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Clean Pulse Technology

Brighten up your images with TOPTICA’s femtosecond fiber lasers

Multi-photon microscopy is a nonlinear microscopy technique where multiple near-infrared photons interact with the sample to create a fluorescent excited state. Thus, the fluorescence brightness and contrast when imaging depend on the peak-power of the excitation laser. The peak-power is determined by the average laser power, the repetition rate, the pulse duration at the sample plane and most importantly the temporal pulse quality.

During in-vivo imaging, the laser average power should be limited at the sample plane to prevent thermal damage and phototoxicity. With the typical throughput of a multi-photon microscope, a Watt-level laser has more than sufficient power for 2-photon excitation. In addition, 80 MHz is a well-established repetition rate yielding appropriate pixel dwell times and synchronization possibilities with galvo and resonant scanners.

High brightness and low sample heating with Clean Pulse Technology

Graph displaying a bad pulse with no 2-photon signal, leading to just heating, accompanied by an image of a cell with 70% brightness. This illustrates the impact of improper pulse shape on two-photon imaging, highlighting decreased signal quality.
Graph showcasing a clean pulse with optimal two-photon signal, resulting in a clear cell image with 100% brightness. This demonstrates the importance of clean pulse shape for high-quality two-photon imaging, ensuring maximum brightness and signal accuracy.

Hence, with laser power and repetition rate fixed by experimental requirements, pulse duration and temporal pulse quality are the crucial factors that can be optimized for fluorescence image quality. Pedestals and side-wings on the temporal pulse profile do not contribute to the non-linear absorption but only unnecessarily heat the sample. Likewise, shorter pulses yield higher fluorescence intensity due to higher peak power. Thus, the combination of a high temporal pulse quality (a “clean pulse”) and a short pulse duration at the sample plane gives the best result in terms of overall 2-photon excitation. This ultimately improves the contrast and brightness of fluorescence images.

The pictures from the article Influence of laser pulse shape and cleanliness on two-photon microscopy by Shau Poh Chong and Peter Török are used with permission of the authors.

The image is a comparative visualization showing the difference between two microscopy techniques. On the left side, labeled "without Clean Pulse Technology," the image appears darker and less detailed. On the right side, labeled "with Clean Pulse Technology," the image is brighter and shows more distinct and clear details of the microscopic sample. A vertical line divides the two halves, with a circular icon featuring arrows pointing left and right at the center, indicating the comparison. The background of both sides is green, highlighting the improvements in image clarity and brightness achieved with Clean Pulse Technology. The image is a comparative visualization showing the difference between two microscopy techniques. On the left side, labeled "without Clean Pulse Technology," the image appears darker and less detailed. On the right side, labeled "with Clean Pulse Technology," the image is brighter and shows more distinct and clear details of the microscopic sample. A vertical line divides the two halves, with a circular icon featuring arrows pointing left and right at the center, indicating the comparison. The background of both sides is green, highlighting the improvements in image clarity and brightness achieved with Clean Pulse Technology.
Two-photon microscopy imaging of gCAMP6s-labeled brain slice without and with Clean Pulse Technology.
The image is a comparative visualization showing the difference between two microscopy techniques. On the left side, labeled "without Clean Pulse Technology," the image appears darker and less detailed. On the right side, labeled "with Clean Pulse Technology," the image is brighter and shows more distinct and clear details of the microscopic sample. A vertical line divides the two halves, with a circular icon featuring arrows pointing left and right at the center, indicating the comparison. The background of both sides is green, highlighting the improvements in image clarity and brightness achieved with Clean Pulse Technology. The image is a comparative visualization showing the difference between two microscopy techniques. On the left side, labeled "without Clean Pulse Technology," the image appears darker and less detailed. On the right side, labeled "with Clean Pulse Technology," the image is brighter and shows more distinct and clear details of the microscopic sample. A vertical line divides the two halves, with a circular icon featuring arrows pointing left and right at the center, indicating the comparison. The background of both sides is green, highlighting the improvements in image clarity and brightness achieved with Clean Pulse Technology.
Zebrafish imaging in vivo in the olfactory bulbs with neurons tagged with gCAMP7f without and with Clean Pulse Technology.

TOPTICA's offer

To achieve the best image quality, TOPTICA’s FemtoFiber ultra 920 incorporates Clean Pulse Technology - a laser design that significantly reduces pedestals and side-wings on the temporal pulse profile compared to other laser amplifier designs. Thus, the full laser power of the FemtoFiber ultra 920 is contained in the main laser pulse to maximize two-photon excitation. With Clean Pulse Technology, the FemtoFiber ultra 920 enables unmatched fluorescence image brightness in non-linear microscopy (see Literature).

Additionally, TOPTICA’s FemtoFiber ultra provides motorized group delay dispersion (GDD) pre-compensation that can be controlled in software.  Due to chromatic dispersion, a femtosecond pulse stretches in time when transmitted through optical material in the microscope. GDD pre-compensation adds the opposite dispersion to circumvent this effect by cancelling it out. TOPTICA’s software-controllable GDD pre-compensation ensures short pulses at the sample plane with user-friendly, repeatable optimization of fluorescence signal strength via the graphical user-interface (GUI).