Jefferis Lab DokuWiki equipment:multiphoton

equipment:multiphoton:2p1_log

Anonymous (anonymous@undisclosed.example.com) — 2021-11-09T16:14:13+00:00

2021-08-18 - Chameleon laser: error 59 Precompensator Homing Fault * this error appeared a while back intermittently, Blair Welsh from Coherent came for a service visit and found that a circuit board was attached incorrectly in the laser, fixing the position of this daughter board took care of the error

equipment:multiphoton:2p2_log

Anonymous (anonymous@undisclosed.example.com) — 2020-02-05T17:49:16+00:00

Install March 2012 Alex (as Sitka Scientific) has been hard at work on the hardware install including splitting the lasers to feed 2P1 and 2P2. He will put a summary/ a few pictures of the new arrangement on a separate hardware page PC/Software Install

equipment:multiphoton:2p2_todo

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

* Cage Light * Decide on objective * Decide on piezo system * Adapter for piezo from workshop * Test out Pockels with real specimens for * blanking * Z series * Calibration curve in scanimage * Buy specimen holder (Sutter from UK distrib)

equipment:multiphoton:2p_dissections

Anonymous (anonymous@undisclosed.example.com) — 2023-05-24T14:49:42+00:00

Prepare the imaging buffer. The pre-made saline will be labeled for 2P Ca+2 imaging. Put a small amount (usually <5mL) in a fresh falcon tube. Bubble in CO2 by using the appropriate cap (labeled Ca+2) and turning the nozzle to let the air through. Close when finished (30 sec - 1 min).

equipment:multiphoton:2p_risk_assessment

Anonymous (anonymous@undisclosed.example.com) — 2019-08-01T11:27:34+00:00

Full risk assessment and SOPs completed 1 August 2019 by Erika Dona, Nick Barry and Greg Jefferis. See 2P Safety for links. Safety Considerations The 2P system is custom-built but is designed to be treated as a closed-box system for laser safety purposes equivalent to a regular commercial confocal.

equipment:multiphoton:basic_use_of_2p_system

Anonymous (anonymous@undisclosed.example.com) — 2020-02-05T17:27:51+00:00

Safety Considerations The 2P system is custom-built but is designed to be treated as a closed-box system for laser safety purposes equivalent to a regular commercial confocal. * Do not attempt to use the system without basic training (from Erika, Istvan, or Greg)

equipment:multiphoton:change_coolant_for_laser

Anonymous (anonymous@undisclosed.example.com) — 2019-01-09T12:18:33+00:00

* Turn Chameleon to Standby. * Switch off chiller and disconnect power line. * Disconnect the coolant lines, ensure that they are labelled so that they are re-connected to the same positions. * Dispose of the spent coolant from the chiller tank into the appropriate waste disposal bottle (stored next to the new coolant). IMPORTANT: it contains Ethylen Glycol, therefore it is harmful for the environment.

equipment:multiphoton:chrimson_led

Anonymous (anonymous@undisclosed.example.com) — 2014-06-12T14:39:49+00:00

Hardware setup A Philips LUXEON Rebel LXM2-PH01-0070 is glued to a microscope slide which in turn is mounted below the dish holder beneath the objective. Two crocodile clips are used to connect the legs to a BNC cable, which is fed through to the Arduino and connected via a further two crocodile clips. Standard colour conventions have been adhered to, such that the red clip connects to the LED's anode.

equipment:multiphoton:config_files

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

Git Repo A git repo with the ScanImage config files used on the SuperFlyGuy user can be cloned from git clone git@flybrain.mrc-lmb.cam.ac.uk:twoppc/ScanImage Location on twoppc \c\Users\superFlyGuy\Documents\MATLAB\ScanImage Or something like that path, Windows is confusing.

equipment:multiphoton:hardware_details_after_move_to_3n390

Anonymous (anonymous@undisclosed.example.com) — 2021-08-19T08:25:10+00:00

Detailed description of 2P1 * This page documents the current state of 2P1, since Alex's 2-photon page became obsolete. To follow the step-by-step history of 2P1 see 2P1 log * Before planning any changes it's worth visiting the most recent version of supported parts list on Vidrio's website

equipment:multiphoton:imaging_2p

Anonymous (anonymous@undisclosed.example.com) — 2021-03-02T12:00:08+00:00

Using the 2-photon microscope * Before use read basic_use_of_2p_system paying particular attention to the safety instructions. * The main shutter of the laser must always be closed (either by the automatic laser interlock but the door of the hood or manually during e.g. alignment procedures)

equipment:multiphoton:mp285

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

The Sutter MP285 consists of a customised 3 axis motor system (2 for xy, 1 for objective focus) and the corresponding controller. Our controller is: Version: WD3.4 Wed Mar 14 11:22:10 2001 Input Device data: 11111111 This sounds old, but seems to be what ScanImage expects

equipment:multiphoton:ni_board_connections

Anonymous (anonymous@undisclosed.example.com) — 2020-02-05T17:46:50+00:00

The information here is mostly obsolete. Easiest way to check this is opening the MachineDataFile in a text editor. Dev1 is the auxiliary board, PCIe-6323 controlling the Piezo and the Pockels cell, receiving external trigger. Dev2 is the imaging board, PCI-6110 controlling the scanners and receiving PMT output.

