The Nanoimager camera records data in two channels, left (green) or right (red), and these channels are generated using a dichroic mirror. The laser-specific emission channel depends on the dichroic split fitted in your Nanoimager microscope. Your Nanoimager can be either fitted with 640 split or 560 split. Please contact support in case you are not sure about the split.

For 3-colour acquisitions, the emission signals of two of the dyes fall in one channel and it is necessary to separate the overlapping emission signals. This can be done by performing sequential imaging by using light program. Light program will allow you to use individual laser in different steps and using group functions to generate virtual channels to separate overlapping emission signal. Click on ‘program’ on the right hand-side menu to set the laser program. 

By default, the laser program has group ‘0’ which will record data as G0-Left (green/Channel 0) and in G0-Right (red/channel 1). Adding a group 1 adds G1-Left (green/Channel 2) and G1-Right (red/Channel 3). Hence, using groups will allow the user to filter and visualize localization data independently. Please follow the instructions below according to the dichroic split in your Nanoimager.

For Nanoimager with 640 split, Use light program with following steps

Step 2. 560 laser, 50% laser power, 1000 times to run and group 0

Step 3. 473 laser, 50% laser power, 1000 times to run and group 1.

For 560 Split, use the following light program

Step1: 640 laser at 30%, 500 frames, Group 0 

Step2: 560 laser at 50%, 1000 frames, Group 1

Step3: 473/488 laser at 50%, 1000 frames, Group 1

A). Acquisition from Acquisition control panel.

Make sure to enter the total number of images as per the light program. In this example we have 500+1000+1000 steps = 2500 images. Enter desired dataset tag and Acquisition tag.

Before you press on Acquire, make sure to select enable light program to start the light program.

Click ‘Acquire’ in Acquisition control panel to start the acquisition.

B) Acquisition using Multiple Acquisition Setup

In Multiple Acquisition Setup, Select Multistep Advanced (Light program).

This will automatically recall the light program. Make sure to enter the repetition of the light program here. No need to enter the number of images as they are already assigned in the light program.

Click ‘Start’ to begin the acquisition.

3). Data visualization - Analysis tab

A). How to see 3 color image: Here we acquire three color data and also generated a virtual set of channels using the group function.

In Analyze tab under viewing options these channels will appear.

You can change the color of the localization for each channel by clicking on the color box and selecting the color of choice for respective channel.

Data Filters: Nimos 1.18 and above highlights the virtual channel numbering. Below image shows the organization of channel in filter window. Please note G0 - left , G0-Right , G1-left and G1- Right corresponds to  Channel 0, Channel 1 and Channel 2 respectively. Each column in filter tab is linked to individual Channels to filter out the noise and record specific information.

Bleed through removal

In this acquisition, We acquired the first data which goes into the following channels

• 640 laser data in G0-Right frames acquired from 1 to 500

• 560 laser data in G0-Left from frames 501 to 1500

• 488 laser data in G1-Left from frame 1501 to 2500
  

Use the frame filter to first get rid of bleed-through using the frame values as mentioned above.

Further, Data filtering can now be applied to each channel separately. Few Filters like localization precision, P-value and sigma are useful to filter out noise.