Measurements of Endosomal pH
in Live Cells by Dual-Excitation
The pH of endosomes can be measured with
minimal interference to cellular function using internalized
ligands or antibodies labeled with pHsensitive
fluorescent probes. Following internalization
of receptor-bound or fluid phase probes, a rapid acidification
can be detected along the endosomal
pathway, with the pH decreasing within minutes by
nearly one pH unit (Fig. 1). Cargo can then recycle back
to the plasma membrane, reach the Trans-Golgi-
Network (TGN) (pH~6.0), or be targeted for degradation
through late endosomes (pH < 6.0) and lysosomes
(pH < 5.5). Determination of the vesicular pH thus
allows one to establish whether, at a specific time point,
the cargo resides within a sorting, recycling, or degradation
compartment (Demaurex, 2002). Repeating
such pH measurements at different times during the
internalization provides a functional readout of the
pathway followed by internalized compounds.
|FIGURE 1 Measuring the pH of endocytic organelles with internalized FITC. Organelles acidify rapidly
as they progress along the endocytic pathway. The pH of endocytic compartments can be measured simply
by allowing cells to internalize FITC-labeled antibodies or ligands (red stars). The fluorescence of the FITClabeled
compartments is then imaged at the pH-dependent (λex = 490nm) and pH-independent (λex = 440nm)
FITC excitation wavelength to determine the pH of the organelle.
The most convenient indicator for such measurements
is Fluorescein Isothiocyanate (FITC), and
numerous specific antibodies and ligands labeled with
FITC are readily available. Covalent labeling of specific
proteins or antibodies with FITC is relatively easy to
perform, at low cost (Grinstein and Furuya, 1988).
From a measurement standpoint, the most important
feature is that the bright fluorescence of FITC is
strongly pH dependent at its peak excitation wavelength
= 490nm, but is almost completely pH
insensitive at λex
= 440nm, thus allowing dualexcitation
ratio fluorescence imaging. The ratio λex
- 440 normalizes the measurements for the
amount of fluorophore, as well as for differences in
refractive index and changes in focus, and truly represents
pH. The fluorescence ratio can be calibrated in
situ by equilibrating the pH of the vesicle with the
extracellular pH using nigericin, a K+
and monensin, a Na+
ionophore (for details, see
Thomas et al.
(1979)). Using a standard fluorescence
microscopy apparatus, such as the one described in
Fig. 2 and the protocol and image analysis techniques
described here, the pH of endocytic compartments can
be measured simply by incubating cells with FITClabeled
ligands or antibodies (Demaurex et al.
Gagescu et al.
, 2000; Piguet et al.
, 1999, 2000).
II. MATERIALS AND
|FIGURE 2 Optical and hardware components used for endosomal pH measurements. The key components
(1) an objective with high (1.4) numerical aperture (2) high-quality filters and dichroic mirrors, and
(3) a fast,
low-noise, highly sensitive CCD camera. The camera is attached at the bottom port of the microscope
to maximize light collection. High-quality optics and a sensitive camera are required to capture the
faint fluorescence of small moving endosomes with sufficient signal to noise.
MES, HEPES, and RPMI 1640 medium (bicarbonate
free) are obtained from Sigma. Ionophores (sodium
salts) are from Fluka (nigericin, Cat. No. 72445; monensin
Cat. No. 69864).
The open perfusion microincubator, Leiden coverslip
holder, and temperature controller are from
Harvard Apparatus (refs. PDMI-2 and TC-202A). Glass
coverslips are "Assistent" circular cover glasses
No. 1001, Ø 25mm, thickness 0.16mm from Karl
Hecht Glaswarenfabrik, D-97647 Sondheim/Rhön,
The setup for ratio fluorescence imaging is illustrated
in Fig. 1. We use a Axiovert S100TV microscope
and a 100X, 1.3 NA oil-immersion objective (Carl Zeiss
AG, Feldbach, Switzerland). Alternate excitation at
440 ± 10 and 490 ± 10nm is achieved using a fast
monochromator (Deltaram, Photon Technology International
Inc., Monmouth Junction, NJ) or a filter wheel
equipped with excitation filters 440DF20 (Cat. No.
XF1010) and 490DF20 (Cat. No. XF1011, both from
Omega Optical, Brattleboro, VT). The light path comprises
a 515DRLPXR dichroic mirror (Cat. No. XF 2058)
and 535DF25 emission filter (Cat. No. XF3001) inserted
into the microscope filter slider. Images are captured
with a back-illuminated CCD frame transfer camera
(MicroMax 512 BFT; Roper Scientific; Trenton, NJ)
attached to the bottom port of the microscope.