equipment:multiphoton:objective_mount

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

We currently (June 2011) have a bayonet style quick exchange objective mount. This works well up to M27 but is not suitable for mounting M32 or piezo objective positioners directly. Our current solutions for those situations result in a Z displacement of the back aperture of the objective about 40 mm. This is bad for 2 reasons:

equipment:multiphoton:piezo_calibrations

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

This is the offset/delay from start (a) and the slope of the interval between sweeps (B) of the Y mirror on the multi-photon system. In short * for AaronCFG (ie 256×128, 0.5 MS/Line, any zoom) the Y mirror sweeps with a period of 94.722ms * If you either change the amount of time it takes to sweep a line or the number of lines/frame the sweeps increase in length in a linear manner

equipment:multiphoton:piezo_objective

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

* Piezo Calibrations * Our current objective is a Zeiss 20x 1.0 NA weighing 285g. * to calculate the resonant frequency, I think one should just be able to use a simple harmonic oscillator approximation * resf=function(k,m) 1/(2*pi)*sqrt(k/m) where k is stiffness in N/m and m is mass in kg.

equipment:multiphoton:problems

Anonymous (anonymous@undisclosed.example.com) — 2019-09-16T14:30:08+00:00

So far we have had problems with the laser * on installation (pump laser not producing full power, replacement installed about 1-2 months after arrival) * slow tuning speed - recommendation was laser exercises switching to range of different wavelengths every month

equipment:multiphoton:recenter_mp285_axes

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

See MP285 Manual “Center axes” is a self-running routine that performs a task that you may need from time to time. First, the routine instructs the manipulator to move to the end of travel on each axis and records the coordinate of these points. Based on this information, the controller then calculates the midpoint of each axis and moves each axis to that location. Finally, with the manipulator still in central position, the controller does a reset of the absolute zero and thus gives the center…

equipment:multiphoton:remove_detector_head

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

I wanted to remove the detector head in order to examine how the objective mount is fixed in place * Turn off the Sutter MP285, PMTs and wide-field/2P servo motor control * Remove the objective! * Undo the connector for the electrical connection to the servo motor that switches the Dichroic from 2P to WF mode.

equipment:multiphoton:retune_scan_mirror_controllers

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

We found out all about this in error after a software configuration issue. Leon has manuals for the 671 control boards that are found (one per mirror) in the Sutter MDR-6 control box. If you hear a loud resonance noise from scanners (often higher pitch than expected) turn off control unit, check software config and if all else fails consider tuning. Leon and Jamie know about this. Leon says that he has had to retune a couple of times for no particularly good reason and the mirrors have gone on …

equipment:multiphoton:sample_view_movement

Anonymous (anonymous@undisclosed.example.com) — 2014-07-29T19:58:52+00:00

Navigation Considering the co-ordinates of the image to have an origin in the top left, movements map as: Image direction Direction on MP285 Label on hand controller -y -x North -x

equipment:multiphoton:scanimage

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

ScanImage 3.7 Some notes about our tests of the 3.7 series which foundered on a problem talking to the Sutter MP285 controller. From: Vijay Iyer Subject: RE: ScanImage 3.7.1 beta Date: 6 September 2011 12:38:47 GMT+01:00 To: Dr Gregory Jefferis Hi Greg, That is very disappointing to hear. There was a bug prior to 3558 that was leading to unreasonably frequent motor communication failures...that's been fixed in r3558. At the moment , …

equipment:multiphoton:set_mp285_home

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

(from MP-285 MICROMANIPULATOR SYSTEM REFERENCE MANUAL – REV. 3.13 (20080229)) To set a New Home position * While in the movement screen, move to the location you wish to establish as HOME * Press <*> to enter the robotics menu * to “New Home” and press . You will see the following screen:

equipment:multiphoton:spatial_calibration

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

2P calibration method from June 29, 2011 Loaded Alexa-568 solution (1uL of 5mM stock in 100uL of water) onto a haemocytometer and imaged using 810nm IR. Images were taken at zoom levels 1X, 2X, 3X, 5X, and 10X, each at 1024×1024, 512×512, and 256×256

equipment:multiphoton:start

Anonymous (anonymous@undisclosed.example.com) — 2024-04-18T05:45:44+00:00

Hardware We currently have one multiphoton system based around a Sutter MOM core assembled and accessorised by Alex. This has been modified, moved, and upgraded by Bruno Pichler's company (INSS) and Istvan. Software control is by ScanImage running under Matlab on a PC, communicating via NI DAQ board. See:

equipment:multiphoton:tune_piezo

Anonymous (anonymous@undisclosed.example.com) — 2013-12-10T11:59:45+00:00

The servo controller module inside the E665 control unit can be tuned to move the PI-726 piezo objective holder as fast as possible. 6ms should be achievable for 10µm steps with the Zeiss 20x objective. * Unplug * Take the lid off (6 screws side and rear)