In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.
1. Align the optics.
The key equipment for an MP-FRAP experiment include: a mode-locked laser source, Pockels Cell (for beam modulation), pulse generator, dichroic, objective lens, fluorescence emission filter, gated photomultiplier tube, and a data recording system (photon counter and multichannel scaler).
2. Determine safe monitor powers.
3. Determine input parameters to the pulse generator and multichannel scaler.
4. Test for excitation saturation.
5. Continue taking MP-FRAP curves.
6. Data Analysis.
7. Representative Results.
Figure 1. Representative bleach and recovery curve for FITC-BSA. A good recovery curve exhibits a strong bleach and smooth recovery, with the fluorescence at full recovery being reported for the latter half of the data.
Figure 2. Normalized recovery curve for FITC-BSA (red) and the associated least-squares fit (black). This fit yields a diffusion coefficient of 52.9 um2/s, consistent with the literature.
The power of multi-photon fluorescence recovery after photobleaching lies in its ability to probe thick samples with 3D resolution. Since its development in the 1990’s, MP-FRAP has been used to determine the diffusion coefficient (or analogous transport parameters) in cell bodies, ex vivo thick tissue slices, and in vivo tissue and interstitium. In this article, we presented the equipment necessary to run an MP-FRAP experiment, as well as the proper procedure for aligning the beam path, setting experimental parameters, taking data, and analyzing recovery curves.
The choices of an appropriate excitation wavelength and emission filter should be guided by the two-photon cross-sections and emission spectra. This information is often included with the technical data for various dyes. Also, in aligning the laser beam, it is important that proper overfilling of the back lens of the objective be achieved. This is often accomplished by adding a beam expander to the optical system. Proper overfilling can be verified by scanning sub-resolution fixed fluorescent beads in both the axial and radial directions and then plotting and fitting the fluorescence profiles to Gaussians to find the 1/e2 widths for comparison with literature values.
It is also important to choose appropriate monitoring and bleaching powers. The power used to monitor the fluorescence pre- and post-bleach should be low enough not to cause appreciable bleaching, but high enough to allow for a good signal-to-noise ratio. The bleaching power must avoid excitation saturation. In an MP-FRAP experiment there is an upper limit to the fluorescence excitation rate, which is proportional to the square of the power incident on the sample. This limit marks the start of the excitation saturation regime. Fluorescence recovery curves produced using bleach powers operating in the excitation saturation regime will yield erroneously low diffusion coefficients.
This work was funded by a Department of Defense Era of Hope Scholar Award (No. W81XWH-05-0396) and a Pew Scholar in the Biomedical Sciences Award to Edward B. Brown III.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Ti:sapphire laser | Spectra Physics | Mai Tai | ||
Pockels Cell | Conoptics | 350-80 | ||
Laser Scanning Microscope | Olympus | Fluoview | ||
Short pass dichroic mirror | Chroma Technologies | 670 DCSX-2P | ||
Photomultiplier tube | Hamamatsu | HC125-02 | ||
Photon counter | Stanford Research Systems | SR 400 | ||
Multi-channel scaler/averager | Stanford Research Systems | SR 430 | ||
Pulse Generator | Stanford Research Systems | DG 535 | ||
FITC-BSA | Invitrogen | — |