Frequently Asked Questions

3D Cell Explorer

What makes the Nanolive technology unique and so innovative?

Nanolive’s technology is unique worldwide and was published on Nature Photonics in 2013.

By combining holography and rotational scanning, it allows to record holograms from different illumination directions on the sample plane determining how light propagates through the cell. By these means, one can measure the cell´s physical properties, called the refractive index (RI). Depending on how the light propagates through the cells, it is possible to determine different RI values for the different parts of the cell. The result is a quantitative cell tomography, in vitro without any invasive sample preparation because the cells do not need to be physically stained.  After the image acquisition, the software STEVE is used to make a quantitative staining based on physical markers (refractive index).

What is the difference to other microscopes that can do 3D imaging?

The measurement of differential interference contrast microscope (DIC) is outside cell TOPOGRAPHY. On the contrary, the 3D Cell Explorer measures the inside cell TOMOGRAPHY.

Rotational scanning allows real 3D reconstruction, noise robustness, and a resolution enhancement. The balanced combination between the high resolution of the system and the high sensitivity for different refractive indices opens opportunities for seeing living cells with nanometric details. It allows measurement of cellular processes in situ with real-time kinetics, enabling multi-parameter analysis at single-cell and sub-cellular scale.   

Because the device does not require special sample preparation (no chemicals, no staining), and thanks to its straightforward handling, the 3D Cell Explorer gives researchers a head start for many applications: in-vitro fertilization, cell division, cell differentiation, bacteria identification, cell death, etc. Perhaps the most exciting application is the potential for monitoring the effect of drugs on patient-specific cells as important part of the development of personal medicine.

Can the 3D Cell Explorer be customized?

Having overcome the inherent resolution limitation of light, we are working now on providing the 3D Cell Explorer to our customers on a secure, global, and affordable scale. Thanks to our new technology, we believe that many new discoveries and publications on living cells will be made. This is the reason why we have designed the 3D Cell Explorer at an affordable price! 

Therefore, to guarantee both – being affordable and the highest quality – we do not offer customization at this point of time. However, should you wish to integrate your own equipment (e.g. top-stage incubator, automated stage, etc.), we can provide you with the exact mechanical dimensions. 

 

Is there an automated stage?

In the future, the stage will be automated. Once we identify the cell of interest, the microscope will be able to follow the cell if it moves from the field of view. Therefore, it will allow the tracking of living cells and also a parallel observation of selected cells within a sample.


Technology

What path is going through the reference lights from the laser for the holographic images?

The reference beam goes through a separate path on the right part of the frame (Mach-Zehnder interferometer).

Which laser type is built into the 3D Cell Explorer and what is the power output?

A Class I laser source at 520 nm with a power output of 0.1 mW. There is no eye protection required with Class I laser.

What is the optical resolution?

The optical resolution is 200nm laterally and 400 nm axially.

What is the 3D image frame rate (acquisition and reconstruction)?

The 3D image frame rate for full acquisition of holograms and with the maximum speed of time-lapse is 0.5 fps.

How long is the self-adjusting time?

The self-adjusting time is less than 90seconds for full self-alignment (depending on optical thickness of the sample).

How does the 3D Cell Explorer senses a new sample on the stage?

The 3D Cell Explorer senses the changes of a sample by measuring the optical thickness of the sample every time.

Does Nanolive have any plans to upgrade the image resolution of the live cells?

We do have a plan to increase the resolution incrementally possibly up to 130nm. Beyond that, a 90nm resolution instrument would necessarily be a custom-build complex instrument which will significantly increase the price and does not fit our current goal to provide every researcher the chance to interact instantly with living cells without damaging them by an affordable price.

Is there more in-depth information to help understand the technology of the 3D Cell Explorer?

Please check our publication on Nature:

For full article please visit: http://www.nature.com/nphoton/journal/v7/n2/full/nphoton.2012.329.html)

What are the dimensions of the 3D Cell Explorer?

Dimensions (width x depth x height in mm): 380 x 170 x 445.

How heavy is the 3D Cell Explorer?

The 3D Cell Explorer weights 8 kg.

How do I store or transport the 3D Cell Explorer?

Please use the provided dust cover to store or transport the 3D Cell Explorer and follow the instructions provided in the user’s guide.

In which permissible ambient temperature can the 3D Cell Explorer operate?

The permissible ambient temperature is between 20°C and 30°C.

What are the voltage ranges of the 3D Cell Explorer?

The voltage ranges are 100-240 VAC / 50-60Hz / 1.0-0.5A

What are the technical specifications of the objective?

The technical specifications of the objective are dry 60x /NA 0.8.

What are the technical specifications of the camera?

