IGC

Technology

Keeping up with researchers needs and improving instrument performance requires technical advancements to take place. The Technology section describes some of the current and past Technical Development Projects of the Imaging Unit involving the Flow Cytometry Core Lab.

The following sections provide information on several current and past technological projects developed at the Imaging Unit, involving the Flow Cytometry Core Facility.

[MoFlo] Fast Drop Delay Determination (FD3) System

Though MoFlo's CYCLONE is a quite reliable drop delay determination system, it can be somewhat time consuming and requires a microscope for bead counting. Based on Becton Dickinson's Accudrop system, and inspired by Andy Riddell's implementation of a similar system in the EMBL core facility's MoFlo and Influx, we implemented a similar solution in our MoFlo. This system allows not only a fast and accurate system to measure the drop delay, but also allows the operator to monitor the quality of the deflected streams during sort (which is extremely difficult in a standard MoFlo when sorting small numbers of particles per second).

  • Material:

    1. 0.1 lux camera with superhad sony CCD.
    2. Custom acrylic support for camera.
    3. 95/5 beam splitter.
    4. Jack connector.
    5. TV Monitor.
    6. Mirror with 3 degrees of freedom.
    7. Focusing lens.
    8. FlowCheck or other high fluorescent beads.
    9. HQ575/150 emission filter for the camera (if using FlowCheck beads).

    Procedures:

    Check MoFlo's Operating Procedures. Videos coming soon.

  • Fast Drop Delay in MoFlo

[MoFlo] 561nm DPSS coupled with fiber optics

In general, red fluorescent proteins (mCherry, RFP, DsRed, etc) are optimally excited by a yellow laser line. To be able to analyze and sort red fluorescent proteins, we installed a 50mW DPSS 561nm laser from CrystaLaser on the MoFlo. Given that there were already 3 lasers installed in the MoFlo we coulpled the yellow laser to fiber optics so that it can be easily switched around from one lens holder to another, replacing any of the 3 existing lasers. To increase alignment stability, we built an acrylic base that secures both the DPSS laser and the kineFLEX fiber optic support. The other end of the fiber optic, can then be placed in any of the three focusing lens holders. Alignment takes <5 min altogether, and

  • Material:

    1. DPSS laser.
    2. kineFLEX from Point Source.
    3. Custom-made acrylic support.
    4. Fiber optics cable.

    Procedures:

    Check MoFlo's Operating Procedures. Videos coming soon.

  • Fiber Optic Couple Laser in MoFlo

[MoFlo] Interchangeable fluidic assemblies

  • Given the large diversity of research areas within the IGC there is a constant need to sort cells ranging from bacteria to mammalian cells. To provide a solution for such a challenge we put together two different fluidic assemblies, that can be setup in the machine in less than 15 min.

    For more information please check "For Flow Operators - Moflo" link.

  • Fluidics Assembly for MoFlo

[CyAn ADP/HyperCyt] Connection between HyperCyt autosampler and CyAn ADP

The solutions provided either by IntelliCyt or Beckman Coulter to connect the HyperCyt to the CyAn ADP involve increasing the tubing length (which is already rather large in the CyAn ADP) and/or disconnecting and reconnecting tubing to switch from the HyperCyt autosampler to 5mL tube sampling mode (or vice-versa). We've overcome these issues by installing an electric three-way pinch-valve just below the flow cell, which is either pinching the CyAn's sample line or the HyperCyt's sample line. This setup maintains almost exactly the same sample line lengths, and the user only needs to press a button to commute from 5mL tube sampling to the HyperCyt autosampler. In case of clogging, we've assured that the CyAn casing and the pinch-valve can easily and rapidly removed.

  • Material:

    1. 3-way pinch-valve from Bio-Chem Valve(P/N 0075P3MP12-10S)
    2. Silicone tubing (0.1inch I.D.) from Bio-Chem Valve (P/N 10025-10S).
      Must be this exact tubing to assure proper pinching.
    3. 3-way union to connect both sample line tubings to the tubing that goes to the flow cell.
    4. 12-Volt power transformer and respective plug.
    5. Electric switch to commute the pinch-valve.
    6. Hook/Holder to be screwed and support the pinch-valve.
  • 3-Way Pinch-Valve CyAn Commuter Button

Detailed Information on how to setup the HyperCyt-CyAn ADP connection is available upon request.

Quality Control .fcs File Analyzer (open source)

  • We have developed a software to extract detectors voltage information from regularly run QC .fcs files.

    This software supports files from:

    1. FACSDiva
    2. Summit
    3. CellQuest.
  • qc_script

To get the right output using the available executable file you should use .fcs files saved with yyyy-mm-dd_comment format.

The software will build a .txt file correctly and incrementally as long as the exact same parameters are selected from acquisition to acquisition. At this time you will have to add manually each of these .fcs files to the build the .txt file.

You can then import this .txt to Excel and build your Levy-Jennings plots or use other methods, such as PHP code and JQplot to build them right on your website (see the example on our own QC webpage).

If you wish to edit or personalize the script yourself, you can download the python script below.

Files:


Laser Control Software

  • We have developed control software for several lasers used in the MoFlo:

    1. Sapphire 488-200 CDRH laser (Coherent)
    2. SLIM-561 DPSS (Oxxius)

    The software is coded and designed using Microsoft Visual C++, and therefore only works in PC's running Windows (tested in XP and Vista). The only requirement is that .Net Framework 2.0 or above is installed. Depending on the laser's capabilities, the laser control software can turn the laser on/off, open/close shutters, and read/write information available through an RS232 connection.

    Material:

    1. Laser
    2. Laser Control Executable
    3. Serial Cable.

    If you have any of the above lasers and would like to use this software to control remotely your laser, please contact us.

  • Laser Control Software