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Zeiss LSM Z.1 | Reservations

  • Z.1

    Location : UIC Room 2 (0B03), Bartolomeu Dias’ Wing

    Manufacturer: Zeiss

    Model: Light Sheet Microscope Z.1

    Year Installed: 2020

    Optics: Light sheet microscopy

    Detectors: 2x PCO Edge 4.2 sCMOS cameras (max 30 fps with 2048x2048 pixels - pixel size 6.5 μm)

    Operating conditions : Temperature, humidity and CO
    2
    control

    Keywords: Light sheet microscopy Z.1, LSM Z.1, LSM Z1, Z1

  • Light sheet fluorescence microscopy (LSFM) is a technique optimized for 3D live imaging of large samples (100-1000um) with good optical sectioning, low phototoxicity and high speed acquisition. In contrast to epifluorescence microscopy, only a thin slice (usually a few micrometers) of the sample is illuminated perpendicularly to the direction of observation. For illumination, a light-sheet is used, i.e. a laser beam which is focused only in one direction (aka SPIM - “selective plane illumination”). Because only the observed plane is illuminated, LSFM reduces the photodamage and stress induced on living samples. Also, the superb optical sectioning capability reduces the background signal and thus creates images with higher contrast than widefield microscopy and can acquire images at speeds 100 to 1000 times faster than laser-scanning microscopy. This Z.1 is equipped with two objectives capable of a fields of view of ~1mm or 500µm with resolutions of 0.15-0.3µm, and is capable of maintaining physiological conditions for observations of live cells and embryos. Note that to use this system, the samples need to be embedded in agarose and, preferably, loaded into a glass/plastic capillary. This system is optimized for live imaging and does NOT have a kit for observation of cleared samples. For those, please consider using the in-house built mesoscope instead. See the facility staff before deciding whether this is the technique for you.


Booking rules:

It is highly advisable to book simultaneously the Light Sheet Microscope Z.1 system and either the Z2 or the Z3 imaging workstations, taking into account these imaging workstations' specifications and installed software to avoid moving data between them.

Suggestion for description in "Materials and Methods":

3D/4D light-sheet datasets were acquired on a Zeiss Z.1 system, equipped with two PCO 4.2 cameras, using a 20x 1.0W (or 40x 1.0W) immersion objective, and the **** fluorescence filter-sets, and excitation with a 455/488/561/647nm laser lines(s). Serial sections were acquired every XX μm and processed for multiview reconstruction with Zeiss’s Zen v3.0/FIJI (Schindelin et al 2012) and the BigStitcher plugin (Hoerl et al 2019).

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).

Setup

General rules of usage of the Z.1:

Users in training should have a basic understanding of the principles of light microscopy, fluorescence and wide-field vs optical sectioning, as well as the use of Agendo AIF Z.1 Users Guide(booking system), and terms of usage of IGC facilities and specifically of the AIF (available in Agendo and in the AIF Z.1 Users Guide). The users must have had completed at least 5 training sessions with the AIF staff, during which they will learn how to assemble the sample chamber, how to load samples and how to operate the multidimentional acquistion Zen software and the basic care for the system components. "In training" users should ask the staff to book on their behalf and plan the experiments in advance. In training users are not allowed to use the Z.1 without supervision.

The following image shows the optical path and major compoenents of the LSM Z.1. The choice of each component is detailed in the following sections namely the lasers, detection optics (objectives), LBFs and filter cubes.

Z1 Setup

Lasers

Excitation lines
Dyes
Laser power
Laser blocking filter (LBF)*
445 nm
CFP (not DAPI!)
20 mW
LBF-445/514/561 (LBF-B)
488 nm
GFP, YFP, FITC
50 mW
LBF-405/488/561/640 (LBF-A)
561 nm
mCherry, RFP, TRITC
50 mW
LBF-405/488/561/640 (LBF-A or B)
638 nm
mRaspberry/Plum, Cy5
75 mW
LBF-405/488/561/640 (LBF-A)

* With these combinations of laser excitation lines and laser blocking filters, one could either use the 488, 561 and 638 OR the 445 and 561 laser excitation lines simultaneously.

