Breakthroughs using multiphoton microscopy

Multiphoton microscopy has revolutionized biological imaging by allowing high-resolution imaging in 3D without the need for dyes or labels. Imagine diagnosing cancer in just six minutes—without slicing, staining, or waiting days for lab results. Thanks to groundbreaking advances in ultrafast laser technology, this vision is rapidly becoming reality.

At the heart of this breakthrough are femtosecond lasers  which have made multiphoton microscopy a valuable tool in the study of biological samples. One key advantage of using femtosecond pulses in multiphoton microscopy is reduced photodamage to the sample. The short pulse duration minimizes the exposure time to light, reducing the amount of photodamage that occurs during imaging. This is particularly important in the study of live samples, as it allows researchers to obtain high-resolution images without affecting the viability of the sample.

The latest generation of compact ultrafast femtosecond lasers can, indeed, simultaneously perform:

• Two-photon fluorescence (2PF)
• Second harmonic generation (SHG)
• Three-photon fluorescence (3PF)
• Third harmonic generation (THG)

This combination unlocks rich, high-contrast images of cells, collagen fibers, and other tissue structures—all in real time.

Why pulse duaration matters in multiphoton imaging?

Shorter laser pulses mean higher peak intensity, which dramatically boosts imaging signals:

• 2PF signals increase fivefold with shorter pulses.
• 3PF signals can increase up to 25 times.

These improvements also reduce heat and photobleaching, making long-term imaging of live cells safer and more effective.

Another advantage of using femtosecond pulses is increased efficiency of two-photon fluorescence. The high peak power of these pulses enhances the nonlinear interaction, resulting in increased fluorescence efficiency. This allows researchers to obtain high-resolution images with fewer photons, reducing the need for high laser power and minimizing photodamage.

In addition, femtosecond pulses provide improved spatial resolution in multiphoton microscopy. The short pulse duration and high peak power allow for high-resolution imaging in 3D, enabling researchers to study samples at the cellular and subcellular level.

Multi photon microscopy image of lung tissue. Co. of Prof ML Groot Vrije Uni Amsterdam

Advantages of ultrashort pulse durations: sub 50 fs

Since the efficiency in multiphoton excitation is strongly dependent on the peak power of the incident light during the pulse, the shorter the pulse, the higher the peak power and the stronger the generated multiphoton signal. Up till now there has been relatively limited ability to generate ultrashort and clean pulses below the 80 fs limit.

The VALO Femtosecond Series offers a novel approach to overcome this limit, resulting in much shorter (sub 50 fs) and clean pulses. Figure 1 illustrates the relationship between the two-photon and three-photon efficiencies and the pulse duration [1, 2]. The efficiency of multiphoton events depends non-linearly on the laser peak power, and the corresponding signal increases with the square and cube of the incident light’s peak power, for two and three photon processes, respectively. For instance, decreasing the pulse duration from 200 fs to 50 fs results in a 4 times higher peak power, the two-photon efficiency is increased by a factor of 4 while the three-photon efficiency increases by a factor of 16.

Figure 1: Two and three photon efficiency vs. pulse duration

However, to experimentally apply this relationship, it is essential to accurately determine the pulse duration at the sample. If dispersion in the microscope optics is not properly compensated for, the pulses will be stretched, and lower peak power will be measured at the sample. In such a case, the relationship between pulse duration and higher-order harmonics generation efficiency, cannot be accurately verified experimentally. To generate sufficient non-linear SHG and THG signals to achieve the required signal-to-noise ratio at average power levels that are gentle to the sample, sub 50 fs pulses and the use of dispersion pre-compensation is essential.

Figure 2a) shows the third harmonic signal from a calibration grid using an average power of 4.7 mW on the sample, with the full bandwidth of the VALO Femtosecond Series laser, yielding ~40 fs pulses. In Figure 2b), the spectral bandwidth of the laser was restricted to 10 nm FWHM around 1 064 nm, resulting in ~160 fs pulses. The image in Figure 2a) and 2b) are identically scaled, but Figure 2b) shows no THG signal. Only after rescaling the lower THG signal from the longer ~160 fs pulses, was it possible to obtain an image above the experimental noise floor, as shown in Figure 2c). In this case, a 2.5 times higher average laser power was needed to achieve a signal to noise ratio comparable to that of THG signals obtained from shorter sub 50 fs pulses [3].

Figure 2: Third harmonics of a calibration grid (Ibidi) with 50 micrometer squares. a) 4.7 mW, full spectrum short pulses (<50 fs; VALO Series). b) 6 mW with laser spectrum limited to 10 nm bandwidth (~160 fs). c) Scaled up contrast for the 6 mW laser spectrum limited to 10 nm bandwidth (~160 fs).

Real-Time Cancer Diagnosis with Multiphoton Microscopy

One of the most promising applications of multiphoton microscopy is in cancer research, and specifically cancer diagnostics. Traditional biopsy analysis can take days, but higher harmonic generation (HHG) imaging can deliver results in minutes. A recent study showed that a biopsy could be imaged and analyzed just six minutes after excision, with 87% accuracy [4] .

This technique is especially valuable during procedures like bronchoscopy, where obtaining sufficient tissue for diagnosis is challenging. HHG imaging provides immediate feedback, potentially eliminating the need for repeat procedures.

Start-up Flash Pathology, spun out of Vrije Universiteit Amsterdam, is developing a compact HHG microscope for clinical use. Their goal? To bring real-time, label-free tissue imaging into hospitals, revolutionizing diagnostics. This is possible because they integrated our VALO femtosecond laser into a mobile microscope that can be transported directly into the surgery room.

Watch Dr. van Huizen from Flash Pathology as she outlines real-life applications of multiphoton microscopy and higher harmonic generation imaging. These are some of the early results and clinical studies from her mobile microscope.

In conclusion, the use of very short femtosecond pulses in multiphoton microscopy has several advantages, including reduced photodamage, increased efficiency, improved spatial resolution, and reduced background signal. In particular, the ultrashort sub 50 fs pulses provide considerably higher pulse peak powers, which results in optimal signal-to-noise ratio images at much lower average power, which in turn reduces photobleaching, and extends cell viability. These advantages have made multiphoton microscopy a valuable tool in the study of biological samples, allowing researchers to obtain high-resolution images with minimal photodamage.

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References

[1] Shuo Tang, Tatiana Krasieva, Zhongping Chen, Gabriel Tempea, Bruce Tromberg (2006), Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy, Journal of Biomedical Optics, 11(2).

[2] Mira Sibai, Hussein Mehidine, Fanny Poulon, Ali Ibrahim, M. Juchaux, J. Pallud, A. Kudlinski,  Darine Haidar (2018), The Impact of Compressed Femtosecond Laser Pulse Durations on Neuronal Tissue Used for Two-Photon Excitation Through an Endoscope, Scientific Reports, 8:11124.

[3] White paper, Sub 50 femtosecond pulse lasers for gentler multiphoton microscopy, HÜBNER Photonics. In publication Feb 2023.

[4] Prochnow Oliver, van Huizen Laura (2025), Unveiling cell secrets with lasers, Photonics Views.

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Learn more about the range of ultrafast femtosecond fiber lasers from HÜBNER Photonics. With outstandingly high pulse peak power levels and computer controlled group velocity dispersion pre-compensation, the VALO femtosecond fiber lasers are ideal for multiphoton imaging.