IGC

DeltaVision CORE system | Reservations

  • DeltaVision
  • The DeltaVision is a fully automated research microscope equipped with a high sensitivity EM-CCD camera, capable of fluorescence and interference contrast (DIC) microscopy. The system has an InsightSSI illumination (7 lines, high-speed), fully motorized fast stage (XYZ) and filters, as well as controlled atmosphere box, and high-resolution optics providing excellent conditions for imaging of cells. The DeltaVision system is also tightly controlled to provide post-acquisition optical sectioning through deconvolution; it is reccomended for quantitative image-processing.

    Location : Bartolomeu Dias wing, UIC Room 2

    Microscope: Olympus AppliedPrecision DeltaVision Core

    Year Installed: 2007

    Camera: Photometrics Cascade II 1024 EM-CCD

    Optics: Fluorescence and DIC + Deconvolution

    Operating: Temperature & atmosphere control (37oC + Humidity)

Suggestion for description in "Materials and Methods":

Images were acquired on an Applied Precision DeltavisionCORE system, mounted on an Olympus inverted microscope, equiped with a Cascade II 2014 EM-CCD camera, using the a 63x 1.4NA Oil immersion objetive, DAPI + CY5 fluorescence filtersets and DIC optics. Images were deconvolved with Applied Precision's softWorx software.

Suggestion for "Acknowledgments":

We would like to thank the technical support of IGC's Advanced Imaging Facility (AIF-UIC), which is supported by the national Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT; Portugal).

Maintenance log:

  • 09-12-2015 - Microscope control computer repaired and re-installed at the microscope.
  • 19-11-2014 - Light Path switcher cleaned of old oil, broken cardan replaced by a bigger and more robust one. Mechanism moves more smoothly.
  • 01-07-2014 - Light Path mechanism replaced for an aluminium one as a more permanet solution.
  • 24-04-2014 - Light Path mechanism cardan broke due to heavy use. Temporarily put together until na aluminium replacement comes.
  • 04-04-2014 - Computer was fixed and is working again
  • 04-02-2014 - Computer is not working.
  • 04-02-2014 - Light Path knob was fixed.
  • 17-03-2014 - Light Path knob was not functional and had to be fixed. Whole microscope was disassembled to reach the light path prisms.
  • 31-01-2014 - Micromanipulation unit has been repaired (Z motor was not moving) and put inside the incubator once more.
  • 11-01-2013 - (in progress) - Installation of LIS brick for accurate Co2 control
  • 31-10-2013 - Adaptation to universal stage insert to accomodate for CO2 "dome"

Available objectives

Below is a list of the objectives and filters available. On the DeltaVision system, the filter cube (dichroic) is manually set and the emission filters are split between two filter wheels that also need to be manually switched. Make sure that both match the excitation and emission filters you want to use.

Move mouse over me!
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm)
Image Pixel size3 (µm)
Pixel size + 1.6 Aux mag3 (µm)
Nyquist sampling @525nm in Z (µm)
Link
10x
UPLan FL N
0.30
-
10
1.068
1.30
0.81
1.601
20x
UPlan SAPO
0.75
-
0.60
0.427
0.65
0.41
0.64
40x
UPlan FL N
1.3
OIL
0.20
0.246
0.32
0.20
0.37
60x
UPlan SAPO
1.2
WATER
0.28
0.267
0.22
0.14
0.40
60x
Plan Apo N
1.42
OIL
0.15
0.226
0.22
0.14
0.34
100x
UPlan SAPO
1.40
OIL
0.13
0.229
0.13
0.08
0.34

(1) NA - Numerical Aperture. For more information, follow this link.

(2) These values are the theoretical optical resolution limits depending on the NA of the objective and were calculated using the Resolution formula adapted to fluorescence as seen in the Olympus micro website: R = 0.61*λ/NA

(3) The pixel size saved automatically with the .dv files. The values on this table were measured with a stage graticule, with and without the 1.6x auxiliary magnification. For more accurate measurements use these values instead. Use Bio-formats to open the images in ImageJ/Fiji (already installed in the Fiji package).

IMPORTANT: For optimal deconvolution you may use the 1.6x auxiliary magnification, however, using this may cause aberration on the edges of the field of view!


