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Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.
The high-resolution microendoscope described here (figure 1a) should be considered as a base configuration with several variations possible in assembly and application. We describe in detail here an embodiment of the platform which is designed to be used with proflavine as a fluorescent contrast agent. Proflavine is a bright nuclear stain with peak absorption and emission wavelengths of 445 nm and 515 nm respectively. The use of other contrast agents will require the user to select excitation, emission, and dichroic filters appropriately. Several elements of the high-resolution microendoscope are generic and may be obtained from multiple vendors. For example, optomechanical positioning components are available from Thorlabs, Newport, Linos among others. Compact CCD cameras are available from companies including Point Grey Research, Prosilica, and Retiga; camera sensitivity should be chosen with consideration of the brightness of the fluorophore to be used, as well as the desired frame rate. High-power light emitting diodes (LEDs) may be obtained from Luxeon, Cree, and Nichia among others. Fiber-optic bundles are available from Sumitomo, Fujikura, and Schott. In selecting components for a specific application, the user should consider the inherent relationships involved in fluorescence microscopy between fluorophore concentration, photobleaching, illumination intensity, camera sensitivity, gain, and exposure time.
The spatial resolution of the microendoscope can be increased by attaching a micro-lens or lens assembly to the distal tip of the fiber bundle. These optics are configured such that instead of placing the bundle tip directly on to the tissue, the tip is imaged onto the tissue surface with demagnification, thereby increasing the spatial sampling frequency imposed by the light-guiding cores of the fiber bundle. The degree of demagnification corresponds to the increase in spatial resolution, and at the same time, to a proportionate decrease in field-of-view. Gradient index (GRIN) lens components are compatible with fiber-optics and are available from GrinTech, NSG, Schott, among others, and can be directly bonded to the distal tip of a fiber bundle.
When assembled correctly, the microendoscope will operate as an epi-fluorescence microscope, relayed through a coherent fiber-optic bundle. For optimal imaging results, attention should be paid to ensuring that three key conditions are met:
Figure 3a demonstrates imaging of 1483 cells in vitro, following labeling with proflavine and light placement of the bare fiber bundle on the sample. Figure 3b demonstrates the improvement in spatial resolution and reduction in field-of-view provided by a 2.5x GRIN lens bonded to the bundle tip. Movie 1 demonstrates in vivo imaging of the mammary fat pad in a mouse model. Here, a fiber bundle with 0.5 mm outer diameter (330 μm field-of-view) was passed through a 21-gauge needle and advanced into the tissue. Fat cells are clearly visible, with motion due to the cardiac cycle apparent in this acquisition at 15 frames per second. Figure 3c demonstrates imaging of the oral mucosa in a healthy human volunteer, this time using a larger fiber bundle with 1.5 mm outer diameter (1.4 mm field-of-view). In all examples shown, proflavine was used as a nuclear labeling fluorescent contrast agent.
Figure 1. Assembling the high-resolution microendoscope (HRME). (a) Schematic diagram of the HRME system. (b) Assembly of the main optomechanical support structure. (c) Addition of optical elements, illumination LED, and CCD camera. (d) Photograph of the HRME system, packaged in a 10" x 8" x 2.5" enclosure.
Figure 2. Setting up the HRME. Examples of imaging with the fiber-optic bundle in (a) poor focus, (b) close to good focus, (c) ideal focus. In (d), a uniform fluorescent target at the bundle's distal tip is imaged under Kohler (uniform) illumination. (e) A uniform fluorescent target imaged under critical illumination, with the source structure apparent on the object. (f) Loose tissue and cells can stick to the fiber bundle face, which is also prone to minor damage at its periphery.
Figure 3. Imaging with the HRME. (a) 1483 cells in vitro, imaged with a bare fiber bundle (IGN-08/30) following labeling with proflavine 0.01% (w/v). (b) The same 1483 cell culture as shown in (a), imaged with a fiber bundle with 2.5x GRIN lens attached. (c) Image of normal human oral mucosa in vivo, following topical application of proflavine 0.01% (w/v).
Movie 1. Imaging the mammary fat pad of a mouse via insertion of a 450 μm outer diameter fiber bundle within the lumen of a 21-gauge needle passed into the tissue. Proflavine 0.01% (w/v) was delivered to the imaging site through the same needle prior to insertion of the imaging fiber. Click here to watch video
The high-resolution microendoscopy technique described here provides researchers in the basic biomedical and clinical research areas with a flexible, robust, and cost-effective method for visualizing cellular detail in situ. We have described a protocol for assembling the imaging system and demonstrated its use in cell culture in vitro, and in animal, and human tissues in vivo. While the imaging results presented here used proflavine as a fluorescent contrast agent, other groups have demonstrated versions of the system with LED illumination wavelengths and filters chosen to match excitation / emission spectra of other dyes 5-7.
Resolution and field-of-view are initially determined by the core-to-core spacing and imaging diameter of the fiber-optic bundle. We have used bundles with approximately 4 μm core-core spacing, and imaging diameters of 330 μm (movie 1), 720 μm (Figure 2, Figure 3a,b), and 1400 μm (Figure 3c). The smaller bundles can be passed through narrower gauge needles and are significantly more flexible than the larger fibers. We and others 8 have, in some cases, noted the appearance of autofluorescence emissions from the fiber bundle itself. When attempting to excite fluorophores at UV wavelengths, or collect emission in the red spectral range, attention should be paid to the level of fiber bundle autofluorescence contributing to the overall measured signal.
While most of the high-resolution microendoscopy work reported to-date has used a bare fiber bundle, additional magnification can be provided by use of GRIN lenses bonded to the distal tip. GRIN lenses offer a straightforward and economical way to increase spatial resolution, though their susceptibility to optical aberrations and limited NA is well recognized. If GRIN lens performance is inadequate for a particular application, hybrid GRIN / spherical lens objectives 9 or miniature objective lens assemblies 10-11 can be employed.
The high-resolution microendoscope described here essentially operates as a wide-field epi-fluorescence microscope; therefore no optical sectioning (as in confocal or nonlinear microscopy) is to be expected. In our experience, using 455 nm excitation light and topical proflavine as a contrast agent, light is primarily collected from a depth corresponding to a few cell layers.
This protocol ought to enable the reader to assemble the high-resolution microendoscope on the benchtop, with a compact footprint of 10" x 8". If desired, the system may be enclosed in a box and the electrical components (LED and camera) powered by a battery pack (Figure 1d). Many compact cameras can be powered by the IEEE-1394 (Firewire) and USB ports of the host computer.
MP and DY have nothing to disclose. RRK holds patents relating to microendoscopic imaging platforms.
This research was partly funded by the National Institutes of Health, grant R01 EB007594, the Department of Defense Breast Cancer Research Program, proposal BCO74699P7, and the Susan G. Komen Foundation grant 26152/98188972.