Detection Optics

Place mouse over Magnification to see the objective.
Magnification
Objective Type
NA1
Immersion
Working distance (mm)
Max Field of View (FOV)
Theoretical resolution2 ⁄ @ Nyquist
Pixel size: @ 1X / max zoom
Observations
Link
20x
Plan-Apochromat
1.0
Water
2.4
1.0
0.32 ⁄ 0.13
0.33 ⁄ **
Correction collar for refractive index 1.32-1.38 (set before loading chamber).
40x
Plan-Apochromat
1.0
Water
2.5
0.5
0.32 ⁄ 0.13
0.16 ⁄ **
-

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

(2) This value is the theoretical optical resolution limit depending on the NA. To achieve optimal lateral (XY) resolution and Nyquist Rate, please speak with the UIC personnel for optimal pixel size.

These values were calculated using the Resolution formula adapted to confocals as seen in the Olympus micro website: R = 0.61*λ/NA with λ = 550nm.

The pixel size in microns of your picture is saved automatically. Use Bio-formats to recognize it with ImageJ/Fiji (already installed in the Fiji package).

Emission Filter Modules

Select. Label
Camera #1 Filter
Camera #2 Filter
Fluorophores (Cam #1 | Cam #2)
Filter Turret
LP 510
BP 460-500
LP 585
CFP, mCherry | RFP
A
LP 560
BP 505-545
BP 565-615
GFP, mCherry | RFP
A
LP 560 (2)
BP 505-545
LP 585
GFP, mCherry | RFP
A
LP 560 (3)
BP 505-545
LP 660
GFP, mPlum | mStrawberry, tdTomato
A
Select. Label
Camera #1 Filter
Camera #2 Filter
Fluorophores (Cam #1 | Cam #2)
Filter Turret
LP 510
BP 460-500
LP 660
CFP, YFP | Cy5
B
LP 580
BP 525-565
LP 585
YFP, mCherry | mPlum
B

In each experiment you can only use Filter Turret A (installed by default) or B. Please let the facility staff know that you want to use filter turret B so that they can install and configure it in advance.

The list of fluorophores presented in both tables is meant to be representative. Use Spectra Viewer or FPBase to determine which set of lasers and emission filters you want to optimize the system to the specific fluorophores you have in your sample.

Setup

Chamber Assembly

The chamber assembly necessary for sample mounting is a complex procedure, and is addressed during the 1st training sessions for the Z.1. A complete guide on how to assemble the chamber is also present on the Z.1 User's Guide.

Turn on procedures

  1. Turn the system, incubation and PC, following this order.

  2. If the computer is already turned on, restart it. Start the system and incubator if you are using either CO2, humidity or/and Peltier modules. Check the humidifier bottle, and replace water if necessary.

  3. Start the Zen Black software (icon in Windows desktop), select the “start system” with the “boot status” option on, and confirm that no errors show up.
  4. If you're controlling temperature,

If you do not start the incubator system, you may receive errors relating to the absence of these systems in ZEN software; however the system should be working as expected. If this is not the case, please contact the facility staff.

Turn off procedures

  1. Place the stage in Load Position and remove your sample.
  2. Close the software and save your files.
    Warning: DO NOT CONNECT external disks/USB flash drives to the workstation!
  3. Shut down the computer once you are done transferring the datasets and will not need to access it remotely. Otherwise, ask the facility staff to turn it off later.
  4. Turn off the system using the control units (PC, System and Incubation).
  5. Remove the chamber, disassemble it, and rinse all the parts that compose it with distilled water. Leave them to dry in the appropriate box with lid opened.
    Warning: If you have been trained and authorised by the AIF staff to handle objectives, make sure to check and store the 2 illumination objs, and to place the detection objective in the "washer". The facility staff will later inspect and clean them to prepare for the next section.
  6. Take your belongings (dishes, gloves, syringe, etc...), and leave the desk ready for the next user.

Extra Info