AdvancedImagingUIC

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• 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

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	lnk
10x		Plan-Neofluar	0.3	-	5.5	1.12 / 0.457	0.44	0.650 / 0.406	N/A	lnk
20x		Plan-Apochromat	0.8	-	0.55	0.42 / 0.17	0.22	0.325 / 0.203	N/A	lnk
40x (CURRENTLY NOT IN USE)		Plan-Neofluar	1.3	OIL	0.21	0.26 / 0,10	0.11	0.163 / 0.102	YES	lnk
63x		Plan-Apochromat	1.4	OIL	0.19	0.24 / 0.10	0.069	0.103 / 0.064	YES	lnk
100x		Plan-Apochromat	1.40	OIL	0.10	0.24 / 0.10	0.044	0.065 / 0.041	YES	lnk

(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!

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

Wait 1-2sec between each step!

1. Turn on the (1) Power Supply.

   

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

   

3. Turn on Computer.

   

4. Login with your agendo account using pGina.

   

5. 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).

   

1. Slide the ApoTome module inside the microscope.

   

2. Verify if it's calibrated.

   

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 power supply.
4. Turn off computer.
5. Done!
6. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipettes, slides, etc...

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 issue has been reported to Zeiss and we are waiting for a fix. We’re sorry for the inconvenience.

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)

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