OpenScan two-photon microscope#

Here we describe a concrete build for a scanning two-photon excitation fluorescence microscope.

Introduction#

TODO: Choices made: use a commercial microscopy body (ergonomic for biologist users, and simplifies integration with other imaging modalities). The limitation is that you don’t have full control over the optics (tube lenses).

Overall layout#

The overall layout of a simple two-photon microscope for fluorescence lifetime imaging is as follows:

flowchart LR %% subgraph Illumination ["Illumination beam line"] Laser["Ti:Sapphire Laser"] --> Isolator; Isolator["Isolator"] --> EOM; EOM("Electro-optic modulator") --> Shutter; Shutter --> Dump[Beam dump]; EOM --> EOMDump[Beam dump]; Shutter --> HWP; HWP("Half wave plate") --> QWP; QWP("Quarter wave plate") --> Mirror1("Mirror M1"); Mirror1_break("Mirror M1") --> Mirror2; Mirror2("Mirror M2") --> Expander; Expander("Beam Expander") --> Tap; Tap["Tap Beam Splitter"] --> Mirror3("Mirror M3"); Mirror3 --> Periscope(("Periscope (M4, M5)")); Periscope_break(("Periscope (M4, M5)")) -->Galvos; Galvos("Galvo mirrors") -->Scan; %% end Scan("Scan Lens") --> EpiTube; %% subgraph MB ["Microscope base"]; EpiTube("Tube Lens") --> EpiDM; EpiDM["Dichroic mirror"] <--> Objective; Objective("Obj") <--> Sample; EpiDM -->PMTTube; %% end PMTTube("Tube Lens") -->Filter; %% subgraph PMTA ["Camera line"] Filter("Filter"); Filter -->PMT; PMT("PMT")-.-Datalines-.-BH("SPC boards"); %% end %% Subgraphs commented because including them causes the diagram to run bottom to top. %% Links to main body sections click Laser href "#ultrafast-pulsed-laser" _blank click Isolator href "#optical-isolator" _blank click EOM href "#intensity-control-electro-optic-modulator-eom" _blank click Shutter href "#shutter-alignment" _blank click HWP href "#polarization-control-wave-plates" _blank click QWP href "#polarization-control-wave-plates" _blank click Mirror1 href "#mirrors-m1-m2" _blank click Mirror2 href "#mirrors-m1-m2" _blank click Expander href "#beam-expander" _blank click Periscope_break href "#mirror-m3-periscope-m4-m5" _blank click Periscope href "#mirror-m3-periscope-m4-m5" _blank click Galvos href "#Galvanometer-scanners" _blank click Scan href "#scan-lens" _blank click EpiTube href "#microscope-body" _blank click PMTTube "#photomultiplier-tubes" _blank click PMT href "#photomultiplier-tubes" _blank click BH href "#electronics" _blank

Optical axes#

Optical axis 1: laser control and modulation#

Parallel to table at ~110 mm height. Established by Ti:Sapphire laser emission.

Optical axis 2: beam expander#

Parallel to table at ~110 mm height. Determined by beam expander placement. Aligned using mirrors M1 and M2.

Optical axis 3: galvanometer scanner input#

Parallel to table. Determined by position of galvo scanners, which in turn depends on optical axis 4 and its components. Aligned using mirrors M3 and M5.

Optical axis 4: microscope epi illumination path#

Parallel to table. Established by microscope body placement.

Overall alignment procedure#

Fix the components that establish optical axes and do not depend on alignment:
- Ti:Sapphire laser (optical axis 1)
- Beam expander (optical axis 2)
- Microscope body (optical axis 4)
Align components on optical axes 1 and 4.
Co-align optical axes 1 and 2 using mirrors M1 and M2.
Align galvanometer scanners to optical axis 4, establishing optical axis 3.
Co-align optical axes 2 and 3 using mirrors M3 and M5.

Photonic/optic components#

Optical table#

4x8-foot, 8-inch thick, with vibration isolation (e.g., TMC). 1-inch-spaced mounting holes tapped for 1/4-20.

TODO: More details; 8-inch hole if using bottom port.

The optical table will very slightly deform when heavy items are placed on it, or when it is floated vs not floated. Alignment should therefore be done after fixing heavy components and floating the table.

