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Zeiss Imager Z2 + ApoTome.2 | Reservations

  • Zeiss Imager Z2 + ApoTome.2
  • The zeiss Imager Z2 + Apotome is a fully automated upright microscope equipped with two cameras: On the left side port is a CMOS color camera (Axiocam 105 color, used for brighfield) and on the top port there’s a high sensitivity CMOS camera (Hamamatsu Orca Flash 4.0 v2, for fluorescence). This microscope is equipped for color brightfield and differential interference contrast (DIC) microscopy and fluorescence. The system has a Colibri 7 light source with 4 high-end LEDs with different excitation wavelengths (385, 475, green-orange and 630 nm). The green-orange LED allows choosing between 555nm or 590nm excitation. The system’s stage is fully motorized (XYZ) capable of z-stacks, multiple positions and tile & stitching acquisition. The imager is also equipped with the ApoTome.2, a module that allows optical sectioning of fluorescent samples using structured illumination. A one-dimensional grid pattern is placed on the illumination path and this pattern is projected onto the sample. Three images are acquired and between each image the grid is moved 1/3 of its cycle. This movement generates images where only the in-focus light is modulated by the grid pattern while out-of-focus light is largely unaffected by the grid. By subtracting pairs of these images, the system removes out-of-focus light and the grid pattern, thus creating an optical section of the sample. This method allows for quantitative microscopy. However, since it’s a subtractive method, it might be less efficient with dim samples. Despite the need to process, it is still faster than a laser scanning confocal, so it should be tested as an alternative for thin samples that require a higher throughput than that possible with the SP5 and LSM980.

    Location : UIC Room 1 (0B02), Bartolomeu Dias Wing

    Microscope: Zeiss Imager Z2 + ApoTome.2

    Year Installed: 2019

    Camera: Left-side port - Axiocam 105 RGB (0.5x adaptor). Top port - Hamamatsu Flash 4.0 v2

    Optics: Color, Fluorescence (with ApoTome for optical sectioning) and DIC

    Keywords: Widefield, ApoTome, optical sectioning, tile & stitching, upright mic., high throughput


Suggestion for description in "Materials and Methods":

Images were acquired on an Zeiss Imager Z2/ApoTome.2 system, equipped with a Hamamatsu Orca Flash 4.0 v2 CMOS camera [/ Axiocam 105 color camera], using the a 63x 1.4NA Oil immersion objective, DAPI + CY5 fluorescence filter-sets and DIC optics. Serial sections were acquired every ** µms in ApoTome mode with 5 phase images and processed for bleach correction with Zeiss’s ZEN v2.6.

Suggestion for "Acknowledgments":

We thank the technical support of IGC's Advanced Imaging Facility, which is supported by 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"

Booking Calendar details

Configuration
Value

Available objectives

Below is a list of the objectives and filters available. Both objectives and filter cubes are controlled by the microscope's software (ZEN application)

Place mouse over Magnification to see the objective.
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @550nm in XY (µm) ⁄ @Nyquist
Pixel size 3 for RGB Camera (µm)
Pixel size 3 for Flash camera ⁄ 1.6x Optovar (µm)
DIC?
Link
2.5x
Plan-Neofluar
0.085
-
8.8
3.95 / 1.58
1.76
2.600 / 1.625
N/A
10x
Plan-Neofluar
0.3
-
5.5
1.12 / 0.457
0.44
0.650 / 0.406
N/A
20x
Plan-Apochromat
0.8
-
0.55
0.42 / 0.17
0.22
0.325 / 0.203
N/A
40x (CURRENTLY NOT IN USE)
Plan-Neofluar
1.3
OIL
0.21
0.26 / 0,10
0.11
0.163 / 0.102
YES
63x
Plan-Apochromat
1.4
OIL
0.19
0.24 / 0.10
0.069
0.103 / 0.064
YES
100x
Plan-Apochromat
1.40
OIL
0.10
0.24 / 0.10
0.044
0.065 / 0.041
YES

(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 as seen in the microscopyu website: R = 0.61*λ/NA

(3) The pixel size is saved automatically with the .czi 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).

