Application Booklet Micro-CT Analysis related instrument Leica EM CPD300

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CRITICAL-POINT DRYING FOR THE PREPARATION OF BIOLOGICAL SAMPLES FOR MICRO-CT ANALYSIS  

Peter Michalik and Elisabeth Lipke 

Zoological Institute and Museum, Ernst‐Moritz‐Arndt University Greifswald, Germany 

 

X‐ray micro‐computed tomography  (micro‐CT)  is a routinely applied non‐invasive technique  for the investigation of the internal anatomy and morphology of organisms. As a result of a micro‐CT scan a stack of grey‐scale  images  is generated  from a series of projections  taken at defined angles during sample rotation. Since several years the number of lab‐based micro‐CT imaging systems  is constantly growing making this technique available to a broad spectrum of researchers and applications. 

Similar to other imaging techniques such as scanning electron microscopy, micro‐CT allows to study biological samples in nearly every condition (e.g. fresh, dried or within preservatives). Micro‐CT is the ideal  technique  for  studying  bones,  teeth  and  shells  in  3D,  but  the  analysis  of  soft  tissue  is significantly  influenced  and  hindered  by  its  low  absorption  contrast  based  on  the  presence  of compounds  with  low‐atomic  number  elements.  In  order  to  overcome  this  limitation,  several approaches  can be applied  including different  staining and/or drying  techniques as well as phase‐related contrast  imaging. However, whenever possible samples should be analyzed  in dry condition as  it provides a significantly higher signal  to noise  ratio compared  to samples scanned  in  liquid.  In order  to dry delicate biological  samples,  critical point drying was proofed  to be  the best method compared to e.g., chemical or air drying as it preserves the structures while minimizing artifacts such as shrinkage of tissue and distortion.

Leica EM CPD300 Application Booklet Micro‐CT Analysis 02/15                Seite  2 

 

MICRO-COMPUTER TOMOGRAPHY PROTOCOLS

1. Micro-CT of Book Scorpion Musculature

Introduction:

Species: Book scorpion (Neobisium sp.)

Critical point drying of book scorpion with subsequent X-ray micro-computed tomography (micro-CT) to detect anatomical features with special regard to the musculature.

Procedure:

Sample Holder:

Sample was transferred to microporous specimen pot and placed into chamber of

Filter Discs and Porous Pots holder.

Fixation and Dehydration:

Ethanol (70%) overnight

Ethanol series: 80%, 90%, 96%, 100% 2x 10 min.

Iodine staining (1% iodine solution in 100% ethanol) overnight

Ethanol: 100% 2x 10 min.

CPD300 auto Program:

Mounting and Scanning:

Dried sample was glued on an insect pin and scanned with an Xradia MicroXCT-200 X-ray imaging system (Carl Zeiss X-ray Microscopy Inc., Pleasanton, USA).

Leica EM CPD300 Application Booklet Micro‐CT Analysis 02/15                Seite  3 

 

Results:

Volume reconstruction of a book scorpion prosoma (outer view and inner view to visualize the musculature).

Courtesy of Elisabeth Lipke and Dr. Peter Michalik, University of Greifswald, Germany.

Leica EM CPD300 Application Booklet Micro‐CT Analysis 02/15                Seite  4 

 

2. Micro-CT of Insect Brain Protocol

Introduction:

Species: Blow fly (Lucilia caesar)

Critical point drying of the blow fly with subsequent X-ray micro-computed tomography (micro-CT) to detect neuroanatomical features.

Procedure:

Sample Holder:

Samples were placed individually in the chambers of Arthropoda holder.

Fixation and Dehydration:

Bouin’s fixative overnight

0.1 M phosphate buffer (1.8% Sucrose, pH 7.2) 3x 10 min.

Ethanol series: 60%, 70%, 80%, 90%, 96%, 100% 2x 10 min.

Iodine staining (1% iodine solution in 100% ethanol) overnight

Ethanol: 100% 2x 10 min.

CPD300 auto Program:

Mounting and Scanning:

Dried samples were glued on insect pins and scanned with an Xradia MicroXCT-200 X-ray imaging system (Carl Zeiss X-ray Microscopy Inc., Pleasanton, USA).

Leica EM CPD300 Application Booklet Micro‐CT Analysis 02/15                Seite  5 

 

Results:

Volume reconstructions and virtual sections of the head and brain of a blow fly.

Courtesy of Elisabeth Lipke and Dr. Peter Michalik, University of Greifswald, Germany.

Leica EM CPD300 Application Booklet Micro‐CT Analysis 02/15                Seite  6 

 

3. Micro-CT of Insect Larva Protocol

Introduction:

Species: red blood worm (midge larva)

Critical point drying of midge larvae with subsequent X-ray micro-computed tomography (micro-CT) to reconstruct the inner anatomy.

Procedure:

Sample Holder:

Sample was placed individually in the chambers of Arthropoda holder.

Fixation and Dehydration:

2.5 % Glutardialdehyde (in 0.1 M phosphate buffer) overnight

0.1 M phosphate buffer (1.8% Sucrose, pH 7.2) 3x 10 min.

Ethanol series: 60%, 70%, 80%, 90%, 96%, 100% 2x 10 min.

Iodine staining (1% iodine solution in 100% ethanol) overnight

Ethanol: 100% 2x 10 min.

CPD300 auto Program:

Mounting and Scanning:

Dried sample was glued on an insect pin and scanned with an Xradia MicroXCT-200 X-ray imaging system (Carl Zeiss X-ray Microscopy Inc., Pleasanton, USA).

Leica EM CPD300 Application Booklet Micro‐CT Analysis 02/15                Seite  7 

 

Results:

Volume reconstructions and virtual section of the red blood worm showing a variety of organ systems.

Courtesy of Elisabeth Lipke and Dr. Peter Michalik, University of Greifswald, Germany.

 

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