Successful Ti: Sapphire laser cell surgery of Phycomyces blakesleeanus cell wall
2018
Аутори:
Pajić, TanjaStevanović, Katarina
Todorović, Nataša
Krmpot, Aleksandar
Rabasović, Mihailo
Lazović, Vladimir
Pantelić, Dejan
Jelenković, Branislav
Živić, Miroslav
Остала ауторства
Majaron, HanaCotar, Petra
Koren, Monika
Golmajer Zima, Neža
Тип документа:
Конференцијски прилог (Објављена верзија)
,
© 2018 by the Slovenian Biophysical Society, Ljubljana, Slovenia
Метаподаци
Приказ свих података о документуАпстракт:
Application of patch-clamp method for investigation of membrane ion channels of filamentous
fungi is nontrivial task due to presence of chitinous cell wall. Complete removal of the
wall patch is needed to make the membrane accessible to glass pipette. We use the model
filamentous fungus organism, Phycomyces blakesleeanus which is undertaken to the cell
surgery procedure by means of tightly focused femtosecond laser beam. The hyphae were
grown on glass coverslips coated with collagen plasmolysed and imaged by homemade nonlinear
laser scanning microscope by detecting two photon excitation fluorescence signal.
Although intrinsic autofluorescence of chitin enables imaging of the cell wall the hyphae
were stained by Calcofluor White dye prior to the imaging in order to improve signal to
noise ratio. Ti:Sa laser, used for both imaging and surgery, was operating at 730nm, with
76MHz repetition rate and 160fs pulse duration. Carl Zeiss, EC Plan-NEOFLUAR, 401.3
oil immersion objective was used for tight focusing of the laser beam and for the collection
of the fluorescence signal. A visible interference filter (415nm - 685nm) was placed in front
of detector in order to remove scattered laser light. The successful cutting of cell wall could
be achieved within the range of laser intensities and cutting speeds (dwell times). The
hyphae were kept in azide throughout the experiment in order to block the regeneration
of the cell wall. After the surgery, hyphae were slowly deplasmolysed to induce exit of a
portion of the protoplast through the laser made incision in the cell wall.
Кључне речи:
Cell surgery; Ti:SapphireФинансирање / пројекти:
- Генерисање и карактеризација нанофотонских функционалних структура у биомедицини и информатици (RS-MESTD-Integrated and Interdisciplinary Research (IIR or III)-45016)
У:
- Majaron H, Cotar P, Koren M, Golmajer Zima N, editors. Book of abstracts: 8th Regional Biophysics Conference: RBC2018; 2018 May 16-20; Zreče, Slovenia. Ljubljana: Slovenian Biophysical Society; 2018. p. PS-43.
URI
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