Boosting volumetric acquisitions

Fast volumetric microscopy is essential to examine various biological aspects in their complexity, ranging from whole animal imaging, to live phenomena, to thick samples to image at the different depth. We compared fast volumetric acquisitions between the Z microscope motor and the Z Piezo nano-positioning system by Mad City Labs (MCL; product code: Nano-Z200) with 200 um of travel and 20 msec of step response. For experimental setups needing higher speed in Z, piezo stages with reduced range (50 um) and higher speed (time response 5 msec) are available, together with the possibility to use the trigger mode to speed it up.

We used two types of samples, each of which require high-speed volumetric acquisitions for specific reasons:

  1. Caenorhabditis elegans: we focused on a transgenic line expressing a pan-neuronal, genetically encoded fluorescent Ca2+ indicator (C. elegans strain was created in the laboratory of Prof. Manuel Zimmer (IMP, Vienna ) and sample courtesy of Silvia Schwartz (IIT, Rome)). Speeding up volume acquisitions is relevant in order to monitor fast dynamics in neuronal activity in living nematodes (Figure A).
  2. cleared mouse kidney tissue: a thick section of 550 um represents a useful tool to test in depth acquisitions and, afterwards, 3D volume tissue reconstruction (Figure B; for further sample details visit SunJin lab website).

Acquisitions have been performed with Nikon Ti2 inverted microscope equipped with: CrestOptics Confocal Spinning Disk X-Light V3, MCL Z Piezo, camera Photometrics 95B (11 um pixel size) and Celesta laser source (Lumencor). In the Table below, Z settings and the total acquisition time required in different experimental conditions are listed.

Globally, with the same camera exposure settings, we managed to halve the duration of acquisition time by using the MCL Z piezo compared to the Z microscope motor, giving significant advantages for acquisitions such as fast live imaging and thick specimens.

Table

Sample and objective Z volume Z step Total acquisition time (sec)
Z microscope motor MCL Z piezo
Caenorhabditis elegans (20x, air) 45 um 0.9 um 2.2 1.2 *
Kidney tissue (20x, air) 200 um 0.9 um 8.0 3.6
Kidney tissue (60x, oil) 150 um 0.3 um 14.8 8.1 **

Figure A

* C. elegans acquisition settings: 20x air objective, 0.75 NA; 4 msec exposure time; Z volume of 45 um with 0.9 um step; field of view of 1604×376 pixels.

3D volume view (threshold)

Maximum Intensity Projection

Figure B

** Kidney section acquisition settings: 60x air objective, 1.4 NA; 1 msec exposure time; Z volume of 150 um with 0.3 um step; field of view of 1604×256 pixels.

        3D volume view

Maximum Intensity Projection

The application note has been prepared in collaboration with Ferdinando Ciceri

Mad City Labs

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