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FAQ’s

General FAQ’S

Does the Nanoimager need an optical table?

No. With the Nanoimager’s unique vibration dampening design, you don’t need an optical table.

Do I need to have specific laser training and a controlled environment to place the Nanoimager in my lab?

No. The Nanoimager is a class 1 laser product and so can be used in a standard lab without specific laser training.

How much does the microscope weigh?

55kg for the entire system.

Sample prep

Which slides are compatible with the Nanoimager?

The Nanoimager works optimally with #1.5H coverslips, which have 170µm thickness. 

The most popular chambers are:

  • ibidi (µ-Slide 8 Well Glass Bottom, No. 1.5) ]
  • Lab-Tek (Nunc™ Lab-Tek™ II Chambered cover-glass, No. 1.5)
  • 35 mm petri dishes with #1.5 cover-glass bottom

Sample chambers/coverslips must be less than 97mm long, 35mm wide and 13mm in height (customizable).

Should the antibodies I use be coupled directly to a fluorophore or can I use a combination of primary and secondary antibodies?

Both methods can be used. Antibodies directly coupled to the fluorochromes are not compulsory, but primary antibodies do have a higher degree of accuracy on the location of proteins. Check out our guide to sample preparation for more information.

Can you do live cell imaging? Can you acquire multi-positions at the same time?

Yes. The Nanoimager is capable of live imaging and acquiring multi-positions data through the following features:

  • Whole microscope unit heating up to 37°C
  • External port that is compatible with microfluidics / CO₂ feed
  • Software support: time-lapse, multi-position and Z-stack

Is there the possibility of working at temperatures other than 37°C?

Yes. The Nanoimager supports temperature in the range of +20°C to 45°C. To warm up from room temperature to 37°C only takes approximately 40 minutes.

Can I do 3 or 4 color imaging?

Yes. The Nanoimager can image up to 4 colors by imaging two channels at the same time; which means you can perform 2-color imaging followed by 2-color imaging in two different channels. You can also program the lasers to run a sequence of on/off for each laser line.

Is it possible to acquire Z-stacks in widefield mode on the Nanoimager?

Yes. The X-Y and the Z position can be scanned through several mm [20 mm x 20 mm x 10 mm (X, Y, Z)].

Can you export the tile overview scan as a high-resolution image?

Yes. The software allows you to export the tiled overview scan at the resolution it was taken.

For smFRET experiments do you use the TIRF mode?

Yes. For smFRET, we typically use TIRF mode to analyze biomolecule interactions on the coverslip surface or the surface of the cell.

Super-resolution imaging

What are the best dyes for super-resolution dSTORM?

We recommend AlexaFluor™ 647, AlexaFluor™ 555, ATTO488, DyLight550 for dSTORM, together with ONI BCubed dSTORM buffer. Check out our guide.

How long does super-resolution image acquisition take?

Imaging time depends on the type of sample and technique, for example:

  • for a typical dSTORM acquisition: 30 ms exposure time for 30,000 frames = 15 minutes
  • for a typical smFRET acquisition: 100 ms for 1000 frames = 100 seconds

Can you use TIRF or HILO mode when acquiring images using dSTORM?

Yes. You can adjust the imaging mode to get the best signal-to-noise ratio for your sample, with a single click.

When you acquire 3D using astigmatism, what is the Z depth you are imaging?

We image up to 1µm depth.

What is the size of each image file?

The size of the video files is generally several GB for standard single-molecule imaging.

Single Particle Tracking

What statistical analysis can I do with the tracking software?

We can look into the distribution of diffusion coefficients, stokes diameter, concentration of species and the type of diffusions (confined/random/directional) by looking at the individual tracks.

How does the tracking algorithm manage with larger, asymmetrical particles/blobby structures?

You can track these particles and structures by adjusting the default localization fitting and filtering parameters.

smFRET

What kind of FRET technique do you perform?

We do single-molecule FRET, which enables identification of single molecules or molecular complexes and measurements of the stoichiometry, distances, reaction kinetics and many others parameters.

What results do you get from a smFRET experiment?

We get values for FRET efficiency, donor and acceptor emission levels, which can be easily exported in an *.csv format.

Hardware

Which objective lens do you use in the system?

We use the oil immersion 100x 1.4 NA from Olympus. Customers can also enquire about other options.

What stage is used within the Nanoimager?

The stage is a closed-loop piezo stage for all X, Y and Z directions. The stage is the most precise on the market and offers 1nm precision and 30nm reproducibility.

What is the maximum weight I can put on the stage?

200g is the maximum weight for the stage.

Which camera is used?

We use the sCMOS Orca flash 4 V3 (in NimS) and Orca flash4  LT+ (in NimX). Check out our models here.

What is the speed of the camera?

100 frames per second with full chip. It can be faster by cropping an area or region of interest.

How many cameras are used in the system?

The Nanoimager supports one camera, and the system is configured to capture two different colors at the same time, with a very large field of view per channel (50µm x80µm).

How many laser lines does the Nanoimager hold?

The Nanoimager holds up to four lasers.

What is the maximum power density I can achieve with the Nanoimager?

The Nanoimager can be configured with lasers such that the power density is > 10kW/cm2

What is the lifetime of the lasers?

This depends on wavelength, but on average the lifetime is 10,000 hours.

Can lasers be swapped out?

Yes. Lasers of the same wavelength can be swapped by opening up the light engine. You will not require a service engineer for this. However, you cannot swap to a different laser wavelength since we have a fixed pre-chosen configuration.

What are the dimensions of each part of the Nanoimager system, is it really that small?

Yes. The Nanoimager (21cm x 15cm x 21cm) and has approximately the same footprint as a piece of A4 paper. The light engine is 21cm x 42cm x 45cm and they are attached to each other by a 1.5m cable.

Software

Can the data acquired by the Nanoimager software be used in other software packages?

Yes. We want users to get the most out of the data, so it is saved for export in standard file formats (*.tiff and *.csv).

Are any other software packages needed for analysis?

No. The Nanoimager software is designed to support all standard analysis needs.

Does the Nanoimager software offer custom scripting?

Yes. A Python interface can be used to write custom scripts to control hardware and analyze data from within the Nanoimager application.

Will the Nanoimager software run on Windows and Mac?

Currently, the Nanoimager software is available for Windows.

Do you require a high-specification computer to run the Nanoimager software?

No, not for analysis. The NimOS software runs on most modern computers, running 64-bit Windows 10. We supply a high-spec laptop with each Nanoimager and this is included in the price.

How can I set up a 3 color acquisition?

Laser combinations can be switched sequentially to allow for 3 color experiments using NimOS.