The camera has a CMOS sensor, provides 1024×1024 pixels at 165 fps (frames per second).

How large the Field-of-view (FoV)?

The FoV is 94 µm but the significant FoV is 80 µm (inaccurate data on the edges).

How big is the Depth-of-field (DoF)?

The DoF is 30 µm.


Biology and Applications

What is the probing volume?

The significant probing volume is of approximately 80x80x30 µm3.

What are the fields of applications or potential applications?

The 3D Cell Explorer is a tool of discovery for cell researchers and biologists in a limitless number of fields, including:

  • Cell division
  • Cell morphology monitoring
  • Visualize and monitor microorganism interaction and internalization
  • Cell differentiation
  • Cell-cell interaction
  • Intracellular trafficking
  • Cellular remodelling processes
  • Cell death (apoptosis or necrosis)
  • Drug monitoring (i.e. monitor cell response to treatments, in term of modification in the cell’s morphology as well as if it is sensible or not to drugs)
  • In vitrofertilization
  • Observe the consequence on cell morphology upon exposure to different types of nanoparticles as well as test the behaviour of them and monitor their location
  • Tissues imaging: histopathological studies, tissue morphological studies, etc.

For further information please visit our web-page: http://nanolive.ch/applications.

What is the recommended liquid height?

It is depending also on the type of liquid. For clear mounting medium, it is recommended to have less than 5 mm of mounting medium height. For scattering medium like red phenol, it is recommended not to have more than 2 mm of liquid height.

Can the user distinguish each and every intracellular organelle?

In principle, yes, we are able to distinguish each and every organelle. The microscope measures the refractive index of the different cell parts with a precision of 0.001 units but to do that you need to define each and every refractive index before. This could be done by sequentially comparing fluorescence images (in which you label your organelles with a targeted antibody or genetically modified living cell) and then look for the exact refractive index ratio with our technology. It is just a matter of doing this at the beginning and then it is possible to assign each part of the cell depending on its precise refractive index signature.

Alternatively, it is possible to use RI-labelled (e.g. gold particles) antibodies that modify the RI of the organelles which they bind, and then compare the RI values before and after the binding (please refer to the blog post http://nanolive.ch/chris-rocks/).

Since this technology is completely new and there are not yet statistical results, for the moment this is the only method to have quantitative results. However, in the future (our team is working on that), it will be possible to directly measure the RI of each part of the cells.

Is it possible to do time lapse studies using the 3D Cell Explorer?

Yes. It is possible to keep the cells at 37°C which means either integrate a top stage incubator (our stage is compatible with classical top-stage incubators). Set the time lapse in a way to take less often images if you are doing an overnight or long-hours time lapse in order to have a less heavy data file afterwards.

We are in the process of testing different top stage incubators with the aim to promote compatible & approved accessories through our web store. 

Neurons are often larger than the field-of-view of 80 x 80 x 30 microns. Is there a way to enlarge the field-of-view?

No, we cannot enlarge the field of view at the moment. However, you could observe part of the neurons and move the stage to get the full view.

What happens if I use additional optical interfaces (e.g. top-stage incubator, dish-lid, etc)?

Additional optical interfaces may lead to longer calibration times and reduced max. mounting medium height. For these additional surfaces the same optical requirements apply, i.e. cleanness, transparency, and thickness.


Software STEVE

Can STEVE provide an image like the one below (yeast cells from NanoLive image gallery), from the current VOL files (Samples A & B)?

Yes, we’ll be able to do that. We got this image using a surface viewer instead of voxel viewer and our team will integrate this function on STEVE.

What are the main software features of STEVE?

Simple and advance microscope control
Single Shot acquisition
Configurable time-lapse acquisition
Object selection using auxiliary bright field mode
Full self-adjustment

Intuitive user interface
Quantitative staining based on physical markers (RI)
Easy management of your digital stains (creation, edit, enable/disable, delete, save)
Playback options for time-lapse data

Comprehensive visualization options
2 visualization modes: Control mode and Viewer mode
2D slice per slice viewer (refractive index and stained data combined)
3D experience/scientific viewers

Multiple data export options
Classical Formats: raw, tiff, obj, png/jpeg
Compressed proprietary formats: vol/vo1x
Screenshot capture for 2D and 3D viewers
Video capture of the 3D viewer (.avi)

Data annotation system

Semi-automatic update of the software

GPU- accelerated 3D processing

What is the 2D view?

The 2D view shows an X-Y slice of the sample’s Refractive Index overlaid with the user’s defined stains.

What is the panel viewer?

The panel viewer represents a 2D space of Refractive Index (horizontal axis) and Index Gradient (vertical axis). Stains are shown as rectangles covering a region in the staining space. Region on the right have a high Refractive Index, regions on the top have a high Index Gradient

What is the 3D view?

