Two-photon microscope upgrade reaches 75 nm resolution, a fivefold gain

A team at Texas A&M University has pushed two-photon microscopy to a lateral resolution of about 75 nanometres, around five times finer than the conventional version of the technique. The approach, named 2PE-ISM-SOFI, adds two established methods to a standard two-photon instrument. It was reported in the Journal of Biomedical Optics by a group led by Aart Verhoef, assistant professor of biophotonics.

The trade-off the method targets

Two-photon microscopy is widely used for living cells and tissue because its excitation light penetrates far into a sample with relatively little damage. Its resolution, however, is limited by diffraction, so fine cellular structure is hard to separate.

Super-resolution methods such as stimulated emission depletion (STED) and photoactivated localisation microscopy (PALM) can resolve features well below 100 nanometres. Their developers shared the 2014 Nobel Prize in Chemistry. In practice, though, they work best close to the sample surface. Researchers have therefore tended to choose between depth and detail.

How the technique works

The name is an abbreviation of two-photon excited image scanning microscopy combined with super-resolution optical fluctuation imaging. Image scanning microscopy records the light from each scanned point with a detector array rather than a single detector. The extra spatial information can then be used to sharpen the image. Optical fluctuation imaging exploits the way fluorescent labels flicker, using statistical analysis of that variation to improve resolution further.

Layering the two on a two-photon microscope is intended to gain resolution while keeping the long-wavelength excitation that gives two-photon imaging its reach. The team reports lateral resolution of about 75 nanometres, which breaks the 100-nanometre barrier named in the paper's title. The first author is Anton Classen. The group laid groundwork for the approach in a 2024 study in PRX Life.

What was tested, and what remains

The demonstration used cultured midbrain dopamine neurons in which the cytoskeletal protein tubulin was labelled with quantum dots. This is a controlled test sample rather than thick, intact tissue. The source text does not report resolution measured at depth in tissue, so the headline figure should be read as a demonstration of the principle.

Co-author Alma Fernandez Gonzalez said that moving the method into fields from neuroscience to plant and animal biology will take time. She noted that more work is needed to improve the technique, but that the team sees a route to do so. She also said colleagues in the Texas A&M College of Medicine are interested in imaging deeper and in more detail.

Why it matters for research

The authors suggest that, together with the earlier study, the work points towards tools that can reach further into plant and soil biology than current methods. Possible targets include root structures, plant–microbe interactions and the cellular progression of disease. Neuroscience and developmental biology, where two-photon microscopy is already routine, are other obvious beneficiaries.

Several questions will decide how useful the method becomes:

• How well the resolution gain holds up as imaging depth increases in scattering tissue.
• Whether labels other than quantum dots, which blink in ways suited to fluctuation analysis, can be used.
• How acquisition time and sample exposure compare with those of existing super-resolution methods.

Independent tests in thicker and more varied specimens would show whether the combination can serve as a general-purpose upgrade for existing two-photon systems.

The study is: Anton Classen et al., Breaking the 100-nm resolution barrier with multiphoton microscopy using image scanning microscopy and optical fluctuation imaging, Journal of Biomedical Optics (2026), DOI: 10.1117/1.jbo.31.8.086501.

Source: Microscopy upgrade sharpens images fivefold without sacrificing tissue imaging depth (Phys.org, 11 November 2026).

Image: AI-generated illustration.