Design & Optical Setup of Fiber Bundle Imaging System
Description of components:
1. Fiber Bundle- FIGH-30-650S
We used the fujikura imaging fiber FIGH-30-650S, providing an imaging diameter of 600um and core-to-core distance of 3.3um. We wanted three fiber bundles to fit into a circle of diameter 2mm, keeping in mind the field of view of a traditional 10X objective. The three fiber bundles were flushed into a single SMA connector at one end.
2. Objective: Nikon CFI Fluor 10X
We used a 10X, 0.5NA objective because of the physical size of the fiber bundles and the need for an NA larger than that of the fiber bundles (NA=0.39).
3. Tube lens: Zeiss tube lens, focal length-165mm
In order to frame project the three fiber bundles onto the 18mm camera sensor, such that the entire sensor area was used, we chose a tube lens with a focal length of 165mm (smaller than the Nikon at 200mm) to achieve a magnification less than 10X.
4. Illumination Arm:
The illumination arm consists of the fiber-coupled 470nm laser (optoengine llc ,BL-III-473/1~100mW), fiber collimator (thorlabs, F230FC-A), and defocusing lens. A 50 mm lens was used to focus the collimated beam at the back focal plane of objective in order to obtain uniform illumination of all three fiber bundles.
5. Camera: Photometrics Prime 95B
Given the high background in wide field imaging and the additional background that the fiber bundle would add to that, a high quantum efficiency and low read noise was required to achieve adequate signal to noise ratio. The photometrics prime 95B provided the highest QE of 95%. The SNR was enhanced to match traditional wide field imaging.
6. Other optical elements : Dichroic mirror (semrock, FF495-Di02-25x36), emission filter (semrock, FF01-520/35-25)
7. Power used : <1 mw
The laser power was maintained to be less than 1 mW at the end of fiber bundle. Effects of photobleaching at higher powers were assessed by comparing population changes in SNR of the calcium responses and through bead fluorescence.
Diagram of components (Parts list below)
* These are 4 inch long, 6mm diameter rods. Thorlabs no longer seems to carry these; an alternative option is to combine shorter rods to reach the desired length. For the rods around the objective (left asterisk), the total length is 4 inches (one rod x4). The rods connecting the filter cube to the camera (holding the tube lens) are 8 inches total in length (two rods attached at the end x4).