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crest Optics X-Light V2 TP Installation And User Manual

Confocal imager with vcs super-resolution module
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CREST OPTICS
X-Light
V2
TP
™
Confocal Imager
VCS
Super-resolution module
Installation and
User's Guide
V1.9
1

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Summary of Contents for crest Optics X-Light V2 TP

  • Page 1 CREST OPTICS X-Light ™ Confocal Imager Super-resolution module Installation and User’s Guide V1.9...
  • Page 2 CREST OPTICS SRL via Mattia Battistini, 184D 00167 Roma www.crestopt.com Tel. +39 066147496 FAX +39 0661662738 E-mail: info@crestopt.com...

  • Page 3: Table Of Contents

    –Installation and user guide CREST OPTICS X-Light V2 X-Light V2 hardware 1.1 X-Light V2 Description 1.2 X-Light V2 Specifications 1.3 X-Light V2 Light path 2.0 X-Light V2 Installation 2.1 X-Light V2 Components 2.2 Filter Wheels Assembly 2.2.1 Filter descriptions for most common applications (filters are not included) 2.2.2...

  • Page 4: Crest Optics X-Light V2

    1.0 X-Light V2 hardware 1.1 X-Light V2 description CREST X-Light V2 is a high throughput full spectrum spinning disk confocal imager with super-resolution add-on module capability. It can be attached to all major inverted epi-fluorescence microscopes allowing full spectrum (360nm-700 nm) confocal imaging. Detailed Specification: •...
  • Page 5 CREST X-Light V2 can be used with most high end EMCCDs, interline or frame transfer CCD cameras, sCMOS cameras together with imaging software including Metamorph, Nis Element and MicroManager. Fig. 1.1 CREST X-Light V2 Fig 1.2 CREST X-Light V2 complete system.
  • Page 6 1.2 CREST X-Light V2 Confocal Imager Specifications Confocal scanner Nipkow spinning Disk (pinholes) with single and double hole pattern Disk scan rate > 2000 scans per second Pinhole diameter 60 micron single pattern standard, 40-70 micron double pattern standard, custom pattern on request 360 nm –...

  • Page 7: X-Light V2

    1.3 CREST X-LIGHT V2 Light Path X-LIGHT V2 Confocal Imager Light path Excitation Filter Wheel: The X-Light V2 has been engineered to work with LEDs and Lasers mainly. Dichroic Filter Wheel: The quick release dichroic wheel has 5, positions and accepts commercially available up to 25.5x36 mm dichroic mirrors.

  • Page 8: Installation

    2.0 CREST X-LIGHT V2 Installation 2.1 CREST X-LIGHT V2 Components Confocal Head Automated dichroic wheel filter holders (5) Camera C-Mount X-Light power supply Power cord USB cable Dichroic Filter extraction tool for automated 5 positions dichroic wheel...
  • Page 9 Emission Filter extraction tool for automated 8 positions filter wheel Bubble level C-mount side-port adapter ring Threaded ring (2)
  • Page 10 2.2 Filter wheels Assembly 2.2.1 Filter descriptions for most common applications (filters are not included) ® Chroma technology DAPI Exciter Dichroic Emission...
  • Page 11 ® EGFP Chroma Technology Exciter Dichroic Emission...
  • Page 12 ® TRITC/Cy3 Chroma Technology Exciter Dichroic Emission...
  • Page 13 2.2.2 Changing emission filters and dichroic mirrors Changing emission filters: In order to change or add new emission filter sets follow the procedure below. Please refer to section 2.3.2 for both dichroic wheel and emission wheel positioning for filter exchange. Figure: Emission Extraction tool Insert the filter:...
  • Page 14 2. Push the emission tool and keeping it pushed place the emission filter in it as shown in figure below (every manufacturer has its proper way to place the filter in the optical path so follow manufacturer instructions in order to mount them correctly.) 3.
  • Page 15 4. Insert the tool all the way down in the slot following the slot entry shape 5. Now start extracting: press the tool and move it in the direction of the red arrow below...
  • Page 16 6. Finally pull out the tool Extract the filter (inverse insertion procedure): 1. Push the top of the extraction tool. 2. Insert the extraction tool in the filter slot of emission wheel. Make sure the extraction tool is completely inserted against the wall indicated by the red arrow (see picture above) 3.
  • Page 17 Changing dichroic filters: Extra precaution is necessary when mounting or removing dichroic filters. Lay the dichroic filter holder flat on a flat surface so that the mounting screws are facing upwards (see figure) Using a Phillips screw driver unscrew the filter holding plate . Now gently remove the holding plate. It is important how the dichroic mirror is mounted on the holder.
  • Page 18 Figure: Dichroic Extraction tool Insert the filter: 1. Locate the dichroic slot circled in the figure below...
  • Page 19 2. Push on the top of the dichroic tool and keeping it pushed place the dichroic mirror in it as shown in figure below (pay attention to mount it with the coated surface toward the excitation light). 3. Release the tool in order to grab the filter in steady position. The two metal hooks (green circle above) must grab the two corresponding hooks on dichroic holder (red circle above)
  • Page 20 4. Insert the tool all the way down in the slot following the slot entry shape 5. Finally, push on the top of the tool again and extract it. The dichroic is correctly positioned into the wheel. In order to extract a filter follow the inverse procedure: Insert the tool with the button on top pressed, once in position all the way down release the button and extract the filter...

