Background Resources#

  1. General principles of microscopy are covered in an excellent book “Fundamentals of Biomedical Optics” sitting in the Wilson Lab library. Nikon Microscopy U by Michael Davidson is also a great resource.
  2. Laser scanning basics are covered in this Thorlabs Laser Scanning Microscopy Tutorial.
  3. Svoboda and Yasuda 2006 has a nice summary of the principles behind two-photon microscopy.

Components#

There are several optics components that make up our two photon systems. Some differences exist between the systems, but the following core components are shared (and differences noted). A convenient way to break them down is into 1) laser and ancillary components, 2) table optics, and 3) the microscope proper.

  1. Multiphoton laser.
  2. Laser. Two-photon imaging requires ultrafast lasers see the resources above for why this is (it’s really cool!). Currently the most popular are Ti:Sapphire lasers (pronounced “tai-saph” or “taitanium-sapphire”). The name refers to the component inside of the laser responsible for the actual “lasing” or emitting of photons. These lasers produce a stream of pulses (periods of high photon flux) with repetition rates of ~100 MHz, corresponding to a pulse duration is on the order of 100 fs, and are therefore termed “femtosecond lasers”. Berg 1 and 2 share a Chameleon Vision-S laser from Coherent, with a tunable excitation wavelength from below 700 nm to above 1000 nm (we use mainly use 940 nm, which excites GCaMP). The other two set ups (soon-to-be Berg 3 and 4) share a Mai-Tai laser from Spectra-Physics. The manual for the Mai-Tai laser is an excellent resource for learning about how tunable, and femtosecond, lasers work, and is largely accessible with a bit of googling and nodding along.
  3. Recirculating chiller. The laser also has a recirculating chiller for maintaining a stable internal laser temperature, it is absolutely critical that this chiller remains functional and regular maintenance is required to prevent laser failure.
  4. Air purification unit. The air inside of the laser cavity must be extremely clean and dry, dust and water vapor can burn on, or damage components as the high energy pulses hit contaminants. There are high-quality filters that remove these impurities and keep the air dry.
  5. Table Optics. On the “table” (the air table) we split and modulate the intensity of the laser beam. From the exit point of the laser to the microscope here are the optical components on the table.
  6. Half-wave plate. A femtosecond laser emits a highly polarized beam, which we take advantage of for beam routing and splitting (and recombination, but not in our systems… yet!). The half-wave plate shifts the polarization direction of the laser beam going through it. We mount the half wave plate in a rotational mount, so that we can point this polarization direction where we want.
  7. Polarizing Beam splitter Often abbreviated PBS, this takes the laser and splits the beam into two beams of orthogonal polarization states, one which is reflected (commonly called “the bounce path”), and one which passes through (“the through path”). By rotating the half-wave plate prior to the PBS, we can effectively divide the laser intensity in half (think about the two polarizations coming out as the vector sum).
  8. Pockels cell. Pockels cells, or P-Cells, are electrically tunable optical elements. More specifically, they are voltage controlled wave plates which operate at very high frequencies (like the previous two components combined). By varying the voltage we deliver to them (which can be changed from the Scanimage console), the polarization beam passing through is rotated different amounts, which lets more or less of the laser through them, and therefore changes the laser power that will eventually reach our sample. Note there are other ways to modulate the laser, some of which are now built in to lasers themselves!
  9. Mirrors. Different set ups will have different number of mirrors, and in different positions. These are used to properly direct the beam towards the scope (see alignment).
  10. Microscope:
  11. Periscope. A pair of mirrors which takes the beam from table level to microscope level. On crowded tables, sometimes they are used to take the beam from a laser stack (on top of another) and move it down to the table level.
  12. Scan head. A system of mirrors that directs the beam to a particular location in the sample (either Galvo-Resonant or Galvo-Galvo).
  13. Flipper mirrors. Motorized mirrors that redirect the beam path. Our two Bergamo microscopes have two flipper mirrors each: 1) one that flips the light source hitting the sample (between the laser and an LED), and 2) one that flips the collection point of the light coming from the sample (between the PMTs and the eyepieces).
  14. Objective. 2-Photon microscopy requires some specific (and often debated) types of objectives, we use standard objectives and stay above the fray.
  15. PMTs. The photomultiplier tubes are very sensitive light detectors. They are at the end of our imaging path and collect the fluorescence light coming from the GCaMP excitation of our sample.

Alignment#

To align the laser, you will need the following components:

  • laser safety goggles (close the curtains, take off your watch and jewelries)
  • laser viewing cards
  • cage plates

Alignment Mode: The Coherent laser can be set to ‘alignment mode’ in the front panel, which tunes the beam to visible red light and reduces the output power, and therefore can be looked at without the safety goggles. The Mai-Tai does not have this feature.

“Walking the beam”: The pdf below has a nice introduction to how to move a laser with a pair of mirrors, reading this and trying for yourself is the best way to learn (it’s not hard! just tedious sometimes).

📎 walking-the-beam.pdf

When adjusting the mirrors on the periscope, use the tilt and the tip. Don’t use the horizon screw! (Thorlabs MM101 manual).

Backward Coherent Vision-S laser ScanImage Setup (Download + Installation) Forward