IGC

Open Spin Wiki

Information on the construction and development of the system (plugin, device adapter, etc...) is available here.

Lightsheet Microscope (oPenSPIN) | Reservations

  • Lightseet Microscope
  • Location Room : UIC Room 6 (0B10), Bartolomeu Dias wing

    Manufacturer : Emilio Gualda and Hugo Pereira

    Model : dSLM (digital Scanning Light Sheet Microscope)

    Keywords: DSLM, Light-Sheet, SPIM, Live Imaging, Multi View


Light Sheet Microscopy (LSM) is a fluorescence microscopy technique, where the illumination is done perpendicularly to the detection. The technique shapes the illumination laser beam into a rectangle and then focuses it down only in one direction, using a cylindrical lens (SPIM) or galvanometric mirrors (DSLM). This forms a thin "sheet of light" right in the focal plane of the detection objective, illuminating the whole sample plane at the same time. A CMOS camera records the fluorescent signal. This allows obtaining images of a big area in a fast way with a good sectioning of the sample and out-of-focus light suppression. LSM is especially well suited for the investigation of the development of large samples to study features (such as gene expression patterns) that require high resolution while being extended over a large volume and a long period of time. It has been successfully used to track developmental processes on Zebra fish, Drosophila fly, C. elegans nematodes or Arabidopsis plants among others.

Booking Calendar details

Configuration
Value
Booking type
Regular reservation
Responsible
Hugo Pereira
Minimum booking time (in min)
60
Schedule tolerance (in days)
5
Detele tolerance (in hours)
1
Booking restriction status
Disabled

Laser Unit

Laser
Excitation lines
MBL
473
Obis
561
Omicron
638

Available objectives

Place mouse over Magnification to see the objective.
Magnification
Objective type
NA
Immersion
Working distance (mm)
Link
2x
Plan Apo
0.10
-
8.5
4x
Plan Fluor
0.13
-
17.4
10x
Plan Fluor
0.30
-
16
16x
LWD
0.8
WATER
3
60x
Fluor
1
WATER
2

Emission FilterWheel Info

Emission filter
eGFP
BP 53570
YFP
BP 580/25
TRITC
BP 620/90
CY5
BP 700/75
eGFP + TRITC
DB 523/40 - 610/52

Spectra, lines & filters:

Setup

Turn on procedures

  1. If you are the first person of the day, turn on the Main Switch power button.

    • Located on the right side of the optical table. Click image to magnify.

    Main Switch

  2. Turn on Laser 473 Controller.

    Laser 473 Controller

  3. Turn on Laser 561 Controller.

    Laser 561 Controller

  4. Turn on Z Stage Controller.

    • Located on the back of the Z stage controller.

    Z Stage Controller Front View

    Z Stage Controller Back View

  5. Turn on galvo mirror controller.

    • Located on the back of the galvo mirror controller.

    Galvo Mirror Controller Front View

    Galvo Mirror Controller Front View

  6. Turn on Laser 473 Key Switch.

    Laser 473 Key Switch

  7. Turn on Laser 561 Key Switch.

    Laser 561 Key Switch

  8. Turn on the Camera.

    mercury

  9. Start the MicroManager "SPIMVi!" software available at the Desktop.

    Note: If you are the first user of the day, you need to "home" the stage motors right after open the software.

    Leica LAS X shortcut

  10. Set up your experiments on the microscope.

Turn off procedures

  1. Close the software. If you are NOT the last user of the day, you don't need to do the following steps.
  2. Turn off the camera.
  3. Turn off Laser 473 and 561 key switchs.
  4. Turn off Galvo Mirror Controller.
  5. Turn off Z Stage Controller.
  6. Turn off Laser 473 and 561 Laser Controllers.
  7. Turn off Main Switch Power.

Reservations

Acquiring images in a LSM microscope is a complex procedure, first time users are required to contact the UIC staff to discuss the use of the microscope, sample preparation and experiment design in general. For reservations please contact Hugo Pereira (hmpereira@igc.gulbenkian.pt).

Acquisition

We have designed a custom Java plugin for fully controlling DSLM/SPIM/OPT microscopes with a single window. It allows the capture of time lapse sequences, multicolor, z stacks and multi-view recordings in an easy and intuitive manner. The shutter state and the excitation/emission filters can be also controlled with this plugin. The Stage Control and the Rotation Control panels help with sample positioning in z and view angle. Two different modes, i.e. DSLM/SPIM or OPT, can be chosen using the Mode Panel. In OPT mode only rotation and time lapse are allowed while in SPIM/DSLM mode the different imaging options (Stack of Images, Channels, Rotation, Time lapse) can be selected through the checkboxes. In the DSLM panel we are able to define the amplitude and speed of a galvo mirror (Move button) or define a specific position (Set button) of the galvanometric mirrors. The camera parameters (exposure time and binning) are controlled with the Micromanager main control, except when the channel option is selected. Then, it can be edited the exposure time, as well as the channel name and the excitation/emission filter combination, for each channel.


Extra Info

In our set-up we included the rotation of the sample allowing obtaining images of the same sample with different view-points every 45 degrees. This permits to have a better insight of the sample features. Those different views could be integrated on a single image or stack using computational methods, called multi-view fusion, avoiding shadow effects and increasing the image quality. To do that beads are added to the agarose solution holding the sample and run the free Fiji plugin MultiView-Registration. Our system is completely designed in open platforms such as Micromanager and Arduino. Source code of the device controllers and plugins can be found here.