Ultrafast pulsed laser#

Component	Manufacturer	Part No
Mode-locked Ti:Sapphire laser	Spectra-Physics	Mai Tai HP

BOM Notes: Mode-locked Ti:Sapphire lasers broadly come in two categories: turn-key systems with automatic control (Spectra-Physics Mai Tai or Coherent Chameleon), and manually tunable optical oscillators (Spectra-Physics Tsunami or Coherent Mira). The necessary pump laser is usually purchased as part of the package.

Laser alignment#

Dependencies: None.

Outgoing beam: Collimated laser beam with linear polarization at 0° (horizontal).

We use the laser emission, as is, to define optical axis 1. It should be parallel to the table at a height of about 110 mm. It is convenient to have the laser be aligned to a row of mounting holes on the table, but this is not essential.

Optical isolator#

Component	Manufacturer	Part No
Faraday isolator	Electro-Optics Technology	BB8-5i
Isolator mount		TODO

BOM Notes: EOTech is now part of Coherent, but this optical isolator is obsolete. It is an isolator designed for Ti:Sapphire lasers. The closest equivalent current model is a Coherent EURYS Broadband Faraday Isolator with a 5 mm aperture.

Isolator alignment#

Dependencies: Optical axis 1.

Incoming beam: Collimated laser beam with linear polarization at 0° (horizontal).

Outgoing beam: Collimated laser beam with linear polarization at 90° (vertical).

Align the device to the pre-existing optical axis 1. See Aligning an EOTech broadband Faraday isolator.

Intensity control: electro-optic modulator (EOM)#

The EOM (consisting of a Pockels cell together with a polarizing beam splitter) provides electronic control over excitation laser power.

Component	Manufacturer	Part No	Name
EOM	Conoptics	350-80	KD*P Modulator
EOM Mount	Conoptics	102
Alignment tool	Conoptics	103
EOM driver	Conoptics	302RM
Beam dump	Thorlabs	LB1	Beam Block, 400 nm - 2 µm, 10 W Max Avg. Power, Pulsed and CW, Includes TR3 Post
Beam dump mount			TODO

BOM Notes: The EOM should be configured for amplitude modulation, with minimum transmission near zero bias voltage.

EOM alignment#

Dependencies: Optical axis 1. Usually the upstream optical isolator should be aligned first.

Incoming beam: Collimated laser beam with linear polarization at 90° (vertical).

Outgoing beam: Collimated laser beam with linear polarization at 0° (horizontal).

Align the device to the pre-existing optical axis 1. See Aligning a Conoptics EOM.

EOM design notes#

For the EOM, Conoptics 350-80LA, which has a larger, 3.5 mm aperture, may be desirable. The 350-80 used here has a 2.7 mm aperture.
The Conoptics EOMs can now be purchased with an integrated beam block.
The EOM can be purchased with input polarizing beam splitter, in case the input beam does not have clean linear polarization. This is not necessary when the immediately previous component is the Faraday isolator (whose last component is a polarizer).
The EOM must have sufficient aperture size for the beam diameter. See manufacturer manual for details.
EOM drivers are available optimized for different applications, e.g., fast switching vs high extinction ratio. For routine power control in TPM, we do not need fast switching. The 302RM is fast enough for laser blanking during retrace (not yet implemented in OpenScan).
The choice of an EOM over an AOM is advantageous for Ti:Sapph lasers which have a wide bandwidth (wavelength distribution). However, power control could also be achieved at significantly lower cost using the combination of a motorized half-wave plate and a polarizing beam splitter, albeit with slower switching.
The EOM should be located before the main shutter in the beam path, so that it does not experience temparature shifts when the shutter is opened or closed.

Shutter#

Component	Manufacturer	Part No
Shutter	Vincent (Uniblitz)	TBD (VS14?)
Shutter mount		TBD
Shutter driver	Vincent (Uniblitz)	TBD (VCM-D1?)
Beam dump	Thorlabs	LB1
Beam dump mount		TBD

Shutter alignment#

Dependencies: Optical axis 1.

Place such that beam passes through near the center of the aperture when the shutter is open. Fix at ~5° from perpendicular to the optical axis, such that the reflected beam (when the shutter is closed) is offset from the incoming beam. Block the reflected beam with the beam dump.

Polarization control: wave plates#

A half-wave plate (HWP) followed by a quarter-wave plate (QWP) allows the excitation beam to be precisely adjusted to have circular polarization at the specimen.

How does this work?

The HWP allows the linear polarization of the incoming beam to be rotated to any chosen angle. The QWP, when aligned, converts the linearly polarized beam to a circularly polarized beam. It may therefore seem that the HWP is unnecessary.

