For questions, comments or concerns please contact Imaging

5. Bruker TruLive3D Finding the sample


Make sure you performed the calibration of the system before lowering your sample in the chamber.

  1. Finding your sample

         

image.png

LED on.png

NOTE: This is not the focus of the detection objective! It brings the sample in focus for the illumination objective the Blackfly camera is looking through. This step is for identification only.

Find the sample in spinview.png

lHIHGMgkse9iErpl-embedded-image-fw7hamcj.png

image.png

image.png

image.png

To acquire the best images from your sample, you need to look at areas that are in the middle of the dish, within the layer of gel that can be imaged. Only the lower 1mm layer of gel at the bottom of the cuvette can be imaged. If your sample is sitting above this, you will not be able to image it. 

The detection objective is fixed, when you move the z stage your are moving the sample dish up and down. The biggest z-stack you  can acquire is around 1mm, that is if you start below the dish and go as far as the working distance of the objective goes. 

The working distance of the objective is what defines your imaging range. However, the images acquired at the limits of this range will not be ideal. If the dish is too high above the detection objective you will be looking at the bottom of the cuvette. If the sample dish is too close from the detection objective, you will be looking far into the gel and this will generate more aberrations.

If you are looking at the edges of the dish, you will also get more aberrations.

The following figures show how to best position the sample and the resulting images with a bead sample.

OptimalSamplePosition.png

SampleTooClose.png

SampleTooFar.png

The LuxBundle software now has the option to perform stage coupling. We can use this feature to couple the z stage with the CR stage (Correction Ring) and optimize spherical aberration correction throughout the entire z-stack. This step should be done once at the working temperature after calibrating the system. We need to use the beads sample for this as beads are the most appropriate object to identify aberrations and correct for them.

Below are the steps to achieve ideal coupling, along with the values used to couple the Z stage and the CR stage at room temperature and 37C. The stage coupling will be discussing and practiced in your training but you can re-use the values presented in this wiki for room temperature or 37C when you introduce a new sample.

a89f2374-6399-4372-ba13-7516fd989893.png

You now have 2 values for stage coupling and the CR stage will extrapolate values to best fit the entire Z range you selected. Below is an overview of the calibration panel with the stage coupling values set for room temperature.


Revision #12
Created 29 February 2024 17:02:23 by Evolene Premillieu
Updated 8 November 2024 19:52:17 by Evolene Premillieu