The zeiss Imager Z2 + Apotome is a fully automated upright microscope equipped with two cameras: On the left port Axiocam105 color (used for brighfield) and on the top port a high sensitivity Hamamatsu Orca Flash 4.0 v2 (for fluorescence); the microscope is equipped for fluorescence, differential interference contrast (DIC) and darkfield. The Colibri 7 light source has 4 LEDs for excitation :385, 475, green-orange (550 or 590 options) and 630 nm. The 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. Multiple images are acquired at different positions of the grid thereby generating images where only the in-focus light is modulated by the grid pattern, while out-of-focus light stays 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. Though the method allows for quantitative microscopy, 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
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.
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).
Below is a list of the objectives and filters available. Both objectives and filter cubes are controlled by the microscope's software (ZEN application).
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 "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.
Wait 1-2sec between each step!
For reference, see images above.
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.
This is an issue that is being addressed by the facility. We’re sorry for the inconvenience.
First time users need to contact the UIC staff for training in order to operate the system, especially for tile and multiple positions acquisition.
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 outside of ZEN software, click on the image for a guide on how to stitch tiles using Fiji.
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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)
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).
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)
