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Biologia

pp 1–8 | Cite as

Optimized cutting laser trajectory for laser capture microdissection

  • Marie Korabecna
  • Zbynek TonarEmail author
  • Zoltan Tomori
  • Erna Demjen
Original Article
  • 18 Downloads

Abstract

Laser capture microdissection (LCM) is an excellent tool using a laser beam for specific selection and harvesting of cells or cell populations from heterogeneous tissue sections prepared on special slides. The aim of our study was to apply mathematical methods for optimizing the planning of laser trajectory in LCM combining positive and negative selection criteria with adjustable size, weight, importance, and security borders. We developed and tested software tool named CutPlanner to be used in a transparent overlay superimposed to the live camera image independently from the manufacturer of the LCM device. Once optimized, quantified and approved by the user, the resulting outline of the region of interest is directly copied to the laser beam cutting trajectory. The software is made publicly available for non-commercial use to the scientific community and provides a versatile tool for effectively minimizing the length of the laser trajectory to obtain the selected cells without destroying surrounding cells and tissue structures. Saving all the settings allows for performing repeating tasks under similar conditions, especially in uniform and routinely performed LCM applications.

Keywords

Quantitative histology Laser capture microdissection Precision medicine Molecular analysis Single cell separation Stereology Trajectory optimization Tumor analysis 

Abbreviations

LCM

Laser capture microdissection

Notes

Acknowledgments

M.K. and Z.T. were supported by the grant TIP I/328 of the Ministry of Industry and Trade of the Czech Republic, M.K. was supported by the Ministry of Education, Youth and Sport of the Czech Republic (grant numbers Progres Q25 and SVV 260 373) and by the Ministry of health of the Czech Republic (grant number RVO/VFN 64165). Zb.T. was supported by the Project NPU I Nr. LO1503 and by the Project No. CZ.02.1.01/0.0/0.0/16_019/0000787 “Fighting INfectious Diseases“, awarded by the MEYS CR and financed from EFRR. Zb.T. also received support from Charles University under the Progres Q39 Project and under the Charles University Research Centre program UNCE/MED/006 “University Center of Clinical and Experimental Liver Surgery”. Zo.T. and E.D. were also supported by Slovak research grant agencies PVV (No 15-0665) and VEGA (No. 2/0175/14 and No. 2/0086/16).

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interests regarding the publication of this paper.

Supplementary material

11756_2019_234_Fig2_ESM.png (4.4 mb)
Supplement 1

Defining and optimizing the laser trajectory. Screenshots from the Drawing module of the CutPlanner software are shown in blank images. Calculation of polygons (A-B) and user-defined settings of Object size (C-D) and Exclusion weight (E,F) are demonstrated. A – Marking objects for positive selection (green points) and negative selection (red points), the Calculate option is off. B – Switching on the Calculate option launches the algorithm producing polygons outlining the area suggested for cutting. C – Small-sized objects separated with long distances result in multiple polygons. D – Using larger objects results in confluence of polygons. E – Minimum Exclusion weight can be used for small-sized negatively selected objects. F – Increasing the Exclusion weight results in reduction of adjacent polygons. (PNG 4488 kb)

11756_2019_234_MOESM1_ESM.tif (18.1 mb)
High Resolution Image (TIF 18521 kb)
11756_2019_234_Fig3_ESM.png (4.3 mb)
Supplement 2

Defining and optimizing the laser trajectory. Screenshots from the Drawing module of the CutPlanner software are shown in blank images. User-defined settings of the Sigma parameter (A-B), Dilate function (C-D), and Convex hull (E,F) are demonstrated. A – Using a small Sigma value increases the polygons. B – Using a greater Sigma value decreases the polygons, because Sigma is proportional to the distance between both positively and negatively selected points. C – Without dilatation, no additional borders are added. D – With dilatation, the polygons homogeneously increase in size to prevent destruction of positively selected objects according to the expected laser spot size. E – Without convex hull, the outline of the polygons is a general shape. F – Switching the Convex hull option on results in reducing the complexity of the laser cutting pathway and in eliminating non-convex regions. (PNG 4429 kb)

11756_2019_234_MOESM2_ESM.tif (18.1 mb)
High Resolution Image (TIF 18552 kb)
11756_2019_234_Fig4_ESM.png (7.7 mb)
Supplement 3

Optimizing the laser trajectory when operating laser microdissection device. A – Software provided by the laser microdissection manufacturer runs in the background on a PC using two monitors. The liver image is on the right side. The CutPlanner software is launched. Further images (B-G) show only cropped regions from the live image window. B – For fast and preliminary quantifications, a stereological grid may be applied. C – Immunopositive cells (dark brown color) are marked as positively selected objects (green). D – Increasing the sigma parameter results in a confluence of multiple regions. E – When adjusting the size and switching the Convex hull option on, two polygons are accepted for cutting (marked red). F – Laser cutting is launched in a freehand mode and one of the polygons is being cut as the CutPlanner software goes to background and takes the mouse pointer control to perform the cutting according to the previously planned polygon. G – The cutting has been finished. Additionally, the results table showing the basic morphometric characteristics of the polygons is shown. (PNG 7933 kb)

11756_2019_234_MOESM3_ESM.tif (29.5 mb)
High Resolution Image (TIF 30196 kb)

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Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019

Authors and Affiliations

  1. 1.Institute of Biology and Medical Genetics, First Faculty of Medicine, Purkyne InstituteCharles UniversityPrague 2Czech Republic
  2. 2.Department of Histology and Embryology and Biomedical Center, Faculty of Medicine in PilsenCharles UniversityPilsenCzech Republic
  3. 3.Department of Biophysics, Institute of Experimental PhysicsSlovak Academy of SciencesKosiceSlovak Republic

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