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Lasers

Various laser launches can be used with diSPIM. Below find notes specific to each one.

In normal diSPIM use the laser launch is controlled using digital TTL inputs. The Tiger controller's PLC card generates those signals, which will turn the lasers on/off at the appropriate times and also selects the correct side if needed. For information on connecting the cables see the appropriate section of the manual. The analog intensity is generally static during the experiment and so can be controlled using the high-level software (Micro-Manager or vendor's software) over a USB or serial connection; there may be some enabling that needs to happen using the high-level software. If analog intensity must be controlled during an experiment then ASI can provide a separate Tiger card which can drive the laser's analog inputs.

In general the laser power requirements are very modest, e.g. a few mW of optical power at the sample at most and often less than 1 mW. The exact power required depends on the sample (fluorophore density, efficiency, required SNR and readout speed to get the biological answers you're seeking). Light sheet microscopy is used precisely because it doesn't need much excitation light, but every sample will bleach eventually so the intensity is often increased to improve SNR until bleaching becomes problematic.

The ASI scanner has a fiber optic input with either an FC/PC or FC/APC connector (specify which when ordering). The focal length of the collimation lens should match the fiber's NA to fill the scanning mirror appropriately, for NA ~0.11 the default 12.5 mm focal length lens works well and for NA ~0.06 order the 20 mm focal length option. Single-mode fiber is required for intended operation.

Getting Dual Outputs

For a dual-view system it is helpful to have a laser launch with dual outputs. That way the entire laser intensity can be switched between the two paths since only one path is used at a time. Such launches are not common but multiple options are commercially available (see below).

If you don't use a laser launch with dual outputs you have two basic approaches: (1) split the beam passively or (2) insert an active switch.

First, you can passively split the beam into two fibers and rely on the unused scanner to blank its beam at any given point in time. The scanner gives decent but incomplete blanking (up to 0.1% bleed-through, which is normally acceptable because when acquisition isn't happened the laser is off anyway). Fiber splitters are inexpensive and compact but the split ratio depends on wavelength so this doesn't work for multi color. You can split into 2 paths in free space on an optical table before fiber coupling, which requires some expertise but allows a 50/50 split across wavelengths. 1) When using an optical table it's possible to insert shutters to fully block the beam. Passively splitting the beam reduces the intensity by at least a factor of 2 (more with losses) but normally that isn't a problem because light sheet imaging usually uses very low intensities. If slit scanning is implemented (not in Micro-manager as of May 2016) there is a possibility of ~2x speedup in acquisition by simultaneously imaging from both sides with a slight offset in beam position, and this potential speedup will require a split beam as both scanners will be illuminating simultaneously.

Second, you can add a galvo or 1×2 switch between the laser output and the two scanners. Leoni makes a commercial switch with male fiber inputs and outputs. You will need to couple the laser's fiber with the switch's input fiber, e.g. with a butt-coupler and its attendant losses (e.g. 90-95%) and possible degradation of the fiber tips over time.2) Note that you should only butt-couple similar fiber terminations, either FC/PC with FC/PC or FC/APC with FC/APC so take care to order the same termination on the Leoni switch as your laser launch has. You could also find a vendor to do fiber splicing to the switch. Another option is to to free-space switching using a galvo or similar on an optical table. There may be other switching options available, if you know of one please add it to this wiki! Active switching usually involves some loss but generally speaking you can keep more than 50% of the input intensity.

Laser Launches Used with diSPIM

Below are notes about commercial laser launches that have been used with diSPIM.

Toptica MLE

Implementation notes

  • dual-output version (integrated switch) available
  • choice of multiple laser lines installed at factory
  • automatic alignment feature makes setup easy even after shipping
  • compact form factor
  • fibers have NA ~0.06
  • 50 ohm SMB inputs for each laser on/off, version with switch has high impedance BNC input for fiber select built into interlock cable

Micro-manager notes

  • Use Toptica MLE device adapter. Fairly full-featured and supported by Toptica.

Vortran VersaLase

Implementation notes

  • choice of multiple laser lines installed at factory
  • compact form factor
  • 50 ohm SMB inputs for each laser on/off
  • integrated switch/split not yet available but being developed

Micro-manager notes

  • Use Vortran VersaLase device adapter. Clunky but undergoing improvement as of May 2016 and supported by Vortran.

Spectral Applied ILE

(no info seems to be available online but similar to LLM5 with dual output option)

Implementation notes

  • has dual output option
  • form factor similar to large desktop computer
  • choice of multiple laser lines installed at factory
  • fibers have NA ~0.12
  • high-impedance inputs for each laser on/off and side select on DB-15 connector (ASI can provide breakout cable to BNC)

Micro-Manager notes

  • use the LLM5 device adapter (not maintained by manufacturer)
  • doesn't support the intensity scale factor but otherwise should be fine
  • switcher shows up as a fake wavelength in property browser

Agilent

Implementation notes

  • dual-output version available
  • quite bulky form factor compared to other launches
  • high impedance inputs for each laser on/off and side select
  • not possible to have intensity selected by software while on/off triggering done by hardware ⇒ added complexity in driving analog inputs

Usage notes

  • Must take care to get master blanking signal correct
  • Because of all-or-nothing external control, must have means to provide analog intensity control (not needed with any other launch)

Micro-manager notes

  • device adapter present??

Coherent OBIS/Galaxy

Implementation notes

  • separate OBIS lasers and Galaxy combiner allow in-field addition or substitution of lasers
  • possibly available with Leoni switch already fiber-spliced

Micro-manager notes

  • Use OBIS device adapter (not maintained by manufacturer)
  • As of Nov 2015, device adapter only works with single-line OBIS; with the multi-line box only the laser in the first position can be controlled so get a single “remote” controller for each laser line

Intelligent Imaging LaserStack

Implementation notes

  • multi-fiber output module available (“FiberSwitcher”)
  • generally sold by 3i intended to be used with their Slidebook software
  • has high impedance inputs for each laser on/off and side select
  • has analog intensity inputs

Micro-manager notes

  • Probably would work if you had way to control intensity, e.g. by DAC card.
1)
this was done on the original diSPIM, see Supplementary “Note 1 SF2, Excitation laser launch for diSPIM” in Supplementary Notes to the Nature Protocols paper
2)
supposedly most problematic with ongoing use of UV wavelengths e.g. 405 nm which can interact with volatile compounds and deposit them on the surface