Pros and cons of characterising an optical translocation setup

Charles Maphanga; Rudzani Malabi; Saturnin Ombinda-Lemboumba; Malik Maaza; Patience Mthunzi-Kufa

doi:10.1117/12.2252426

15 February 2017 Pros and cons of characterising an optical translocation setup

Charles Maphanga, Rudzani Malabi, Saturnin Ombinda-Lemboumba, Malik Maaza, Patience Mthunzi-Kufa

Proceedings Volume 10062, Optical Interactions with Tissue and Cells XXVIII; 100621A (2017) https://doi.org/10.1117/12.2252426
Event: SPIE BiOS, 2017, San Francisco, California, United States

Abstract

The delivery of genetic material and drugs into mammalian cells using femtosecond (fs) laser pulses is escalating rapidly. This novel light based technique achieved through a precise focusing of a laser beam on the plasma membrane is called photoporation. This technique is attained using ultrashort laser pulses to irradiate plasma membrane of mammalian cells, thus resulting in the accumulation of a vast amount of free electrons. These generated electrons react photochemically with the cell membrane, resulting in the generation of sub-microscopic pores on the cell membrane enabling a variety of extracellular media to diffuse into the cell. This study is aimed at critically analysing the “do’s and don’ts” of designing, assembling, and characterising an optical translocation setup using a femtosecond legend titanium sapphire regenerative amplifier pulsed laser (Gaussian beam, 800 nm, 1 kHz, 113 fs, and an output power of 850 mW). The main objective in our study is to determine optical phototranslocation parameters which are compatible to the plasma membrane and cell viability. Such parameters included beam profiling, testing a range of laser fluencies suitable for photoporation, assessment of the beam quality and laser-cell interaction time. In our study, Chinese Hamster Ovary-K1 (CHO-K1) cells were photoporated in the presence of trypan blue to determine optimal parameters for photoporation experiment. An average power of 4.5 μW, exposure time of 7 ms, with a laser beam spot of ~1.1 μm diameter at the focus worked optimally without any sign of cell stress and cytoplasmic bleeding. Cellular responses post laser treatment were analysed using cell morphology studies.

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Charles Maphanga, Rudzani Malabi, Saturnin Ombinda-Lemboumba, Malik Maaza, and Patience Mthunzi-Kufa "Pros and cons of characterising an optical translocation setup", Proc. SPIE 10062, Optical Interactions with Tissue and Cells XXVIII, 100621A (15 February 2017); https://doi.org/10.1117/12.2252426

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KEYWORDS

Femtosecond phenomena

Objectives

Camera shutters

Microscopes

Plasma

Mirrors

Pulsed laser operation

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