  • Page 21: Power Supply For X-Light V2

    2.3 Power supply for X-Light V2 and connectors Check that the plug-socket 110/220Vac has got the earth plate. Attach the connector of power supply to the X-Light main power connector. Attach one end of the USB communication cable to the X-LIGHT unit and the other end to a USB port of the computer.

  • Page 22: Laser Safety Interlock

    2.3.3 Laser safety interlock As the top panel is removed, laser source power is cut off if connected to the X-light V2 laser safety interlock...

  • Page 23: Attaching X-Light V2

    2.4 Attaching X-LIGHT V2 to the microscope You will need the specific microscope brand side-port C-mount (1x) adapter in order to attach the X-LIGHT . (see figure; Nikon adapter example). Mount the side port-adapter on the C-Mount side-port ring Place the adapter in its housing (disk box side) and the screw the threaded ring to block it in position Mount the Confocal head to the side port (camera port) of the microscope.
  • Page 24 position is the same as it was when placed on the stage of the microscope. Now tighten microscope side port holding screws firmly.

  • Page 25: Spinning Disk Box Plug In/Plug Out

    2.5 Spinning disk box Plug IN/Plug OUT The following procedure will allow to easily switch between different spinning disk boxes you may have. Be sure that the system is disconnected from the power. Unscrew the four screws on the top of the spinning box: two for each side and remove the cover Disconnect the flat cable and unscrew the two hex screws that keep the disk blocked in the housing...

  • Page 26: Attaching The Camera

    You can finally lift the spinning disk box 2.6 Attaching the camera A range of CCD/sCMOS cameras can be used with the X-LIGHT V2 . Mount the CCD/sCMOS to the C- mount confocal head adapter. You will need to mount the c-mount and the ring at the same time (see pictures).
  • Page 27 2.7 X-LIGHT alignment Focusing the pinholes The spinning disk is in a plane conjugate to the image plane. As it spins, it scans the microscopic fluorescent image point by point in real-time. It is essential that the CCD/sCMOS cameras be precisely focused on the image plane (the pinholes) of the spinning disk in order to obtain a sharp image.
  • Page 28 Pinholes not in focus Pinholes in focus When mounting the confocal head be sure it is levelled on the same microscope plane...
  • Page 29 WHENEVER YOU ARE USING UV ILLUMINATION OR LASER SOURCE IS ABSOLUTELY NEEDED TO PROTECT YOUR EYES BEFORE TO DO THIS OPERATION. PLEASE WEAR PROTECTIVE GOOGLES. Adjusting the excitation light path The X-Light excitation adapter is pre-aligned by factory. Nevertheless some further fine adjustments may be required in order to optimize illumination efficiency.
  • Page 30 ALIGNMENT PROCEDURE: Excitation angle 1. Connect your excitation source, to the excitation SMA adapter...
  • Page 31 2. Unloose the three hexagonal screws circled in red (see picture)
  • Page 32 3. Adjust the three rods circled in yellow (see picture) in sequence until you can center the illumination spot on the spinning disk box window...
  • Page 33 4. Tighten hexagonal screws in sequence to fix the gimbal mount excitation position. Make sure your light path stays aligned during this step.
  • Page 34 XY adjustment Additionally, verify the beam spot alignment at the entrance of the objective through the XY Gimbal adjustment. 1. You have to adjust one axis at a time. You have 4 screws for X axis (circled in red) and 4 screws for the Y axis (circled in yellow).
  • Page 35 2. Adjust Gimbal overall position <XY> until you center the spot at the entrance of the objective Tighten XY adjustment screws to fix the gimbal mount excitation position. N.B. An iteration of both angle adjustment and XY adjustment could be needed to optimize alignment...

  • Page 36: Maintenance

    2.8 Maintenance The system doesn’t need maintenance. In case of malfunctioning the system shall be shipped to the supplier to be checked by our authorized engineers. Not being permitted the opening of the system and the access to its internal components, the system hasn’t got risk for the user.