- Recording solution: 140mM NaCl, 5 mM KCl, 1 mM MgCl2, 1mM CaCl2, 10 mM glucose, 20 mM HEPES,
pH 7.4, with NaOH. To make 100ml, add 14ml NaCl
1M, 0.5 ml KCl 1M, 0.1 ml MgCl2 1M, 0.1 ml CaCl2 1M,
0.5 ml 40% glucose, 2ml HEPES 1M, and complete to
100ml with distilled water.
- Calibration solution: 140mM KCl, 1 mM MgCl2,
0.2mM EGTA, 20mM HEPES (pH 7.0-8.0) or MES
(pH 5.5-6.5), 5µg/ml nigericin, 5µM monensin. To
make 50ml of each, add 28 ml KCl 1M, 4ml MES 1M,
200µl MgCI2 1M, 200µl EGTA 0.4M, and complete to
200ml with distilled water. Divide in 50-ml aliquots
labeled "5.5," "6.0," and "6.5." Repeat the same procedure
using HEPES instead of MES as buffer and divide
in 50-ml aliquots labeled "7.0," "7.5," and "8.0." Adjust
the pH to 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 with KOH
or HCl using a pH electrode precalibrated at 37°C under continuous magnetic stirring. These solutions
can be kept at 4°C for 1-2 weeks. On the day of
the experiment, take 10ml of each calibration solution
and add 10µl nigericin (stock 5mg/ml) and 1µl
monensin (stock 50mM) to each vial. Measure the pH
precisely with the pH electrode (at 37°C) and write
down the pH value to two decimals (e.g., pH 5.54; 5.92;
6.02; 6.54; 7.04). These solutions must be used the same
- Nigericin stock: Add 1 ml ethanol 100% to 5mg
nigericin and store at -20°C for no more than 3
- Monensin stock: Add 1.44ml ethanol 100% to
50mg monensin and store at -20°C for no more than
B. Loading Cells with the FITC.Labeled
Ligand or Antibody
- In a hood, wash the coverslips separately with
ethanol and rinse with distilled water. Place coverslips
into a tank filled with distilled water. Autoclave. Clean
coverslips can be stored in sterile water for 1-2
- If necessary, coat the coverslips with an agent
that promotes attachment of weakly adherent cells.
Poly-L-lysine and Cell Tak (collaborative Research Inc)
have been used extensively. Wash excess adhesive
- Place each coverslip into a 35-mm petri culture
dish (single or multiwell). Close the lid and leave for
20min under UV illumination. The culture dishes
"loaded" with clean coverslips can be used for 1-2 weeks.
- Two to 3 days prior to the measurements, plate
cells on the glass coverslip at low density (~104 cells).
Fill the petri dish with 3 ml of culture medium.
- Optional: One to 2 days before measurements,
transfect cells with the receptor or recombinant protein
bearing the cognate epitope for the FITC-labeled
ligand or antibody.
The example given here is for endosomal pH measurements
with internalized FITC-transferrin. Optimal
concentrations, requirement for serum depletion, and
incubation times have to be determined for each particular
ligand or antibody.
C. Measurements of FITC Fluorescence Ratio
- Incubate cells in their appropriate culture
medium with 25µg/ml of FITC-labeled ligand for
20min at 37°C.
- Wash the cells twice with phosphate-buffered
saline (PBS) to remove extracellular FITC. Maintain
cells in recording medium until use. Measurements
should be performed within 30min of loading.
- If necessary, wash the cells twice with acidic solution
(pH 4.0) at 4°C to remove FITC bound to the
- Using fine tweezers, place the coverslip into the
Leiden chamber, orienting the coverslip so that cells
face upward. Ensure that a good seal exists between
the coverslip and the O ring by wiping away excess
medium from the underside of the coverslip and checking for leaks. The underside should remain dry
and clean to allow oil-immersion microscopy.
- Add 2ml of recording solution to the dish and
proceed to the experiment.
D. Calibration of pH vs Fluorescence
- Place a drop of oil on the immersion objective
and position the loaded Leiden dish in an open perfusion
microincubator over the objective.
