depth of anesthesia by perform the following checks on reflexes i.e. toe pinching, eyelids blinking, respiration and heart rate.
  • 36|
    Close the incision with a nylon suture and give analgesics such as buprenorphine (0.1 mg/kg; subcutaneously; every 12 hours for 3 days), if needed. For side-view imaging through natural openings, there is no need for suturing.
    CRITICAL STEP In the case of longitudinal studies, it is critical to minimize the stress and infection to the animals due to repeated anesthesia and surgical intervention.

? TROUBLESHOOTING

Troubleshooting advice can be found in Table 1.

TABLE 1
Troubleshooting table.

● TIMING

  • Steps 1–6, Assembling a coupling lens to a relay lens: 30 min
  • Steps 7–14, Polishing an imaging lens to the designed length (for side-view probe only; inexperienced experimenters may need more time): 1 h
  • Steps 15 and 16, Assembling an imaging lens to a relay lens: 30 min
  • Steps 17–19, Assembling a prism mirror (for side-view probe only): 30 min
  • Steps 20–22, Inserting a prepared metal sleeve: 3 h
  • Steps 23–25, Integration of probe into a microscope: 1 h
  • Steps 26–31, Animal preparation: 30 min
  • Steps 32–36, Performing mouse imaging: 1–2 h
  • Box 1, Optical characterization of the probe: 0.5–1 h
  • Box 2, Integration of the probe into a commercial microscope: 1–4 h

ANTICIPATED RESULTS

Front-view probe

We show fluorescence images taken using the front-view optical probe at different sites in various murine organs in vivo. In Fig. 5a–f, we used a transgenic mouse, in which green fluorescent protein (GFP) is expressed under the direction of the major histocompatibility complex (MHC) class II promoter. MHC-II+ GFP+ antigen presenting cells are observed in green, and the vasculature is visualized by intravenously injected dye-dextran conjugates. In Fig. 5g, h, a Tie2-GFP transgenic mouse was imaged, where the endothelial cells of blood vessels are visualized by GFP fluorescence. With the front-view probe, we also performed repeated imaging of immune cells in a mouse model of renal transplantation (Miyajima et al. 2011). A donor kidney was harvested from a MHC class II-GFP mouse in C57BL/6 background strain and then transplanted into a wild-type mouse in BALB/C background strain. The front-view optical probe and the imaging procedures described earlier were used to monitor the donor GFP-expressing antigen-presentation cells in the kidney allograft. The tissues in the kidney expressed relatively strong auto fluorescence, which visualizes kidney tubules in the cortex. At Day 1 after transplantation, a significant number of dendritic shaped GFP+ donor APCs are observed (Fig. 6a). At Day 3, most dendritic GFP cells have entered the capsules, which indicates the process of clearance (Fig. 6b). Time-lapse imaging can allow the migration of immune cells to be tracked in the kidney (Supplementary Fig. 5).

Figure 5
In vivo fluorescence images obtained by a front-view probe. (a–f) Images of MHC Class-II+ GFP+ cells in intestine, spleen, kidney, liver, bladder, and ureter, as indicated. Vasculature is visualized by the fluorescence (red) from the TAMRA-dextran ...
Figure 6
Time-lapse imaging of kidney graft with front-view probe. (a, b) Monitoring of donor-origin MHC Class-II+ GFP+ cells in kidney graft after allogeneic transplantation at day 1 and day 3. Renal tubules are visualized by red autofluorescence. Scale bars, ...

Side-view probe

In Fig. 7a-c, we show fluorescence images taken using the side-view optical probe in the normal mucosa of the descending colon, which show MHC-II+ GFP+ cells, epithelial cells lining the crypt structure, and the blood vasculature visualized by FITC-dextran in the blood stream. A three-dimensional dataset of the vasculature was acquired by scanning the probe over a wide area of the tissue at a speed of 100–200 μm/sec. Rotation and registration of individual images allow a large-area mosaic image to be constructed (Kim et al. 2010; Fig. 7d). The dataset can be rendered in various modes, such as a fly-through presentation (Fig. 7e). Using a similar procedure, vasculature and cellular images can be obtained from the upper gastrointestinal tracts and respiratory tracts (Kim et al. 2010).

