Optimized cutting laser trajectory for laser capture microdissection
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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.
KeywordsQuantitative histology Laser capture microdissection Precision medicine Molecular analysis Single cell separation Stereology Trajectory optimization Tumor analysis
Laser capture microdissection
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).
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Conflict of interests
The authors declare that they have no conflict of interests regarding the publication of this paper.
- Baddeley AJ, Gundersen HJ, Cruz-Orive LM (1986) Estimation of surface area from vertical sections. J Microsc 142:259–276. https://doi.org/10.1111/j.1365-2818.1986.tb04282.x CrossRefGoogle Scholar
- Gundersen HJG (1977) Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc 111:219–223. https://doi.org/10.1111/j.1365-2818.1977.tb00062.x CrossRefGoogle Scholar
- Gundersen HJG, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147:229–263. https://doi.org/10.1111/j.1365-2818.1987.tb02837.x CrossRefGoogle Scholar
- Howard CV, Reed MG (2005) Unbiased stereology. Three-dimensional measurement in microscopy (advanced methods). Garland, New YorkGoogle Scholar
- Korabečná M, Tonar Z, Tomori Z, Demjén E (2016). Optimization method of laser trajectory at laser microdissection”. Charles University in Prague, Faculty of Medicine in Pilsen. National patent no. CZ 305856 issued by the industrial property Office of the Czech RepublicGoogle Scholar
- Legres LG, Janin A, Masselon C, Bertheau P (2014) Beyond laser microdissection technology: follow the yellow brick road for cancer research. Am J Cancer Res 4:1–28Google Scholar
- Mouton PR (2002) Principles and practices of unbiased stereology. In: An introduction for bioscientists. John Hopkins University Press, BaltimoreGoogle Scholar
- Russ JC, Neal FB (2016) The image processing handbook. CRC Press, Boca RatonGoogle Scholar
- Schmidt F, Efferth T (2016) Tumor heterogeneity, single-cell sequencing, and drug resistance pharmaceuticals (Basel) 9:E33. https://doi.org/10.3390/ph9020033
- Tonar Z, Kochova P, Holecek M, Janacek J (2007) Stereological assessment, mechanical measurement and computer modelling of smooth muscle. Mater Sci Forum 567-568:353–356. https://doi.org/10.4028/www.scientific.net/MSF.567-568.353 CrossRefGoogle Scholar