Physics · the two-photon microscope
Seeing deep
A two-photon microscope has no camera. It moves one tiny glowing spot across the sample and counts the photons that come back, one pixel at a time. Watch it draw a picture, then take it deep into tissue next to a confocal microscope.
The next step after the femtosecond laser and the photomultiplier and one photon or two.
Part of Work · What I've done
A two-photon microscope is the two instruments from my lab put together: a femtosecond laser like the 800 nm one from my PhD, and photomultipliers like the ones from my postdoc. Two mirrors sweep the laser's focus across the sample, and a photomultiplier counts the glow at each point. Only the focus glows, so a photon that comes back belongs to that pixel, even after bouncing around in tissue.
That is why the image stays sharp about three times deeper than a confocal microscope's, deep enough for neuroscientists to watch living neurons half a millimetre inside the brain. Change the dwell time to see photon counts become an image, then push the focus deeper and watch which image breaks up first.
Terms used here
- Scanning mirrors
- Two small mirrors that tilt very fast, one for left and right, one for up and down. They move the focus across the sample.
- Dwell time
- How long the focus rests on one pixel while its photons are counted, a few millionths of a second.
- Dichroic mirror
- A mirror that lets one colour through and reflects another. Here the red laser passes and the green glow turns towards the detector.
- Confocal microscope
- Excites the sample with visible light and puts a pinhole in front of the detector, so only light coming straight back from the focus gets through.
- Scattering length
- How far light travels in tissue, on average, before it bounces off course. In the brain it is about 60 µm for visible light and 200 µm for near-infrared.
- Shot noise
- Photons arrive at random, so a pixel expecting n photons gets about n ± √n. Ten photons are uncertain by 30%, a hundred by 10%.
01 · Drawing a picture
one pixel at a time
A picture made of photon counts
The mirrors move the focus along a line, then step down to the next one. At each pixel the photomultiplier counts photons for a few microseconds: more photons, brighter pixel. The scan is slowed down so you can follow it. Press on the image to take over: the focus stays under your finger, and the longer you hold a spot, the cleaner it gets.
Look at the cells: at 0.5 µs the image is grainy because each pixel catches only one or two photons. At 20 µs it is clean, but a full frame takes five seconds, too slow to follow a neuron firing.
A bright pixel sends 3 photons per microsecond to the detector here (a typical order for a well-labelled sample). Frames of 512 × 512 pixels; the simulation shows 64 × 64 so you can see each pixel.
02 · Going deeper
confocal or two-photon
Keeping the scattered photons
Light leaving the focus bounces around in tissue. Green photons come straight back and both microscopes keep them; blue ones bounced, and only the two-photon microscope keeps them. A confocal microscope's pinhole only passes photons that come straight back, so it throws the bounced ones away. In a two-photon microscope only the focus glows, so a wide detector keeps all the photons that reach the objective. Wherever they bounced, they came from that pixel.
Both images are turned up to fill the screen, the way you would raise the detector gain. Look for the moment the confocal image turns to speckle: the cells are still there, but too few photons come back to draw them.
Scattering lengths in brain tissue: 60 µm visible, 200 µm near-infrared (Helmchen and Denk, 2005). Confocal: the 488 nm light must reach the focus unscattered and the glow must come back unscattered, signal ∝ e−z/60 · e−z/60. Two-photon: only the 800 nm light must arrive unscattered, and the glow goes as its square, signal ∝ e−2z/200; the wide detector is taken to keep every photon. Real microscopes raise the laser power with depth, which pushes two-photon imaging towards a millimetre.
Sources
- The first two-photon microscope: W. Denk, J. H. Strickler and W. W. Webb, Science 248, 73 (1990).
- Depth, scattering and collecting scattered light: F. Helmchen and W. Denk, Nature Methods 2, 932 (2005).
- The depth limit of two-photon imaging: P. Theer and W. Denk, J. Opt. Soc. Am. A 23, 3139 (2006).