What Are Optical pH Sensors and How Do They Work?

What are optical pH sensors? 

Everyone who’s been involved in cell culture for more than a few days knows that maintaining proper pH is one of the most important elements in obtaining high quality repeatable results. What’s not always so clear is how to measure pH, and for that matter, where the measurement should happen. 

Optical pH sensors are made up of a variety of different thin layers sandwiched together. The uppermost layer of the optical sensor, the only layer that comes in contact with cells, is made up of a halochromic chemical compound suspended in a hydrogel. While each layer is critically important for one reason or another, be it structural or adhesive, it is the hydrophilic layer impregnated with immobilized dye that is arguably the most important. An external reader sends a signal through the cell culture vessel, bouncing it off the optical sensor inside. The difference in the strength of the return signal is all the information the reader needs to measure the pH. 


How do optical pH sensors work? 

There are a few different types of optical pH sensors, but the best and most reliable tend to be those that contain a fluorescent dye that experiences a dual excitation maxima. The external reader emits a blue light followed by a violet light to excite the dye at both its excitation wavelengths. The dye then emits light back at a different wavelength. When the sensor is in a solution with a higher pH, more of the deprotonated form (conjugate base) will be present, which leads to increased intensity of the blue light (457nm) and decreased intensity of violet light (405nm). When the solution is more acidic, the exact opposite happens. The most popular readers use a ratiometric fluorescence measuring technique in which the ratio of the intensities at the two excitation wavelengths reveals changes in the pH of the sensor’s local environment. 


What’s the best application for optical pH sensors? 

For small-scale cell culture work, whether it’s in an Erlenmeyer flask, a T-flask, or other vessel, optical sensors are proving to be one of the best options for pH measurement. They provide noninvasive measurements of pH at the pericellular level. The low profile of optical sensors (< 0.33mm), allows them to effectively become part of the cell culture device. Because cells can easily grow over the top of the sensor, measurements are made at the most important location of the cell culture, where the cells actually are. 

Without a doubt, cells are the most important element of cell culture, and it only makes sense that measuring pH should be happening where the cells actually live. Since cells tend to live on the bottom or sides of the flask, that is the optimal place for pH measurements. 


What are the benefits of optical pH sensors? 

Minimal Maintenance 

The optical sensor spot is the only part of the device that comes in contact with cells or media and they are designed to be single use, affordable, and disposable.  


No Need for Calibration 

High quality optical pH sensors come pre-calibrated and do not need to be adjusted before use. This limits any possible down-time during experiments. 



Small, low profile sensor spots are able to effectively become part of the cell culture vessel. The cells are able to grow on top of the sensor and because they are so small, they do not disrupt the flow of media the same way traditional probe style sensors do.  


Immune to Electric Interference 

The nature of a sensor that uses a fluorescent dye suspended in a hydrogel and a dual excitation maxima to calculate readings means that there is no electric interaction, and thus no electric interference. 


Pericellular Sensing 

The small nature of optical sensor spots allows them to take readings at the most important place in cell culture. Where the cells actually are. The discrete location of the sensor allows for highly localized readings. 


What are the limitations of optical pH sensors? 

Sensor Spot Sensitivity 

Optical sensor spots are small, delicate, and must be handled with care. Exposure to high concentrations of alcohol or certain organic compounds can also cause damage.


New Technology 

Despite having been discovered more than 20 years ago, optical sensor technology is still relatively new to the market and many users are not familiar with how it works. 


Limited Range 

Optical pH sensors currently on the market have a limited pH range. For this reason, most companies have designed multiple sensor spots with a variety of ranges. The most common is pH 6.00 to 8.00 for cell culture in the mammalian physiological range. 


Continuous Measurements 

Optical sensors must send and receive signals causing a small lag in continuous measurements. Even though readings can be taken for long periods of time, it takes anywhere from 8 to 18 seconds per individual reading.

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