In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.
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 situ 1. 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 vivo 2,7. 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 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.
The authors have nothing to disclose.
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
CCD camera | Point Grey Research | GRAS-14S5M | ||
LED | Thorlabs | M455L2 | Selected for use with proflavine – other fluorophores may require different parts | |
Excitation filter | Semrock | 452/45 | Selected for use with proflavine – other fluorophores may require different parts | |
Emission filter | Semrock | 550/88 | Selected for use with proflavine – other fluorophores may require different parts | |
Dichroic mirror | Chroma | 485 DCLP | Selected for use with proflavine – other fluorophores may require different parts | |
Objective lens | Thorlabs (Olympus) | RMS 10X | ||
Tube lens | Thorlabs | AC-254-150-A1 | Select focal length to achieve required magnification to CCD | |
Condenser lens | Thorlabs | ACL2520 | ||
Cage cube unit | Thorlabs | C6W, B1C, B3C, B5C, SM1CP2 | ||
Cage rods and plates | Thorlabs | ER05 (x4), ER1.5 (x2), ER2 (x2), ER6 (x2), CP02 (x3) | ||
Fold mirror unit | Thorlabs | KCB1, PF10-03-G01 | ||
Lens tubes | Thorlabs | SM1L05, SM1L30, SM1V05 (or SM1Z) | ||
Adapters / couplers | Thorlabs | SM1A3, SM1A9, SM1T2 (x2) | ||
SMA connectors | Thorlabs | SM1SMA, 11040A | ||
LED driver | Thorlabs | LEDD1B TPS001 | ||
Fiber optic bundle | Sumitomo | IGN-08/30 | Larger or smaller bundles are available (Sumitomo / Fujikura) |