Figure 7
In vivo fluorescence images by side-view probe. (a–c) Single field of view images taken in the descending colon. (a) MHC Class-II+ GFP+ cells in the colonic mucosa. (b) Epithelial cells stained with systemically delivered Evans Blue. (c) Vasculature ...

The side-view optical probe is well suited to track the development of colorectal tumors in a spontaneous colorectal cancer mouse model. We used a genetically modified mouse model, in which the adenomatous polyposis coli (Apc) gene in the intestinal cells in the colonic mucosa can be knocked out by administration of adenoviral Cre. The inactivation of Apc is followed by the activation of reporter gene of GFP to enable the long-term tracking and quantifying the growth of Apc-mutated cells in the colon by side-view fluorescence endomicroscopy (Kim et al. 2010). Using the side-view optical probe and the imaging procedures described earlier, we observed multiple GFP expressing polyps at 4 weeks after the viral delivery of Cre (Fig. 8a). Blood vessels are simultaneously visualized by intravenously injected tetramethylrhodamine (TAMRA)-Dextran. At this stage, two micro-nodules with diameters of 50–150 μm are detected, where noticeable changes in vasculature are observed. Two weeks later, the same area can be identified at approximately the same distance from anus, and it is found that one of the nodules has grown to approximately 400 μm in diameter (Fig. 8b). Dilation of blood vessels surrounding the nodules is clearly observed. The injected dye can leak out of the permeable vessels in tumors, which makes it difficult to image the vasculature repeatedly at a short time interval.

Figure 8
Time-lapse imaging of colorectal tumorgenesis with side view probe (a) GFP+ polyps and vasculature at 4 weeks after Apc inactivation by delivery of adenoviral Cre in to the colon. (b) Same region at 6 weeks. Scale bars, 100 μm.

Box 3

Glossary

Conjugate plane:One spatial point is relayed onto a second conjugate/reciprocal spatial point through a set of optical lenses. For optimal laser beam steering, a 4f conjugate lens system is used to relay the pivot point on the beam steering mirrors onto the center of back pupil plane of the microscope objective lens.

Field of view (FOV) refers the extent of observable area through the optical system.

Focal plane indicates the plane, lying on the focal length away from the center of an optical arrangement, in which an image is formed for light focused from infinity.

Numerical aperture (NA) is a dimensionless number that indicates the resolving power of a microscope objective. The number is obtained by multiplying the index of refraction of the medium in front of the microscope objective and sine of half-angle over which the system can accept or emit light.

Optical aberration is a distortion in an optical system that reduces the optical performance as compared to an ideal system. They can arise due to the misalignment or physical imperfection of optical components such as lenses and mirrors. Chromatic aberration refers to a shift of focal plane due to a change in wavelength.

Pitch is defined as the length of the GRIN lens within which a single ray will propagate a full sinusoidal cycle.

Spatial resolution is defined as the shortest distance between two spatially separated points on a specimen that can be resolved by an imaging system as separate entities.

Temporal resolution is defined as the shortest time difference between two separate points captured in succession.

Supplementary Material

Supplementary Figures

Acknowledgments

The authors thank Robert Colvin and Cuffy Chase for providing the mice with renal transplantation, Raju Kucherlapati and Ken Hung for the mouse model of spontaneous colorectal tumor, Charles Lin, Daniel Cote, Lee Kaplan, Mauro Ferrari, and Gou Young Koh for discussions, and David Stevenson for proof-reading of the manuscript. This work was supported by grants from National Institutes of Health (R21AI081010, RC1DK086242, RC2DK088661, U54CA143837, R01AI081734), Department of Defense (FA9550-10-1-0537), and National Research Foundation of Korea (R31-2008-000-10071-0, 2009-352-C00042, 2011-0009503).

Footnotes

AUTHOR CONTRIBUTIONS

JKK, WML, PK, MC, SHY designed and wrote the protocol. KJ and SK contributed figures.

COMPETING FINANTIAL INTERESTS

The authors declare no competing financial interests.

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