- Turn off room lights or use a dark curtain around
the microscope to eliminate stray light from entering
the optical system. Red light can be used for dim
illumination of the room if a red-blocking filter
(550CFSP-Omega Optical) is mounted in front of the
- Turn on the lamp power supply and then start
the arc lamp. It is important to start the lamp before
turning the computer on to eliminate possible damage
due to power surges.
- Turn on the computer and other external hardware
devices. Verify that the temperature controller of
the microincubator is set to the appropriate value (e.g.,
- Launch the software designed for time-lapse
ratio fluorescence measurements and initiate a new
experiment. We use Metafluor 5.0 from Universal
Imaging Corp. Using the illumination control panel,
install the correct hardware drivers for the external
devices, and configure them with the appropriate settings.
Through the use of "Metadevices," Metafluor
allows the user to create a variety of hardware configurations
with different names.
- In the "configure acquisition" window set the
experiment-specific parameters such as exposure time,
pixel binning, and Metadevice selection for each wavelength.
Typical exposure times range from 0.5 to l s
and should not exceed 2 s (see Section V). Keep in mind
that wavelength 1 is always the numerator of ratio 1.
After configuring these settings, save them as a protocol
file that can be retrieved for subsequent use.
- In the "configure experiment" menu, choose the
wavelengths to acquire (W1 and W2) and ratio to be
calculated (W1 over W2). Make sure that the acquired
images and calculated ratio are displayed and updated
- Observe the sample using transmission light to
bring the cells in focus and then switch to fluorescence
illumination to select cells that express the internalized
probe correctly. Open the camera port and acquire
a set of images. If necessary, reset the "configure
acquisition" parameters to optimize the exposure
- In the "reference image" menu, acquire a set of
images with the illumination shutter closed to subtract
the background noise of the camera. Then reopen the
shutter and acquire updated images with background
- Use the "region" tool to define regions of interest
around individual cells. For each region, spatially
averaged fluorescence and ratio data will be graphed
only for pixels with absolute values higher than the
preset threshold. Once the regions are defined, return
to the image windows and set the threshold levels to
separate the labeled structures from the background
- From the "experiment control panel," select
"save image" to save the fluorescence images. Always
save the original fluorescence images during acquisition,
not the calculated ratios.
- Open the "event mark" window to define and
reuse markers associated with a specific experiment.
Markers applied during drug addition and calibration
facilitate the analysis of the experiment during
- Acquire a series of fluorescence images from
several individual cells (~50) to obtain the steady-state
ratio value of the labeled organelle. Do not perform
more than two to three image acquisitions per field to
avoid bleaching of FITC. Check on the ratio graph that
the spatially averaged ratio values are homogeneous
E. Image Analysis: Determining the pH of
- At the end of the acquisition period, select one
representative field and perfuse the dish sequentially
with the calibration solutions. Monitor the fluorescence
to ensure establishment of a stable ratio, indicating
equilibration of the vesicular pH with the
- Once a steady-state ratio has been collected,
change to the next solution and repeat step 1. Continue
until fluorescence ratios have been recorded for all the
- Using the Metamorph software, create a titration
calibration table to convert fluorescence ratio values to
pH. The calibration table can be used to plot real-time
pH change and to create a pH "map" of the labeled
Using Metamorph, create the following analysis
"Journals," "table," and "state file":
1. Journal "Initialize Ratio.jnl"
2. Calibration Table "256.GRY"
||Load gray calibration table
||Apply gray calibration table
||Rescale the 8-bit
from 256 digits
into ratio units
||Image: current at start Threshold: 1; 255; inclusive
||Select labeled structures on the ratio image
||Integrated morphometry-load state
||Load predefined object selection and measurement criteria
Image depth: 8 bit; gray vs calibration values:
0.5; 256 →
3.5; interpolation: linear
3. State file "Ratio.IMA"
Setup parameters for classifying: Enable: pixel area:
10 ≤ N ≤ 500; hole area: 0 ≤ N ≤ 0
Setup parameters for measuring: Enable: area; average
gray value; format: #.###
Configure log: Enable: image name; image plane; object
#; average gray value; area
Preferences: Measure objects: Disable
: "warn user
when measurements data will be erased"
4. Journal "Measure ratio.Jnl"
|Image: current at start Plane: current
||Measure all thresholded objects meeting criteria on
|Image: current at start
||Log measurement data to spreadsheet
- Using the MetaFluor program, open a previous
experiment and create a series of ratio images. In the
"Image display controls," check that the minimal and
maximal ratio values are set to 0.5 and 3.5, respectively.
