Imaging Intracellular pH in Living Cells
Intracellular pH is important in many functions that take place in a cell. For example, pH
affects protein structure and the function of lysosomes, mitochondria, and other organelles.
Changes in energy metabolism also often correlate with pH changes, and so scientists
would like to monitor ATP and pH in a cell simultaneously.
The Andor Revolution DSD confocal microscope is helping Dr. Gary
Yellen’s lab at Harvard Medical School develop genetically encoded
fluorescent sensors for monitoring metabolism of single cells. “We
are interested in sub-cellular compartmentalization of metabolites,
and the optical sectioning afforded by the DSD allows us to assess
the distribution of our sensors when they are genetically targeted to
different cellular locations (e.g., plasma membrane, mitochondria,
etc; Figures 1a and 1b),” he says.
Dr. Yellen’s research team recently developed a genetically encoded
pH sensor that could be used with GFP-based ATP sensors (Figure
1.) This type of probe was needed to monitor ATP and pH changes
in a cell and to correct for the pH sensitivity of ATP sensors. They
used the red fluorescent protein mKeima as a basis to engineer a
genetically encoded ratiometric pH sensor they named pHRed.
The sensor has a fluorescence emission peak at 610 nm and dual
excitation peaks at 440 and 585 nm that allow ratiometric imaging.
Postdoctoral research fellow Mathew Tantama imaged energy-
dependent changes in cytosolic and mitochondrial pH with the
Revolution DSD, demonstrating pHRed’s capability to monitor
intracellular pH in living cells. “The Andor Revolution DSD offered
us a practical, cost-effective solution for optical sectioning with the
flexibility of a white light source,” Dr. Yellen says. The microscope’s
broadband prefiltered light source and user-selectable filters gave
the researchers access to a wide variety of excitation wavelengths.
“During the process of sensor development, color requirements
often change, making the fixed wavelengths of laser-based systems
constraining or cost-prohibitive for an individual lab,” he adds. The
unique optical architecture of the Revolution DSD enables capture of
both conventional and confocal fluorescence images simultaneously.
This feature provides further flexibility for the researchers who
can choose between the sensitivity of widefield and the resolution
of confocal imaging according to conditions and experimental
requirements.
The researchers transfected the pHRed sensor and the mVenus-
based Perceval ATP/ADP sensor into live cells. For intensity ratio
imaging they imaged the cells on an inverted microscope equipped
with a Revolution DSD system controlled by Andor iQ software.Ratio imaging of the two sensors used excitation filters 445/20 nm,
482/18 nm, or 578/16 nm filters. The DSD unit contained a 59022bs
dichroic, 492 nm short pass, 490 nm short pass, and 590 nm short
pass filters. Emission light was passed through 525/39 nm or 629/56
nm filters. They typically acquired images every 10 seconds with
2x2 binning and 50 to 400 ms exposure times using a 20X/0.75NA
objective. The speed performance of the Revolution DSD allowed
imaging of several wavelengths at multiple XY positions while
maintaining an adequate temporal resolution.
After capturing the images, they subtracted background and bleed-
through of Perceval fluorescence into the pHRed channel before calculating ratios. At the shared wavelength of 445 nm there was
little bleedthrough during excitation. The researchers calculated
pixel-by-pixel ratios, identified regions of interest around cells, and
calculated averaged measurements using thresholding with ImageJ
software. They found that pHRed’s intensity ratio responds with an
apparent pKa of 6.6 and a greater than 10-fold dynamic range.
“This is the first ratiometric sensor based on a single red fluorescent
protein that can be used in multiple imaging modalities,” Dr. Yellen
says. “There are several other genetically-encoded pH sensors that
utilize the blue to yellow color range or that provide intensity-based
readouts only. Our red-colored pH sensor can be used in conjunction
with other genetically-encoded sensors with the possibility of
minimal color channel crosstalk.”
Research Paper:
Imaging Intracellular pH in Live Cells with a
Genetically Encoded Red Fluorescent Protein Sensor, Journal of the
American Chemical Society, 2011, 133 (26), pp 10034–10037, DOI:
10.1021/ja202902d.