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Hypereon for multiphoton microscopy
Weak fluorescence and large differences in signal intensity place demanding requirements on optical detection. Hypereon combines high sensitivity, low readout noise, and a wide dynamic range. The examples below compare image quality with conventional GaAsP-PMT-based detection in multiphoton fluorescence imaging.
Weak and bright structures in a single image
In multiphoton fluorescence imaging, the visibility of fine neuronal structures depends on how clearly weak signals can be distinguished from the background. The images below compare Hypereon with a conventional large-area GaAsP PMT at two gain settings. Overview and enlarged images are shown to compare the visibility of both the overall tissue structure and fine neuronal features.
With a GaAsP PMT, lowering the gain avoids saturation but leaves more background noise, while raising the gain brings out weak structures but saturates bright areas. In this example, the Hypereon image shows weak and bright structures together with lower background noise, without gain adjustment.
Hypereon (Hy-GaAsP)
GaAsP PMT, low gain
GaAsP PMT, high gain
Image courtesy of Keisuke Isobe, RIKEN Center for Advanced Photonics, and Takayuki Michikawa, Tatsushi Yokoyama and Itaru Imayoshi, Institute for Life and Medical Sciences, Kyoto University.
Measurement condition
| Items | Hypereon | GaAsP PMT (low gain) | GaAsP PMT (high gain) |
|---|---|---|---|
| Detectors | Hypereon, Hy-GaAsP | H15460, large-area GaAsP PMT | |
| Effective area | φ5 mm | 14 mm × 14 mm | |
| Gain setting | No gain adjustment | Adjusted to avoid saturation | High gain |
| Excitation wavelength | 920 nm | ||
| Average excitation power at the sample | 8 mW | ||
| Sample | HaloTag Oregon Green Ligand-labeled mouse cerebral cortex tissue section | ||
| Field of view | 500 μm x 500 μm, 3 x 3 tiled image | ||
| Image size | 512 pixels x 512 pixels | ||
| Frame rate | 15 fps | ||
| Frame averaging | None | ||
Multiphoton imaging without frame averaging
The images below compare results obtained with a Hypereon-based imaging system and a commercial multiphoton microscope. Both images were acquired at an average excitation power of 4 mW without frame averaging.
Under these low-excitation-power conditions, fluorescent structures are clearly visible in the image acquired with the Hypereon-based system. The two systems operated at different excitation repetition rates: 8 MHz for the Hypereon-based system and 80 MHz for the commercial multiphoton microscope.
The comparison therefore shows the overall imaging results obtained with the two system configurations rather than detector performance alone.
Hypereon-based system
No frame averaging
Commercial multiphoton microscope
No frame averaging
Image courtesy of Keisuke Isobe, RIKEN Center for Advanced Photonics, and Takayuki Michikawa, Tatsushi Yokoyama and Itaru Imayoshi, Institute for Life and Medical Sciences, Kyoto University.
Measurement condition
| Items | Hypereon-based system | Commercial multiphoton microscope |
|---|---|---|
| Detectors | Hypereon, Hy-GaAsP | GaAsP PMT |
| Excitation repetition rate | 8 MHz | 80 MHz |
| Excitation wavelength | 920 nm | |
| Pulse duration | 250 fs | |
| Average excitation power at the sample | 4 mW | |
| Sample | Alexa Fluor 488-labeled mouse cerebral cortex tissue section | |
| Field of view | 200 µm × 200 µm | |
| Image size | 512 pixels × 512 pixels | |
| Frame rate | 15 fps | |
| Frame averaging | None | |
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