Calibration protocols for caged compounds
1) A general approach for pretty good estimates of uncaging efficiency:
If FITC fluorescence viewing is available and the spectral characteristics of the uncaging
illumination are known, then caged fluorescein dextran can be used to estimate the uncaging
efficiency of the system.
Step 1. Prepare a dried caged fluorescein dextran solution by making 1 mg/mL caged fluorescein
dextran (MW 3000) in any pH-buffered solution, for instance PBS. Put 1 uL of this on a
microscope slide, smear it around, and let it evaporate. Drop a cover slip on top and seal all
around with Krazy Glue.
Step 2. Put the slide on the setup and set your flash intensity to whatever you plan on using in a
biology experiment.
Step 3. Focus on the slide by looking for fluorescent particles. Take a fluorescence picture to use
as background.
Step 4. Give a flash, wait 5 seconds, and take a fluorescence picture. Repeat this step until the
fluorescence does not increase any more. Note that the wait is necessary because dried caged
fluorescein uncages very slowly.
Step 5. Measure the fluorescence at the uncaging region as a function of flash number. This
should be an approximately exponential curve; when the endpoints are normalized to 0 and 1, the
first data point will tell you what fraction of caged fluorescein was photolyzed. Note that if you
flash too many times the spot will begin to bleach.
Step 6. To extrapolate to other compounds, use the fact that the fraction of compound uncaged is
expected to be proportional to the “uncagability index,” defined as   (see lecture notes). These
values, especially , must be taken at the same wavelength as that used by the flash. You can
then scale the uncaging efficiency measured for caged fluorescein to predict performance for
other compounds. For instance, if Cage X has a value of   half as large, its uncaging efficiency
will also be half as large.
Specific points on Step 6:
- If your source is monochromatic (i.e. a laser) then calculating   is simple.
- If you have some other source such as a flashlamp and the caging group is the same as the caged
fluorescein (i.e. DMNB-caged-fluorescein and DMNB-caged-glutamate), then just use  and  for
a wavelength near the peak of the flashlamp’s spectrum.
- If you are using different caging groups then it is necessary, strictly speaking, to convolve the
wavelength distribution of the flash source and the extinction spectrum to come up with an
effective . How accurately this will turn out is dependent on the particular situation.
A second, more accurate procedure can be applied in the case of caged phosphates.
2) Caged phosphates, modified from correspondence with Kamran Khodakhah:
Caged phosphates, including caged InsP3 and caged ATP, have similar  and .
Any member of the family can therefore be used to calibrate all (Walker et al., 1988).
UNCAGING CALIBRATION (non-focal)
Test solution:
50 M BCECF (pH indicator)
3 mM MgCl2
I mM dithiothreitol (HPLC grade, Calbiochem), to mop up insoluble photolytic byproduct
100 M caged ATP, disodium salt
Handling BCECF: BCECF must be titrated with KOH to bring it into solution.
Bring pH to 7.0 with KOH (if caged ATP is free acid, you must adjust pH after adding
it!). It comes as a disodium salt, but unless it is exactly right then free acid may acidify the
solution. For small volumes this is a pain.
If you are putting the test solution in the bath (i.e. not in a 50 m capillary tubing
made of quartz), use low concentrations. Otherwise you can really suffer from UV loss
and it will cost a mint as well. Caged compounds have a bloody good extinction
coefficient in the UV and thus with the light traveling through a 1.3 mm of a 9 mM
solution what gets to the focal point of the objective is much less than what left the
objective (Beer-Lambert law).
Calibration measurements:
1) Measure the absolute light intensity from the test solution in the chamber/cuvet.
2) Give a light pulse, record the change in light intensity (a decrease in fluorescence), and
remix the solution. Remixing should give the same fluorescence before the UV pulse since
in most configurations the quantity of caged ATP photolyzed will be insignificant
compared to the 1 ml of the total solution. Repeat until you are happy with the
consistency of the results.
3) Successively add aliquots of cone. HCl to the 1 ml solution and measure the
fluorescence, thus making a nice calibration curve. This calibration curve can be used to
calculate the exact number of protons released by the light pulses applied in (2). You will
also get the minimum fluorescence of the dye when you have saturated the solution with
protons (Fmax, at max. H+).· You can also get the maximum fluorescence if you add tons of
conc. KOH (Fmin, at min. H+).· Note that Fmin > Fmax, at 488 nm excitation.
4) An alternate method of determining the change in [H7 is to use the HendersonHasselbalch equation, [H+]= (F  Fmin)/(Fmax  F). This method and the method in (3)
should agree well.
...yet another approach to calibration is to take successive absorption spectra of the test
solution after light flashes. In this approach BCECF is not necessary.
For BCECF, the optimum excitation wavelength is 505 nm (Fmin/Fmax ~ 17) but for
fluorescein excitation by an Ar laser at 488 nm, Fmin/Fmax ~ 7.
UNCAGING CALIBRATION (focal)
On a focal uncaging setup, the path length of the test solution should be much
smaller. Put the solution but between cover slips. Because the signal is therefore much
smaller -- and also dissipates in aqueous solution -- one must acquire at video rate or
suspend droplets of test solution in oil. Also, since the path length is shorter, one should
use more dye.
In this configuration the total extinction is insignificant Note, however, that
addition of HCl to the test solution is hard. Therefore Fmin and Fmax, must be achieved
using the caged compound alone. The test solution must therefore begin at very high pH
and there must at first be a large excess of caged ATP.
Test solution:
2 mM BCECF
13 mM MgCl2
10 mM dithiothreitol
10 mM caged ATP, disodium salt
2 mM KOH in addition to what was in the BCECF stock
Make sure that the solution is initially alkaline (pH > 9); if not, add more KOH.
1) Suspend the solution in ~4 parts light mineral oil and vortex to get small droplets.
2) Sandwich the suspension under a cover slip; glue down all round with Krazy Glue.
Find droplets that are equal in size to the area to be calibrated (typically 2-5 m wide).
3) Monitor fluorescence with successive flashes, or with continuous exposure to low-level
UV. Because BCECF bleaches, take a 30 second baseline; also compare UV responses
with a test solution with no caged ATP.
4) Take the starting fluorescence as Fmin, and the ending fluorescence as Fmax. Calculate
changes in [H+] as in (4) of the non-focal protocol.