The 3D view shows the stained data in three dimensions

How does the self-adjustment work?

At the start of an acquisition, the system performs a check on sample conditions, and if necessary (for example of sample conditions have changed beyond threshold) the system performs a full self-adjustment so you can be sure you are getting the best possible images of your cells.

How do I exactly prepare for a measurement with the 3D Cell Explorer?
  1. Take the sample previously prepared (for details on how to prepare the sample, please refer to the “sample preparation protocol”) and place it on the 3D Cell Explorer stage.
  2. Open STEVE and click on the white light button. On the left panel of STEVE you can visualize the white light mode. Use the XY and Z knobs of the 3D Cell Explorer to search for the cell of interest and focus on it.
  3. Now you can choose to either do a single-frame image acquisition or a time-lapse acquisition.
How do I prepare to do a single-frame image acquisition?

To perform a single shot, click on the 3D button or on the Single Shot from the Microscope menu.

How do I do a time-lapse acquisition?

A time lapse acquisition is an extended measurement that results in several instantaneous images of the object taken at different times.

To perform a time lapse, the speed and the duration of the acquisition needs to be set.

The time lapse can be performed either at the maximum speed or by setting the time interval between two consecutive frames.

The duration of the time lapse acquisition can be determined either by stopping it manually (by clicking on the Stop Acquiring button) or by setting the ending time for it to stop automatically.

At the end of the acquisition, the software STEVE will enable to digitally stain the data and interactively explore the cell and its subcomponents.

What limits time lapse acquisition?

The live cell imaging feature is only limited by

  1. Maximum available space on the hard-drive.
  2. The live cell conditions (e.g. top-stage incubator, medium, etc.).
How can I save my data obtained with STEVE?

The data can be saved by clicking on the Save icon which allows to save a 3D or a 4D .vol file (readable only with STEVE).

Alternatively, it is possible to export the data in other file formats by opening the export dialog.

The data can be exported by choosing

  • the dimension: 2D (current 2D image in the left panel), 3D (current 3D frame) or 4D (all the time-lapse or the range between the markers A and B).
  • the type: index (the refractive index) or stained.
  • the format of the file: Raw, Vol/Vol1x,Tiff, Obj, Png/Jpeg (available depending on the above selections)
How do I work with data annotation?

For each measurement you can visualize and modify the measurement properties, called also data annotation. On File menu the “Properties” menu open the annotation dialog. All the visible properties, marked with “Eye icon” will be added to the current measurement after the button “Apply” is pushed. The invisible properties, marked with “Close Eye icon” will not be saved. The first property entries, such as: “X size”, “X resolution”, “Acquisition Time”, “Microscope”,etc. are not editable, you cannot modify them, but they will always be saved on the measurement. You can add new entries by clicking on the “Add Entry” button or you can delete the selected entry by clicking on “Delete Entry”. By deleting an entry, that entry will never be shown again on the annotation dialog, you will have deleted it permanently. Also, you can edit each entry by clicking on the “Edit icon” button on the right side of the entry.

How do I update the software STEVE?

Go to Help Menu in Steve and click on “Update STEVE” menu. On Steve Maintenance Tool choose “Update components” and click “Next” button. Follow the messages provide by the update dialog.

How much space do I need for time-lapse acquisition?

Every tomographic frame takes 25mb of space after saving. During acquisition the user is warned by STEVE in case the system is running out of space leading to a stop of the acquisition.

Prior to 4D acquisition the maximum number of frames is automatically estimated by STEVE.

What to do in case of abnormal termination of Steve? Can I recover the acquired data?

During the normal functioning of Steve, all acquired data is continuously stored on the hard drive. In case of abnormal termination, the acquired data should be saved in the file “C:\Users\\AppData\Roaming\Nanolive\data\volume.vof”. Please copy this file somewhere else on your system and rename it to your liking, keeping the file extension intact. You can then open this file in Steve and save it permanently as you wish to. The “AppData” folder is hidden on most computers. Please ask your system administrator if you need help to find it.

What if I accidentally move the arms when the device is power supplied?

It will create a loss of synchronization which is automatically solved by restarting the software STEVE.


General Questions


Does Nanolive offer a suitable PC in their product portfolio?

We do not offer PC’s in our product portfolio but we have a recommendation which PC is best suitable to work with the 3D Cell Explorer. Please check our web site at: http://nanolive.ch/wp-content/uploads/PC-Recommendation_sb.pdf.

Does Nanolive include all needed cables such as USB and power plug cables?

Yes, we do send along the needed cables and power plugs for each different countries.

How can I obtain additional consumables (cables, cleaning swabs, dust cover, power supply)?

Please contact us.