Traditionally biological and clinical studies that require microscopic analysis at cellular level resolution have been performed by running multiple experiments in parallel with individual samples removed from the study at sequential time Points for evaluation by light microscopy while confocal, multiphoton and second harmonic microscopy are all useful techniques for imaging in situ. The required infrastructure is complex and expensive involving scanning laser systems and complex light sources. Here we demonstrate how to build a high resolution fiber optic micro endoscope system in a single day, using off the shelf components for under 5, 000 US dollars.
The platform offers flexibility in terms of image resolution, field of view, and operating wavelength. First, a simple epi fluorescence microscope frame is assembled to accommodate a light source optical filters lenses, a fiber optic bundle, and a CCD camera. Next, the imaging lenses are installed, which form a magnified image of the fiber optic bundle on the CCD camera.
An LED source is then installed with illumination optics in order to provide fluorescent excitation light to the sample. Finally, with the assembled micro endoscope, real-time imaging reveals cellular level details of fluorescently labeled samples, including in vitro cultures, in vivo animal models, and human subjects. The main advantage of this technique compared to existing methods like confocal or multiphoton microscopy, is that it's sufficiently simple and inexpensive so that researchers working in a diverse range of fields can quickly implement it and customize it to meet their specific needs.
The high resolution micro endoscope described here should be considered as a base configuration with several variations possible in assembly and application. In selecting components for a specific application, consider the inherent relationships involved in fluorescence microscopy between fluorol concentration, photobleaching illumination, intensity, camera sensitivity, gain, and exposure time. This platform is designed to be used with pro flavin as a fluorescent contrast agent, but can be configured to work with other fluorescent dyes.
Pro flavin is a bright nucleus stain with peak absorption and emission wavelengths of 445 nanometers and 515 nanometers respectively. The use of other contrast agents will require the user to select excitation emission, and dichroic filters appropriately to assemble the micro endoscope system begin by connecting two six inch cage rods to two 1.5 inch cage rods to form a pair of 7.5 inch long rods. Screw the rods into one face of the fold mirror unit, then install a one inch diameter mirror.
Next, screw four 0.5 inch rods into the opposite face of the fold mirror unit. Slide the cage cube onto the 7.5 inch cage rods via the two lower through holes. Screw the two inch rods into the side face of the cage cube.
Using a C mount to SM one adapter, attach the camera to a cage plate. Secure the cage plate on the 0.5 inch cage rods flush with the face of the fold mirror unit. Select a one inch diameter tube lens to form an image of the fiber optic bundle on the camera.
The focal length of the lens should be selected such that the cause of the fiber optic bundle are sampled by at least two pixels. When imaged onto the camera, insert the lens into a three inch long lens tube and secure the lens with a retaining ring. A spanner wrench is helpful for screwing, retaining rings into long lens tubes.
Drop the emission filter into the three inch lens tube onto top of the first retaining ring and add another ring to secure the filter in place. Ensure that the filter orientation satisfies the convention indicated by the filter manufacturer. Screw the lens tube into the side of the cage cube nearest to the CCD camera.
This position is also shown here. Note that in the diagram, this lens and filter is shown without three inch lens tube. For clarity, connect the camera to a computer and view the image on screen.
Direct the cage assembly at a distant object and slide the cage cube along the rails until an image of the object appears in focus. Use a screwdriver to tighten the four screws located on the sides of the cage cube. These screws grip the rails when tightened holding the cube in position.
This will ensure that the tube lenss will form a focused image of the fiber bundle on the camera when combined with the infinity corrected objective lens. Next, insert the diic mirror into the holder and place it at 45 degrees in the cage cube. Screw the objective lens via an RMS to SM one thread adapter and an adjustable lens tube into the face of the cage cube opposite the tube lens holder.
After that, screw an SMA connector into a cage plate and mount this on the rods approximately at the working distance of the objective lens. Add the base plate and end cap to the open sides of the cage cube. Then mount an LED on a cage plate and slide it onto the end of the two inch rods.
Add a lens to a 0.5 inch lens tube such that when this tube is screwed into the side of the cage cube, it'll form an image of the LED that fills the back aperture of the objective lens. Also, add the excitation filter to the lens tube. At this stage, an LED driver can be used to control the illuminating light intensity using epoxy.
