Lithuanian Journal of Physics article / Lietuvos fizikos žurnalo straipsnis

OPTIMIZATION OF WIDE-FIELD SECOND-HARMONIC GENERATION MICROSCOPY FOR FAST IMAGING OF LARGE SAMPLE AREAS IN BIOLOGICAL TISSUES
Andrej Dementjev^a, Romualdas Rudys^b,c, Renata Karpicz^a, and Danielis Rutkauskas^a
^aInstitute of Physics, Center for Physical Sciences and Technology, Savanorių 231, 02300 Vilnius, Lithuania
^b Department of Biomodels, State Research Institute Centre for Innovative Medicine, Santariškių 5, 08406 Vilnius, Lithuania
^cPhotosana, UAB, Mokslininkų 11, 08412 Vilnius, Lithuania
Email: danielis.rutkauskas@ftmc.lt

Received 19 May 2020; revised 1 July 2020; accepted 1 July 2020

Second-harmonic generation (SHG) microscopy is a label-free imaging method that can be used to visualize the detailed arrangement of collagen structures in biological tissues. Here, we sought to optimize the speed of microscopic SHG image acquisition of macroscopic fixed tissue sample areas by employing the wide-field imaging with a high power and medium, 1 MHz pulse repetition frequency laser in combination with a mechanical sample scanning. Unlike in the conventional laser-scanning microscopy, the optimum of the wide-field acquisition entails an interplay between the size of the illuminated area and the intensity of the generated signal. We delineate quantitative procedures to set the image parameters for the maximum speed of the tiled image acquisition, and also describe the possible optimization of the laser parameters for further enhancement of the speed of acquisition.

Keywords: microscopy, second-harmonic generation, wide-field, collagen, tissue samples
PACS: 87.64.mn, 42.65.Ky, 87.14.em

PLATAUS LAUKO ANTROSIOS HARMONIKOS GENERAVIMO MIKROSKOPO OPTIMIZAVIMAS GREITAM DIDELIŲ BIOLOGINIŲ AUDINIŲ PLOTŲ VAIZDINIMUI
Andrej Dementjev^a, Romualdas Rudys^b,c, Renata Karpicz^a, Danielis Rutkauskas^a

^aFizinių ir technologijos mokslų centras, Vilnius, Lietuva
^bInovatyvios medicinos centras, Vilnius, Lietuva
^cUAB Photosana, Vilnius, Lietuva

Antrosios harmonikos generavimo (AHG) mikroskopija yra papildomų žymių nereikalaujantis vaizdinimo metodas, kurį naudojant įmanoma atvaizduoti detalų kolageno darinių išsidėstymą biologiniuose bandiniuose. Šiame darbe optimizuota makroskopinių fiksuoto audinio bandinių plotų mikroskopinių AHG vaizdų registravimo sparta naudojant plataus lauko vaizdinimą su didelės galios ir vidutinio, 1 MHz impulso pasikartojimo dažnio, lazeriu bei mechaniniu bandinio skenavimu. Skirtingai nei įprastos skenuojančios mikroskopijos atveju, plataus lauko vaizdinimo optimumą lemia apšviečiamo bandinio ploto dydžio ir generuojamo signalo intensyvumo kombinacija. Darbe aprašomos kiekybinės procedūros parenkant vaizdo parametrus maksimaliai sudurtinio vaizdo registravimo spartai. Taip pat apibūdinamas galimas lazerio parametrų optimizavimas siekiant tolesnio tos spartos padinimo.

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