2 photon Laser optical path (Will Grimes June 3, 2013) (page 1 of 10)

polarized mirror

from laser

Goals and Principles: • The laser puts out up to 3W, which is dangerous, and more than twice what is needed for excitation of a biological sample. • To split the laser into two 1W paths or to dump one of the paths into a beam dump. • Optical components before the pockel cell need to be of special grade to handle the power of the laser (GVD group velocity dispersion, lower number # < 0.4 (check?) • The longer the light path, the more alignment is needed. Want to keep the light path short so that changing the wavelength of the laser does not require re-aligning the path. The Rieke lab rig path is about 6-7ft long in total • To keep dust off the optics. Dust will burn and damage the optics. Ideally cover all optical components.

mirror

iris to prevent light from back scattering into the cavity of the laser and shutting the laser down (Thor labs SM1D12, held by LMR1) waveplate to polarize the beam and send part of the beam of one polarization to a second scope or to a beam dump

beam dump (Thor labs BT610)

mirror (not necessary but

helpful for having an extra place for

adjustment)

pockel cell: crystal polarizers that change orientation with voltage. want to orient the polarizers so that the maximum range can be obtained. hole out of the side of the pockel cell where extra laser light gets dumped so be careful where this points. (ConOptics model 350-80 EO modulator)

mechanical shutter: allows you to change the pockel cell and measure the light before opening the shutter to the prep (Vincent Associates, LS6ZM2, Uniblitz VMM-D1 Shutter Driver)

glass slide or mirror that mostly transmits (holder from Thor labs FH2)

power meter

bottom periscope mirror

periscope (Thor labs RS99)

into microscope scan head

2 photon Laser optical path (Will Grimes June 3, 2013) (page 2 of 10) simplified optical path

polarized mirror (Thor labs

PSB052) or 50/50 cube (BS005)

from laser

iris (Thor lab, ID25SS) iris to prevent

light from back scattering into the cavity of the laser and shutting the laser down (Thor labs SM1D12, held by LMR1)

waveplate to polarize the beam and send part of the beam of one polarization to a second scope or to a beam dump (Thor labs WPH05M-808, PRM05)

beam dump (Thor labs BT610)

pockel cell

mechanical shutter: allows you to change the pockel cell and measure the light before opening the shutter to the prep (Vincent Associates, LS6ZM2, Uniblitz VMM-D1 Shutter Driver)

glass slide or mirror that mostly transmits (holder from Thor labs FH2)

power meter using one from Spectra Physics, but I haven’t determined if it’s sensitive enough for picking off such a small portion of the beam. That detector is usually placed directly in front of the beam.

bottom periscope mirror

periscope (Thor labs RS99)

into microscope scan head

beam dump for backscatter light (Thor labs, LB1)

Other necessary materials: !•Edmund IR detector card •Paper business card that you can see through •Protective eyeware !!

iris (to help with alignment)

iris

Conoptics

Aligning the 2 photon laser optical path (Will Grimes June 7, 2013) (page 3 of 10)

Goals and Principles: • Align the pockel cell to the laser path. Get the laser path aligned before introducing the pockel cell • Especially useful to have a power meter that you can place at different steps in the path (Thor labs PM100D, S130C) • Would like about 100mW going down the main path. The pockel cell will allow 2 log units of attenuation. The rest of the optical path will reduce the light by 50% once it reaches the output of the objective. Would like <1mW to work with at the prep. Will orient the waveplate and pockel cell to achieve these light levels • Check the power output at 860nm and 910nm, where the laser will be used most frequently. Need to adjust the power meter to read at these wavelengths. Turn the waveplate until you get the approximate right power out. • Align the laser at 750nm so that the light is visible • Wear goggles to protect your eyes. Some goggles will not allow you to see the red light at all. •Avoid backscatter of the beam into the cavity of the laser source. This will shut down the laser when you open the shutter !• (1) Start by making sure your components are in the approximate correct location. You can do this by measuring the distance from the table to the laser output. Distance from the table to the scan head. Align the periscope mirrors to match these two locations as best as possible. The beam should be hitting the center of the mirror. • (2) Align the first part of the optical path: iris, waveplate, mirror, beam dump. Check that the backscatter is getting caught by a beam dump (doesn’t need to be rated for as high power as the main beam dump) • (3) Align the last part of the optic path: iris (if needed), glass slide and meter, shutter, bottom periscope mirror and upper periscope mirror • (4) Place the dummy pockel cell in place. Put the power meter in front of the pockel cell to check that is is approximately 100mW. Move the pockel cell controls until the beam goes through the center of the hole. Some of the light will be lost so what comes out of the alignment pockel cell will be less • (5) Place the light meter after the alignment pockel cell to make sure you are maximizing the output. • Step after this involves turning on the pockel cell. (Paused to replace the fuse) continues on the next page... !

power meter

beam dump waveplate

polarizing mirror

beam dump

pockel cell

pockel cell holder iris holder for coverslip

mechanical shutter

bottom periscope mirror

dump from pockel cell

1”

2-photon laser optical path (page 4 of 10)