Attach an SMA connector for a 1030 micron fiber to the 720 micron field of view fiber optic bundle. For details on fiber optic connectorization, refer to T Thor labs, part number FN nine six. A guide to connectorization and polishing of optical fibers.
Screw the SMA connector eyes bundle into the SMA receptacle mounted on the cage Rods direct the distal end of the bundle towards a broadband light source fluorescent lighting will suffice. Then observe the image of the bundle proximal face on the CCD camera. Finally, adjust the position of the objective lens by screwing in or out of the cage cube.
Until the fiber bundle image appears in focus, the individual cause should be clearly visible. The spatial resolution of the micro endoscope 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 onto the tissue, the tip is imaged onto the tissue surface with dem magnification.
Thereby increasing the spatial sampling frequency imposed by the light guiding cause of the fiber bundle. The degree of dem magnification corresponds to the increase in spatial resolution and at the same time, to a proportionate decrease in field of view. Select a grin lens with desired magnification and working distance for your application.
Ensure that the diameter of the lens matches that of the fiber bundle. You plan to work with. Mount the fiber bundle and grin lens on separate three axis manual positioning stages under a low power microscope or stereoscope for accurate alignment.
Next place a drop of optical adhesive on either the lens or bundle face. Using the manual positioners, bring the two components into contact. Expose the interface to UV light for the dose recommended by the manufacturer to cure the adhesive.
To protect the grin lens and the bonded interface, use a short length of aluminum capillary tubing to enclose the joint. Slide the tubing over the joint and secure in place with the drop of epoxy at each end of the tubing, use heat shrink tubing to finish the assembly. Prior to micro endoscopic imaging, apply the contrast agent to the cells or tissue to be imaged to image cells and culture.
First, remove growth media by peppe. Then add pro flavin solution by gently pipetting into the well. After a brief incubation of up to one minute, remove the pro flavin solution by peppe and discard.
Rinse the cells by adding and removing fresh PBS solution to the well mount the distal end of the fiber bundle in a secure fixture with manual positioning stages on x, Y, Z axis for stability. During imaging following application of the dye to cells or tissue, place the fiber optic bundle in light contact with the sample. View the image of the fiber optic bundle on the computer screen, either using the software supplied by the camera manufacturer or any custom software which you may wish to develop.
Adjust the exposure time and gain settings of the camera to obtain an image that is of acceptable brightness. When assembled correctly, the micro endoscope 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.
First, the proximal face of the fiber bundle should be imaged onto the CCD camera without defocus in order to achieve the full resolution of the system.Here. A portion of a fiber bundle is imaged with poor focus, slightly focus and good focus. The optimum focus is found by adjusting the actual position of the objective lens relative to the bundle face.
Second, the proximal face of the fiber bundle should be uniformly illuminated over its full diameter. As described earlier, this is achieved by configuring the illumination optics for cola illumination. The image shown here was acquired when a uniform fluorescent sample is illuminated in this preferred configuration.
The corresponding result under critical illumination is shown here. The structure of the LED is imaged onto the fiber bundle face resulting in the appearance of this unwanted pattern superimposed upon the true sample structure. Third, it is important to ensure that both proximal and distal faces of the fiber bundle should be clean and free of scratches and chips.
In this example, image debris is attached to the bundle face and damage in the form of a small chip at five o'clock is seen on the fiber perimeter. Debris can be removed from the bundle faces by cleaning in the same fashion as conventional fiber optic connectors with lens paper and isopropanol or standard fiber optic cleaning tools. 1483 cells were labeled with pro flavin and the bare fiber bundle was lightly placed on the sample.
In vitro imaging is shown here when a 2.5 x grin lens was bonded to the bundle tip, an improvement in spatial resolution and reduction in field of view was achieved. In vivo imaging in which a fiber bundle with a 0.5 millimeter outer diameter was passed through a 21 gauge needle and advanced into a mouse mammary fat pad tissue was performed as seen in this movie, adipocytes are clearly visible with motion due to the cardiac cycle. In this example, imaging of the oral mucosa in a healthy human volunteer was performed using a larger fiber bundle with a 1.5 millimeter outer diameter.
In all examples, shown pro flavin was used as a nuclear labeling fluorescent contrast agent. After watching this video, you should have a good understanding of how to design and assemble a fiber optic micro endoscope for in C two fluorescence imaging of cells and tissues.