If you want to use the 40xoil objective, please ask the staff.

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!

Filter sets

Filter "name"
Colibri7 Excitation lines (LED)
Excitation filter
Emission filter
FPBase* (spectra)
Description (use)
BFP
385 nm
390/40 nm
450/40 nm
DAPI, CFP or Hoechst filter
GFP
475 nm
470/40 nm
525/50 nm
GFP or FITC filter
Alexa 555
555 nm
550/25 nm
605/70 nm
RFP or TRITC (LongPass)
Cy5
630 nm
640/30 nm
690/50 nm
Cy5/Far red Filter
DIC
Transmitted Light
<800 nm
Polarizer
mPlum
590 nm
587/25 nm
629/62 nm
Alexa 594
Quad
385 + 475 + 555 + 630 nm
385/30 + 469/38 + 555/30 + 631/33 nm
425/30 + 514/30 + 592/25 + 709/100 nm
Quadruple filter (DAPI, GFP, RFP, Cy5)

Note: * FPBase link collum has the zeiss reference number for the corresponding filters.

Setup

Turning system ON

Wait 1-2sec between each step!

  1. Turn on the (1) Power Supply, (2) Stage controller and (3) ApoTome power supply.

    Microscope, Stage and Apotome Power Supply

  2. Turn on Microscope (button in the left side, back bottom).

    Microscope Turn-on Button

  3. Turn on Hamamatsu camera.

    Hamamatsu graphic image

  4. Turn on Computer.

    Computer Turn-on Button

  5. Login with your agendo account using pGina.

    pGina login

  6. Start ZEN software. Click on ZEN icon (1), choose ZEN pro (2) and let the system calibrate (3 and 4) (it should calibrate both the stage and the focus).

    ZEN desktop icon


Using the ApoTome

  1. Slide the ApoTome module inside the microscope.

    apotome in

  2. Verify if it's calibrated.

    apotome live mode
    To see the ApoTome grid, make sure "Live mode" as the "Grid Visible" option


Turning OFF

For reference, see images above.

  1. Check the reservation system that no one else is coming today. If there is someone else later, simply close ZEN software, clean up the objectives and log-out from your pGina account (same as agendo).
  2. Turn off microscope using the microscope button.
  3. Turn off apotome power supply, stage controller and power supply.
  4. Turn off hamamatsu camera.
  5. Turn off computer.
  6. Done!
  7. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipettes, slides, etc...

Warning

If you mistakenly switch from a dry objective to an oil objective (or vice versa), do not click the “Back” button to go back to the previous objective. Doing so will move you to the next objective on the revolver. Instead, remove your sample or move the stage so that there’s no slide beneath the objective. Then, click “Done”. Afterwards, switch back to the objective you want to use.

Screen Changing Objectives

This is an issue that is being addressed by the facility. We’re sorry for the inconvenience.

Acquisition

First time users need to contact the UIC staff for training in order to operate the system, especially for tile and multiple positions acquisition.

  • Tiles and Multiple Positions

    It’s possible to acquire tiles (sometimes called grids) and multiple positions in this system via ZEN software. For the detailed protocol, check the Imager Z2 user’s guide. If you plan to stitch images after acquisition, click on the image for a guide on how to stitch tiles using Fiji.

  • 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 either before or after acquiring the images. (SHADING BEFORE ACQUISITION FOR FLUORESCENCE IS STILL BEING OPTIMIZED)

  • Flat field correction


Saving and getting your files

The Zeiss Imager Z2 tends to create large files because of the capability to perform imaging with multiple channels, z-stacks, tiles and multiple positions. As such, please save your data in the "Users_data" folder (path is "D:⁄Users_data" and there's a shortcut to it in the Desktop).

Afterwards, you can transfer your data to IGC server (shortcut in Desktop).


Extra Info

Click here to access this microscope's manual -> Link to guide.

ImageJ/Fiji recognizes and opens ZEN image files (.czi), 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)