Adjust the threshold on the 440-nm image (W2) to separate
the fluorescent vesicles from the background. If
possible, the same threshold should be used for all
images. From the "experiment control panel," select
"save ratios" to save the ratio images as 8-bit TIF images. Make sure to save only one ratio image for
each field acquired during the steady-state measurement
- Launch the MetaMorph program. In the "File"
menu, use the function "Build stack: numbered name"
to load a stack of ratio image from the desired experiment.
Use the "Stack: Montage" function to create and
print an image gallery. Check for duplicates.
- In the "Journal" menu, use the function "Run"
with journal "Initialize Ratio.jnl" to rescale the 8-bit
ratio image and load the object selection and measurement
criteria. Tip: Journal playback can be simplifled
by defining journals as button on the "Journal
- In the "Log" menu, use the function "Open object
log" to create the connection with the spreadsheet
program (e.g., Microsoft Excel).
- In the "Journal" menu, use the function "Loop
for all planes," with journal "Measure ratio.Jnl." This
will extract the average ratio value of all objects that
meet the criteria (area size between 10 and 500 pixels,
devoid of holes) in every image from the ratio stack.
Check the quality of the segmentation by comparing
the measurement images with the original fluorescence
images. Most individual vesicles should be
included as well as aggregates of <10 vesicles. Spots
and very large compound structures should not be
included (Fig. 3).
- Switch to the spreadsheet program. Check the
quality of the measurements by displaying ratio data
as a histogram. The distribution should be gaussian if
the population of vesicles has a homogeneous pH.
- Using the spreadsheet program, create a calibration
curve for the experiment. Enter the ratio values
measured during the calibration procedure as X
column and the pH values of the calibration solutions
(measured at two decimals) as Y column. Display the
resulting points on a dot graph. Fit a linear equation
to the points and copy the parameters. Use these
values to apply a calibration equation to ratio data. A
pH histogram can now be generated and the average
pH values of the population of vesicles determined.
|FIGURE 3 Fluorescence and ratio images of early endosomes in MDCK cells. MCDK cells were allowed
to internalize FITC-labeled transferrin for 5 min at 37°C. (Top left) A 490-nm fluorescence image, exposure
time 1 s. (Top right) A 490/440-nm ratio image. A low-intensity threshold of 30/4095 was used to separate
fluorescent vesicles from background. (Bottom left) Vesicles recognized as individual endosomes by the image
analysis routine. Seventy-three individual objects met selection criteria (pixel area 10-500, no holes). (Bottom
right) Composite image of the vesicles selected by the image analysis routine and the original fluorescence
Even for experienced users, a few minutes are
required to collect enough images of FITC-labeled cells
to construct a pH histogram. Within the acquisitioln
period, the pH of endosomes can change significantly,
particularly during the early phases of endocytosis.
For this reason, it is important to check that the pH is
at steady state by graphing the ratio values during the acquisition period. In any case, the acquisition period
should be kept as short as possible and should not
exceed 20 min.
When counting individual vesicles, small and large
structures have the same "weight" in the pH histogram.
This biases the readout toward the pH value
of the smallest, most numerous vesicles. If the vesicles
are highly heterogeneous in size, the pH values should
be normalized to the vesicle area.
Failure to wash carefully extracellular FITC might
result in a large contribution of the plasma membrane
to the ratio images. These structures are normally
excluded by the size criteria, but will otherwise appear
as a second peak around pH 7.4 on the histogram.
- Do not use exposure times longer than 2s to
avoid distortions caused by the movement of endosomes. The division of two fluorescence images
acquired sequentially will invariably produce "hot"
or "cool" pixels on the ratio images, mainly at the
organelle edges. Most irrelevant pixels are removed by
the intensity threshold, but this procedure discards
many small moving vesicles, resulting in a significant
loss of information. Using short exposures is the best
way to capture the rapidly moving endosomes and to
avoid such distortions. The gain in resolution allows
to better quantify the ratio images so that a fast, sensitive
camera is highly desirable to obtain accurate pH
- Use minimal illumination to prevent photobleaching
and cellular damage. Do not make repeated
measurements of the same field, but instead perform
separate measurements on different fields. The best
procedure is to select the cells at random, using brightfield
illumination. If only a few cells are labeled with
the FITC ligand, the labeled cells should be selected
using 440-nm illumination to avoid selection bias
induced by the pH dependency of FITC-a bright 490nm fluorescence reflecting not only a successful labeling,
but also an elevated pH.
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