Jul 10, 2023
Version 1
Modified Frame-projected Independent Fiber Photometry (FIP) System_Hardware V.1
This protocol is a draft, published without a DOI.
- 1Allen Institute for Neural Dynamics
Protocol Citation: Kenta M. Hagihara 2023. Modified Frame-projected Independent Fiber Photometry (FIP) System_Hardware. protocols.io https://protocols.io/view/modified-frame-projected-independent-fiber-photome-cn96vh9e
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 11, 2023
Last Modified: June 18, 2024
Protocol Integer ID: 76830
Keywords: fiber photometry, calcium imaging, neuromodulator imaging, CMOS-based photometry
Funders Acknowledgement:
Allen Institute
Abstract
This is a step-by-step protocol to build a modified FIP (Frame-projected Independent FP) system. FIP was first implemented and reported by Kim et al. (2016). Their protocol is available here.
We modified previous design so that it can be built mostly with Thorlabs products (still some products from other manufactures are required), which are off-the-shelf and easy to be purchased within a reasonable lead time. The system described in this protocol is designed to record signals from 1)GFP-based sensors, 2)mApple-based sensors, and 3) iso sbestic signals from up to 4 sites. When applied to measurements of other fluorescent protein-based sensors or spectrally shifted sensors, selection of optical filters and/or excitation light sources should be modified accordingly. Also, the selection
of fiber patch cable should be based on the number of simultaneously recorded locations, and diameter/NA of fiber implants.
We thank Takeo Katsuki Ph.D., (Thorlabs Japan) and Sho Yagishita M.D., Ph.D., (U.Tokyo, Japan) for sharing their galvano-mirror-based photometry design described in Iino et al. (2020). TK also kindly provided CAD model used in this protocol.
Materials
Thorlabs
| product# | Description | # | |
| Excitation | |||
| M415F3 | LED415nm | 1 | |
| M470F3 | LED470nm | 1 | |
| M565F3 | LED565nm | 1 | |
| LEDD1B | t-cube LED driver(1200mA) | 3 | |
| KPS201 | PowerUnit for T-cube | 3 | |
| FB410-10 | 410 bandpass | 1 | |
| FB470-10 | 470 bandpass | 1 | |
| FB560-10 | 560 bandpass | 1 | |
| M59L01 | SMA-SMA 1000um 0.48NA fiber | 3 | |
| KAD11F | collimator mount (kinematic) | 2 | |
| AD15F | collimator mount | 1 | |
| F220SM-532 | collimator | 2 | |
| F950SMA-A | HighNA collimator | 1 | |
| General cage system (30mm cage) | |||
| DFM1 | kinematic filter cube | 4 | |
| RS1P | post | 4 | |
| CP33 | cage plate | 5 | |
| ER025 | rod | 16 | |
| ER2-P4 | rod | 2 | |
| ER4-P4 | rod | 1 | |
| SM1CP2 | Externally SM1-Threaded End Cap | 4 | |
| SM1L05 | LensTube | 4 | |
| SM1L03 | LensTube | 2 | |
| - | Breadboard with a proper size | 1 | |
| Emission | |||
| SM1A12 | M25*0.75 Objective adaptor | 1 | |
| AC254-080-A-ML | achromatic lens f=80 | 2 | |
| SM1A9 | C-Mount/SMA adaptor | 2 | |
| SM1AB2 | C-Mount/SMA adaptor | 2 | |
| SM1P1 | Optic mount | 2 | |
| Fine-tunable Fiber Mount | |||
| CXYZ05 | xyz fiber mount | 1 | |
| SM05L03 | lens tube | 1 | |
| SM05FC | fiber adapter | 1 | |
| SM05T2 | capla for lens tube | 1 |
Note, Thorlabs products can get obsolete routinely; replace them with corresponding newer products.
Semrock
| product# | Description | |
| FF493/574-Di01-25x36 | 493/574 nm BrightLine® dual-edge standard epi-fluorescence dichroic beamsplitter | |
| FF01-520/35-25 | 520/35 nm BrightLine® single-band bandpass filter | |
| FF01-630/69-25 | 630/69 nm BrightLine® single-band bandpass filter | |
| FF562-Di03-25x36 | 562 nm edge BrightLine® single-edge standard epi-fluorescence dichroic |
Nikon
| product# | Description | |
| MRD70170 | CFI60 PLAN APOCHROMAT LAMBDA D 10X |
Edmund Optics
| product# | Description | |
| #69-899 | 500nm, 25.2 x 35.6mm, Dichroic Longpass Filter | |
| #69-898 | 450nm, 25.2 x 35.6mm, Dichroic Longpass Filter |
Doric
Fiber patch cable should be selected based on application.We recommend "Low-Autofluorescence Bundle Branching Fiber-optic Patch Cord" from Doric for standard applications.
FLIR-Teledyne
CMOS sensors should be selected based on required frame rate and sensitivity. We use "BFS-U3-20S4M-C", which is based on Sony IMX422 sensor, as default CMOSs.
Safety warnings
Although resultant FIP setup will be a fairly optically closed system, in particular during building, experimenter(s) could be exposed to stray light from LEDs. In general, inappropriate use of any Fiber-Coupled LEDs may result in permanent eye damage. To prevent injury, use the LEDs in accordance with the International Standard “Photobiological Safety of Lamps &
Lamp Systems” IEC 62471. The LEDs used in this system fall under RG2 - Moderate Risk Group in accordance to the
standard IEC 62471:2006.
System Overview
System Overview
Fig. 1. System Overview
Fig. 2. Design diagram
See the Materials section for products used in the setup.
Fig. 3. Spectral features of LEDs, optical filters, and genetic sensor emission signals.
a, Normalized intensity of excitation LEDs (L415M, L470M, L565M), and transmission of excitation band-pass filters (FB410, FB470, FB560) and the dual dichroic filter (FF493_574).
b, Transmission of the dual dichroic filter (duplicated from a) separating emission spectrum from excitation spectrum (Fig. 1), and normalized intensity of GCaMP6f and jRGECO1a. Note, this design would be suited for most cpGFP- and cpmApple-based sensors.
c, Transmission of the dichroic filter separating green and red emission signals (FF562), and emission band-pass filters (FF01_520_35 for green and FF01_630_69 for red)
Assemblying optical parts
Assemblying optical parts
2h
2h
Assemblying the cubes
(This step-by-step part will have corresponding CAD models later.)
30m
Adding optical filters, collimators, and the objective lens
30m
Assemblysing the fiber positioning module
15m
Assemblysing the CMOS modules
30m
Connecting excitation light sources with fibers
15m
Alignment
Alignment
30m
30m
Adjusting the fiber location so that fiber end makes a crisp image on CMOSs
Adjusting angle of collimators so that excitation light coming from fiber patch cables would get roughly uniform.
30m
Protocol references
Kim, C., Yang, S., Pichamoorthy, N. et al. Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain. Nat Methods 13, 325–328 (2016). https://doi.org/10.1038/nmeth.3770
Iino, Y., Sawada, T., Yamaguchi, K. et al. Dopamine D2 receptors in discrimination learning and spine enlargement. Nature 579, 555–560 (2020). https://doi.org/10.1038/s41586-020-2115-1