ISSN Print 2500–1094
ISSN Online 2542–1204
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
ORIGINAL RESEARCH
Exploration of the femtosecond laser pulse thermal effects on the mouse embryos during the assisted hatching procedure
About authors
1 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
2 Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia
Correspondence should be addressed: Marina V. Kubekina
Vavilova, 34/5, 119334, Moscow, Russia; moc.liamg@ymukyram
About paper
Funding: the procedures involving manipulating embryos using a laser and assessment of expression of the genes responsible for synthesis of heat shock proteins were supported by RSF (project 23-19-00424) and conducted using the equipment of the UNU "Laser Terawatt Femtosecond Complex", which was a part of the Center for Collective Usage "Laser Femtosecond Complex" of the Joint Institute for High Temperatures RAS. The procedures to acquire embryos were supported by the UNU “Transgenbank” grant (№ 075-15-2021-668 of July 29, 2021).
Author contribution: Kubekina MV — immunofluorescence staining and assessment of the heat shock protein expression levels, manuscript writing; Filatov MA — handling embryos, statistical processing, manuscript writing; Silaeva YuYu — general management of the experiment; Sitnikov DS — laser miscrosurgery, data processing, manuscript writing; all authors — discussion and manuscript editing.
Compliance with ethical standards: the study was approved by the Ethics Committee of the Institute of Gene Biology RAS (protocol № 1 dated 25 September 2023) and conducted in strict compliance with the provisions of the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Received: 2023-11-07
Accepted: 2023-12-07
Published online: 2023-12-26
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Fig. 1. Femtosecond laser scalpel scheme. 1 — fs laser, 2 — attenuator, 3 — KDP crystal, 4 — glass plate, 5 — photodiode, 6 — telescope, 7 — laser beam shutter, 8 — mirrors for the laser beam wavelength, 9 — microlens, 10 — motorized microscope stage, 11 — Petri dish with embryos, 12 — substage condenser, 13 — substage lamp, 14 — video camera, 15 — inverted microscope
Fig. 2. Images of the embryos obtained before (A) and after (B) the zona pellucida microsurgery. The scale bar is 20 µm
Fig. 3. Fluorescence staining of different groups of embryos. Viable embryos were stained with Calcein-AM, non-viable embryos with Propidium Iodide, nuclei of viable and non-viable embryos with Hoechst 33342
Fig. 4. Graphic representation of heat shock protein gene expression in the negative and positive control groups, as well as in the experimental group of embryos. Expression of genes Hsp90aa1 (A) and Hspa5 (B) encoding heat shock proteins
Fig. 5. Radial temperature profiles at different times for the millisecond (A) and femtosecond (B) pulse. Laser beam size at 1/е level is marked with arrows. Time (t) is counted down from the beginning of the pulse
Table. Primers used in the study (sequences are shown in the 5' to 3' direction)
