In confocal microscopy, a pinhole is used to remove extra light generated outside the focal point on the sample and to improve optical resolution and contrast. However, the amount of light that reaches the detector is reduced by the pinhole. So the detector needs higher sensitivity and lower dark current to achieve a better signal-to-noise ratio, resulting in higher dynamic range and in better contrast between the sample’s structure and the background.
Then how about multiphoton microscopy? Multiphoton microscopy does not require a pinhole like confocal microscopy, and it can observe deeper into the sample. Despite the light not being dimmed by a pinhole, the amount of signal is still very small because the multiphoton absorption process is a phenomenon that occurs with a very low probability. So a detector for multiphoton microscopy requires high sensitivity and low noise performance, just like confocal microscopy.
Hamamatsu offers both PMTs and MPPCs (SiPMs) as detectors in confocal and multiphoton microscopy, but the best detector depends on light intensity, sample thickness, scan speed, and many other factors. Please feel free to contact us regarding the selection of detectors.
A compound-semiconductor photocathode for PMTs is designed to further improve the photocathode’s crystallinity and increase its sensitivity in the visible and near-infrared regions. To produce compound-semiconductor photocathodes, a crystal made of gallium arsenide (GaAs) or gallium arsenide phosphide (GaAsP) is adhered to glass, thinned to several µm thickness, and then reacted with cesium to achieve high quantum efficiency (QE). These compound-semiconductor photocathodes, which achieve high sensitivity, are superior to alkali photocathodes in the following ways:
Figure 1. Typical spectral response curves for PMTs with GaAs and GaAsP photocathodes
As shown in the spectral response graph (Fig. 1), GaAs/GaAsP photocathodes are highly sensitive not only in the visible region but also in the red region (such as at 700 nm and 800 nm), which tends to have a smaller amount of fluorescence.
Although GaAs/GaAsP photocathodes have high QE, their disadvantage in the past was their low damage threshold. However, Hamamatsu has recently improved the durability of these photocathodes, and now offers several PMT modules (Fig. 2) that incorporate the improved photocathodes. Click on a part number below to learn more about these modules.
Among the modules listed above, the GaAsP PMT modules (-40 models) have improved maximum output current from 2 µA to 40 µA. The output current value of GaAs PMT modules (-50 models) has not been finalized, but it has improved.
In the H15460-40 GaAsP PMT module, the maximum current output has been further improved to 100 µA, the same value as a PMT with a typical alkali photocathode. The H15460-40 is discussed in more detail in the next question below.
Figure 3. H15460-40 PMT module with a large 14 mm square effective area
To improve light collection in a multiphoton microscope, Hamamatsu offers the H15460-40 PMT module. This module’s 14 mm square effective area is larger than the 5 mm dia. of the PMT modules mentioned in the previous question (Fig. 3).
As described in a previous question, multiphoton microscopy obtains signals from a deeper part of the sample. The signals are diffused in the sample, and a detector with a wider field of view is required to collect as much of the signal as possible.
Also, when designing a microscope, it is difficult to balance the field of view, magnification, numerical aperture (NA), etc. For example, if you just want to widen the field of view, you can use a low-magnification objective lens, but this objective lens has a small NA. A detector with a large effective area helps facilitate such designs.
In addition to its larger area, the H15460-40 has a built-in amplifier to reduce noise between the detector and electronic devices, improving the overall sensitivity. This will improve the image contrast.
Table 1. Gating function specifications of H11706-40 and H12056-40 PMT modules
A gating function temporarily switches the distribution of the applied voltage to the dynode, preventing the electrons from moving to the subsequent stage. Please note that the gating function is effective only when the timing of the excess light is known in advance; it has no effect on accidental overexposure to light. See Table 1 for the gating specifications of the H11706-40 and H12056-40 PMT modules.
Hiro Furuhashi is a marketing engineer in Hamamatsu’s San Jose, CA, office. He had been designing PMT modules for 9 years before moving to the US from Japan in 2019. He currently conducts market research on microscopy, in vitro diagnostics (IVD), and flow cytometry (FCM), and he also provides technical support as a PMT and PMT module specialist. He is a very active person, loves traveling, loves sports particularly surfing, and goes to the beach to surf almost every weekend.
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