Installed filtersets

Fluorescence filterset
SSI Excitation lines
Excitation filter
Emission filter
Dichroic*
Emission wheel
DAPI
381-399 nm
390/18
457/50
1
A
FITC
461-489 nm
475/28
528/38
TRITC
529-556 nm
542/28
617/73
Cy5
621-643 nm
632/22
685/40
CFP
426-450 nm
430/24
470/24
2
B
YFP
505-515 nm
500/20
535/30
GFP
461-489 nm
475/28
525/50
3
mCherry
563-588 nm
575/25
632/60

* - This is the optimal dichroich, may work with other filters but with the possibility of having lower signal intensity. Speak with the Advanced Imaging personnel for further info.

for more info on the InsightSSI fluorescence illuminator click here


Spectra, lines & filters:


Setup

Turning system ON

  1. Check that the Linux computer is running (small green light in the middle should be on).

    Main PC

  2. Switch ON the main power switch at the bottom of the rack (orange light will come on).

    Main PC

  3. Briefly press the button on Instrument controller to initiate startup (green light will come on after a few seconds).

    Main PC

  4. Log-in to the computer with the igcuser account.
  5. Click "Start softworx" to start the software.
  6. Remember to lower the objective.
  7. Go to File>Acquire (Resolve 3D).

Switching filter wheels

  1. On the software, change the Emission to "OPEN".
  2. Unplug the cable from the current eyepiece filter wheel.
  3. Switch the camera emission filter wheel between A and B or vice-versa. Don't force the knob.

    Main PC

  4. Plug the cable to the corresponding Eyepiece filter wheel.
  5. The system will recognize the change and will need some 30 seconds to readjust.

Turning OFF

  1. Check the reservation system that no one else is coming today. If there is someone else later, simply click Stop Softworx, clean up the objectives and log-out.
  2. Find the "Stop Softworx" button on your desktop and click it.
  3. Logout from the computer.
  4. While the computer Logs out, BRIEFLY press the glowing green button. It'll take a few seconds to shutdown.
  5. When the green button turns off, click the orange button.
  6. Leave the little green button (which is the linux computer) alone.
  7. Done!
  8. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipettes, slides, etc...

Acquisition

Acquiring images in a spinning disk confocal microscope is a complex procedure, first time users are required to contact the UIC staff for training. If you need assistance don’t forget to press “with assistance” at reservation time.

  • Grid Colection / Panels

    It is possible to acquire grids and tiles of big samples through Deltavision SoftWorx using the Panels tab. For a more detailed protocol, please check the following link or click in the image to the right.

  • Tile Scan

  • Illumination Correction

    Most microscopes have a problem of field illumination heterogeneity where the middle of the field of view is properly illuminated but decays to the edges, creating problems when both quantifying or qualifying samples. There are some ways to correct this problem after acquiring the images.

  • Flat field correction


Getting your files

The DeltaVision tends to create large files because of the capability to perform live imaging with multiple channels and multiple positions. As such it has been setup as an FTP server so users can download the files from the comfort of their desk. Learn how to below.

  1. Install the File-Zilla Client.
  2. Quick connection, fill the top bar:

    Host:

    • Deltaivision microsocpe: dvm
    • Deltavision workstation: dvw

    username & password

    • username: igcuser
    • corresponding password
    • Port: 22
  3. After you click connect, you should get some messages on the top text window and a new file tree should appear on the right; these are your DeltaVision files.
  4. Find a folder you want to save your files to on the list on the left.
  5. To transfer files, just drag from the right to the left.
  6. Done!

If you want to learn more timesaving features of FileZilla, visit their website.


Extra Info

ImageJ/Fiji recognize and open DeltaVision .dv files, but you will need to use the Bio-Formats plugin. This plugin is available by default in the Fiji distribution.

To install the plugin in ImageJ, download the latest stable release of Bio-formats and drag it into the plugins folder. Once you start ImageJ again it will appear on the plugins menu. Go to "Plugins>Bio-formats>Bio-formats importer" or drag-and-drop a file in ImageJ/Fiji to open it.

Differential Interference Contrast (DIC)