Twice the Challenge, but Double the Opportunity

par | Août 15, 2023

High-speed multiphoton (MP) imaging is often used to visualize dynamic changes in live tissue or cellular preparations. For neuroscience applications, electrophysiology often is combined with imaging, and allows for the simultaneous acquisition and/or control of electrical activity within the living sample. Though powerful, integrating electrophysiology (often termed “ephys”) and MP imaging requires additional considerations when compared to using each technique alone.


Probe Placement 

It is important to work out where the electrophysiology probes used will be placed relative to the objective and sample. These probes are narrow diameter pipettes and/or electrodes which must be introduced into the sample from the side. Placing these probes in a sample underneath a large objective lens can be challenging

As a starting point, it is important to check for sufficient clearance around the objective when introducing the probe. High-resolution (high numerical aperture, NA) objectives often have short working distance and wider barrels, and can end up interfering with the placement of the probe (fig, left). Often it is better to choose a lens with longer working distance, as it allows for a steeper probe angle and more room for maneuvering your probe around the imaging region (fig right). It is good practice to test an objective if you are not sure whether it will be compatible with the required electrophysiological measurements. 

Quick tip: Often vendors will have “demo” objectives set aside just for this purpose, so do not hesitate to reach out in advance of purchase; as an added advantage, vendors also can direct you to the objective that best suits your needs.

The importance of the DIC light path 

Differential Interference Contrast (DIC) is often used in combined electrophysiology and MP systems, as it allows  electrophysiologists to accurately position the probes relative to the imaged regions. The DIC light path is different from the MP light path and requires distinct  adjustments. DIC is typically used in transmission, with the condenser and objective arranged on opposite sides of the sample. The DIC light source, commonly a LED,  is chosen to emit in the near-infrared (near-IR). Just as with MP imaging, the longer wavelengths minimize absorption and scatter, allowing for deeper penetration into tissue. The near-IR wavelengths mean that the sample is visualized with a camera rather than through the eyepiece, as near-IR light is difficult to perceive by eye. 

In addition to a condenser and objective, the DIC path will include a prism and analyzer for manipulating polarized light, and sometimes will have additional controls for adjusting contrast.  For best results, it is important to set up proper Köehler illumination, as well as following the specific instructions for adjusting the additional optical components, as there are numerous ways of configuring DIC light paths. 

Quick tip: Imaging through plastic (such as a dish or chamber bottom) may disrupt the polarization of your light and degrade the contrast observed in DIC. If this proves to be a problem, consider switching to a glass-bottom holder or chamber or another type of white-light imaging altogether.

Optical path switching and troubleshooting

As the DIC and MP imaging use similar near-IR wavelengths, it can lead to odd results when the light paths are not set up properly. Be systematic when switching between modalities so you don’t inadvertently leave a filter or shutter in the wrong position. Check-lists are your friend here too.

Quick tip: If you notice odd interference or lower contrast when using multiphoton, try to image with the DIC LED unplugged as LEDs sometimes have low light leakage and will be “on” slightly even if the DIC system is not active.

Buffer leaks may damage microscope components

The live samples used for multi-modal electrophysiology and imaging experiments often require buffer or media to be perfused over the sample to maintain metabolic function. If the system is an upright microscope, fluid can leak onto the condenser and mechanical components located beneath the stage (or worse, the much more expensive objective and filters for inverted systems). Even minor leaks can lead to major issues long-term, so it is important to take care to minimize leaks and to inspect the microscope after each use. A silicone sheet with cutouts or other waterproofing measures are recommended to avoid equipment damage from the inevitable leaks that come from working with a flow system.

Quick tip: Dental suppliers often sell latex sheets that can be draped over the optics below the stage. Ensure that the sheeting is low-static or otherwise you may damage any electrical components in the system.

Placement of support hardware and electronics

Combining electrophysiology with imaging also requires more hardware and the system can quickly become a maze of boxes and wires. As you set up a system, it is advisable to take the time to set up your equipment for easy access. Equipment should be mounted in a standard rack or similar, with color-coded or labeled velcro ties for organizing cabling. Some cable ties are designed to be anchored to optical table tops or attached to hard surfaces to support and organize longer runs of cable.

Quick tip: Labels for everything and capture photos of the various connections. This will help you if you have to disassemble or reassemble the system at some point and also prevent tangled wires and tubing. 

Do you have any tips to share? Please feel free to share them in the Comments box below.

Additional References:

Differential Interference Contrast Microscopy (DIC) – The Canadian Nature Photographer – Robert Berdan
Electrophysiology Fundamentals, Membrane Potential and Electrophysiological Techniques | Technology Networks
Intro To Electrophysiology (
Neuropixels Probes Can Record from Hundreds of Neurons in Different Brain Areas at Once | Technology Networks
Comparing Micromanipulators for Electrophysiology | Features | Apr 2015 | BioPhotonics
What is Koehler illumination? —
Optical Microscopy Application: Differential Interference Contrast (
Photoelasticity | Harvard Natural Sciences Lecture Demonstrations
Open Ephys (
19-inch rack – Wikipedia

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