In practice, even if the QWP produces perfectly circularly polarized light, anisotropy in the optics between the QWP and the specimen will cause the beam to end up elliptically polarized. We can compensate for this effect by adjusting the QWP such that it emits a beam that is elliptically polarized in the orthogonal direction, so that it will end up circularly polarized at the specimen. However, in order to adjust the QWP in such a way, we need to be able to supply it with a linearly polarized beam at an angle of our choosing, which is why we need the HWP.

The wave plates can also be adjusted to obtain linearly polarized light for applications such as fluorescence anisotropy measurements (the QWP is not necessary for this).

Component	Manufacturer	Part No	Name
Half-wave plate	Thorlabs	AHWP05M-980	Ø1/2” Mounted Achromatic Half-Wave Plate, Ø1” Mount, 690 - 1200 nm
HWP mount	Thorlabs	CRM1P	Precision Cage Rotation Mount with Micrometer Drive, Ø1” Optics, 8-32 Tap
Quarter-wave plate	Thorlabs	AQWP05M-980	Ø1/2” Mounted Achromatic Quarter-Wave Plate, Ø1” Mount, 690 - 1200 nm
QWP mount	Thorlabs	PRM1	High-Precision Rotation Mount for Ø1” (25.4 mm) Optics
Posts and clamps		TBD

BOM Notes: Thorlabs CRM1P is obsolete and superseded by CRM1PT “Precision Cage Rotation Mount with Micrometer Drive, Ø1” Optics, 8-32 Tap”.

Wave plate alignment#

Dependencies: Optical axis 1. (Final polarization adjustment requires the full system to be built so that specimens can be imaged.)

Place the two wave plates perpendicular to optical axis 1, centered around the beam.

TODO: Back reflection must overlap with the beam (link to alignment section)

TODO: Obtaining circularly polarized light at the QWP (use PBS, power meter, and mirror; link to alignment section)

TODO: Polarization adjustment procedure using giant unilamellar vesicles.

Mirrors M1, M2#

These mirrors affort beam steering to establish optical axis 2.

M1-M2 alignment#

Dependencies: Optical axis 1 and optical axis 2. These mirrors co-align the two optical axes.

See TODO (beam steering with two kinematic mirrors) and TODO (aligning a laser beam into a beam expander).

Beam expander#

Component	Manufacturer	Part No	Name
Beam expander	Special Optics	56-30-2-8X-1064	Variable Zoom Beam Expander
Beam expander mount		TBD

Beam expander alignment#

Dependencies: None. Optical axis 2 will be determined by the beam expander.

TODO: Magnification adjustment to fill objective back aperture.

Mirror M3; periscope M4-M5#

M3-M5 design notes#

The Thorlabs periscope assembly has fine tilt adjustment screws only on the upper mirror, so we use M3 and M5 to steer the laser beam onto optical axis 3. Some periscopes (e.g. Newport) have fine tilt adjustment also on the lower mirror, in which case M3 is not necessary (adjustment can be done with the periscope mirrors M4 and M5).

M3-M5 alignment#

Dependencies: Optical axis 2 and optical axis 3-4.

See TODO (beam steering with two kinematic mirrors) and TODO.

Galvanometer scanners#

TODO BOM. Mount should have XYZ translation (no need for micrometers)

Galvo alignment#

Dependencies: Optical axis 4 and all its components.

TODO.

Scan lens#

TBD.

Scan lens design notes#

The scan lens should focus a collimated beam at the epi field diaphragm (slider slot) position in the LAPP main branch. This requires a scan lens with a focal length of at least ~120 mm (exact requirement for mechanical clearance may be slightly larger).

Scan lens alignment#

Dependencies: Optical axis 4.

See TODO (aligning a scan lens using transmitted illumination).

Microscope body#

Component	Manufacturer	Part No
Inverted microscope body	Nikon Instruments	Eclipse Ti2-E/B

TODO: LAPP details, fixing plate. Filter cube.

BOM Notes: The Ti2 can be configured with a large array of options. We use the LAPP Main Branch in the epi illumination port; this includes the tube lens for the excitation light. Details TBD.

Microscopy body alignment#

Dependencies: None. Placement determines optical axis 4.

It is conveninet to fix the microscope so that its front-back center axis is aligned to a row of mounting holes on the optical table.

Emission beam splitter#