  • Page 37: X-Light V2

    3.0 X-Light V2 options 3.1 VCS – Video Confocal Super-resolution Fig 1.2 CREST X-Light V2 with VCS complete system. 3.1.1 Crest VCS description Crest VCS is an add-on module for X-Light V2 resolution enhancement. Specifications VCS scanner Glass mask with double square grid hole pattern ...
  • Page 38 Observation EMCCD cameras or sCMOS camera port with fixed position; camera pixel ≤ 6.5um for higher sampling Microscope Compatibility Inverted fluorescence microscopes with 100% camera port Control USB serial control Software Metamorph, Nis-Elements Power* 15V Vdc 1.5A, 47-63Hz *If you use external power, specifics are: Vin 110-240 Vac, Vout 15Vdc, 2.5A Key features: •...
  • Page 39 3.1.2 VCS Connectors: To power the unit just insert the connector. The power Led will flash at the start and during the whole hardware initialization procedure Power Led Programming connector Power connector USB connector 15 Vdc 2,5 A...
  • Page 40 3.1.3 Alignment procedure: VCS module has three hexagonal captive screws that allow the connection to the X-light V2. Screws are already mounted inside the module (see picture below) VCS module is also provided with two pins. During the VCS module connection, before screwing, be sure to insert the pins in the corresponding holes on the X-light V2 side (see below).
  • Page 41 Once inserted you can screw...
  • Page 42 VCS module has four major degrees of freedom to accomplish the alignment:  Traslational degree in X and Y with respect to the X-light V2  Angular degree in sagittal and tagential plane with center of rotation coincident with the center of the dichroic mirror The following procedure shows how to perform the alignment: Remove the X-light V2 top and verify to have a clear visibility of the spinning box window...
  • Page 43 Loosen the rear hexagonal screw you see in picture below. A quarter of one complete turn is required.
  • Page 44 Loosen the 2 little grub screws (see picture below circled in red). No more than a complete turn is required. Do not turn more than a complete turn.
  • Page 45 XY adjustment: The hexagonal screws for X e Y adjustament are respectively shown in the two pictures below...
  • Page 46 By the XY adjustament try to center the beam at the objective entrance...
  • Page 47 Angle adjustment The two angles are adjustable by the two screws you see in picture below. Sagittal plane angle Tangential plane angle...
  • Page 48 By angle adjustament try to center the beam on the spinning box window (see figure below)
  • Page 49 If at the end of the procedure the spot at the entrance of the objective has been decentered, iterate the procedure. When the illumination is aligned, tighten back the rear hexagonal screw and the 2 little grub screws...
  • Page 50 Supporting foot for VCS balancing VCS module is equipped with a balancing foot which must be propped against the table by screwing a hexagonal screw inside the unit (see pictures below). The hex key that can be used is the long 5mm hex key shipped in the tool box...
  • Page 51 3.1.4 VCS Mask replacement VCS module will be shipped with a mask that is separate from the unit and with the following specifications Double pattern mask: 13um pinholes 78um pitch (grid) 13um pinholes 78um pitch (slits)
  • Page 52 Replacement procedure Use gloves for the entire procedure Insertion: Put the piezo motors group in the fourth position ( command “D3”, maintanance mode) The glass mask has an arrow on the side. It must be inserted with the arrow pointing toward the light source. Extraction: Put the piezo motors group in the fourth position ( command “D3”, maintanance mode) Gently push the mask upwards on the outer side.
  • Page 53 Pull out the mask 4.0 X-light command sets X-light Driver Manual 1.02B...
  • Page 54 X-light V2 Regardless of the controlling application, the settings for the COM port used to connect to the X-LIGHT are as follows: 9600 baud, 8 data bits, 1 stop bit, No parity, and no flow control. Devices The X-LIGHT has three individual devices that can be actuated to affect the light path. Each device has a single capital letter name (Device ID), which is used to address the individual device with the commands that are described later in this manual.
  • Page 55 Command Formats In the following command descriptions the Format describes how the command should look as an individual command. Parameters describe any characters that must accompany the command or are returned with the echo of the command. The Return Value describes how the echo of the individual command will look.
  • Page 56 DEVICE ID – Move Device (C , D , N) Format <device ID><position><CR> Parameters <device ID> ID assigned to a device (see table on page 1) <position> Desired position or state Return Value <device ID><position><CR> End of action execution: <device ID><position><CR> D1<CR>...
  • Page 57 – Rehome all devices Format H<CR> Parameters NULL Return Value setting: HB1C1D0N0<CR> H<CR>  HB1C1D0N0<CR> Examples – Response Activation: Turn on/off responses to Host Format R<active><CR> Parameters <active> 0 = no response sent to host (no echo) 1 = responses sent to host Return Value If <active>...
  • Page 58 – Query State of All Devices Format q<CR> q <B’N’><C’N’><D’N’><N’N’> <CR> Return Value q<CR>  B1C1D1N1<CR> Examples – Read Current Position of Individual Device Format r<device ID><CR> Parameters <device ID> ID assigned to a device (see table on page 1) <position>...

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