• (6) Place the light meter after the pockel cell, turn on the pockel cell and set to zero, and rotate the pockel cell and aiming for 100mW coming out. Test at the wavelengths used most: 810nm and 960nm. • (7) Apply a voltage to the pockel cell: +50, -50, +100, -100 (max is +/- 400). Aiming for 20mW at the maximal extinction. The orientation of the pockel cell to get maximal extinction doesn’t depend on the waveplate but depends on the polarization of the beam coming from the laser. There is a dump coming off the pockel cell, so be careful about what direction that dump points and make sure to place a beam dump device in front. • (8) Set the waveplate so that ~100mW is coming out of the pockel cell. • (9) Now place the detector post-objective underneath the 60x objective. Point scan and zoom in 10.0-fold • (10) Adjust the periscope mirrors to maximize the power coming out of the objective. • (11) Adjusted the wave plate to get ~20mW coming out of the objective at the max of the pockel cell (400 bias voltage). Figured out the waveplate location for both wavelengths (810, 960nm). The optimal position of the mirrors will be off between both wavelengths, but optimize for the lower power wavelength (910nm). • (12) Adjusted the pockel cell to get post-objective power of 1mW, 5mW, lowest, and highest • (13) Test the alignment with small beads. !!!!!!!!!!!

Aligning the 2 photon Laser optical path (Will Grimes June 24, 2013) (page 5 of 10)

wave plate: 160 deg 810nm 960nm

lowest pockel cell: 45. power: 0.25mW pockel cell: 35. power: 0.1mW

1mW pockel cell: 85 pockel cell: 124

5mW pockel cell: 147 pockel cell: 250

highest pockel cell: 400. power: 37mW pockel cell: 400. power: 11.7mW

• Could not image the cells during the experiment, so we turned the laser to 760nm and Rachel saw that the path was going to the side of the scan head • We put in the target (beam splitter with a paper target from BioRad) and the spot could be seen at the top of the target. So at least the laser was hitting the galvos. • With the laser at 760nm, Rachel looked at the target while I adjusted the bottom mirror controls. We got the beam back on target. • Switched to the regular objectives and imaged a sample of fluorescent paper. The hot spot was slightly above center. Moving the slide around, the same spot got brighter as we moved it to the center. • Tried a slide with bipolar cells labeled. Rachel decided that the adjustment wasn’t worth it. But it would be fine to dry and adjust the knobs again. !•Recommendations. To place an iris right before the bottom mirror to help with alignment. Place two irises as far apart from each other as possible after the top mirror to help with alignment. !Aligning the laser with Luca Della Santina (October 21, 2013) • put in the target but only say lines that were moving across the target • the rest of the path seemed to be in place with the iris • moved the bottom mirror until the beam was hitting the top mirror approximately in the middle • then looked inside the scan head to look for the beam. the beam was off the aperture in the front compartment, so much that instead of a spot there was a line • moved the top mirror until the line became a beam • then looked for the galvos in the right front compartment of the scan head and saw which direction to move the beam so that the galvos were hit • once the beam was hitting the galvos, I looked back at the target and now there was a spot of light. The last movements were only the top mirrors and we got the spot in the middle !• Also tried to put in the detector in the laser path, but there may be something wrong with the display. check the calibrations on the meter. !!!!!!!!!!!!

Aligning the 2 photon Laser optical path (Rachel Wong September 15, 2013) (page 6 of 10)

Scientifica Setup: Setting up the Laser Path (October 20-22, 2014)

Purpose: !• To set the launch optics for the laser as safely as possible

Materials: !• Remove all reflective surfaces: watches, rings,

Procedure: !• Turn on the laser to alignment mode (750nm, 300mW still strong enough to cause damage so you want to attenuate the beam as soon as possible • Start with the 50/50 beam splitter and two beam dumps as close as possible to the output to attenuate the laser right away. Make sure you understand the orientation of the beam splitter. There are arrows drawn into the glass. • Back off the bottom two threads of the pockel cell holder all the way. • Place in the dummy pockel cell and align so that the beam goes straight through the center of the pockel cell. Looking for a clean spot to come out of the other end of the pockel cell. • Place in the real pockel cell. Want to start with the orientation of maximal extinction, which usually ends up being with the dump facing straight up. To anticipate this, place a beam dump on posts that with a 90 deg angle. • Plug in the cables for the pockel cell so that you can modulate the power (PM goes to the modulator). With the offset at 0, place a meter at the output end of the pocket cell and rotate the pockel cell until you get the minimum power out. Turn up the offset so that you have a visible beam to align for the rest of the path. The power will be about 4mW. As a sanity check, the power before the pockel cell is about 130mW, so a 3W beam is getting split 50-50. • Start with an iris before the pockel cell to get the approximate height and then get a few other irises to sit at that same height. You will use these throughout to check the alignment. You can check if the beam is straight by placing two irises in a row. Place an iris at the front end of the pockel cell to prevent backscatter into the laser cavity. Set irises between every critical component to use as alignment checks. • Align the beam through the center of the shutter. • Align the beam to hit the bottom mirror of the periscope. Want to check that the beam is hitting the center of the mirror. • Place the beam close to, but not yet shooting into the laser. !• Check the minimum offset of pockel cell by placing a meter in front of the pockel cell. 80uW at -8 offset. Have one person adjust the offset while the other person reads the meter.

beam dump

beam dump

laser

beam dump facing the pockel cell from above

iris

beam dumpplaced another beam dump because we saw back scatter in this direction

pockel cell in orientation of maximal extinction

beam dump on top of the pockel cell

45 deg mirror

irisshutter

iris

45 deg mirror

iris

periscope