Abstract
Combined two-photon fluorescence microscopy and femtosecond laser microsurgery has many potential biomedical applications as a powerful "seek-and-treat" tool. Towards developing such a tool, we demonstrate a miniaturized probe which combines these techniques in a compact housing. The device is 10 x 15 x 40 mm(3) in size and uses an aircore photonic crystal fiber to deliver femtosecond laser pulses at 80 MHz repetition rate for imaging and 1 kHz for microsurgery. A fast two-axis microelectromechanical system scanning mirror is driven at resonance to produce Lissajous beam scanning at 10 frames per second. Field of view is 310 microm in diameter and the lateral and axial resolutions are 1.64 microm and 16.4 microm, respectively. Combined imaging and microsurgery is demonstrated using live cancer cells.
Highlights
In recent years, femtosecond laser microsurgery (FLMS) has emerged as a superior technique for ablation of cells and subcellular structures and offers the highest precision for microsurgery in three-dimensional (3D) tissue [1,2,3,4]
The major components of the probe are an air-core photonic crystal fiber (PCF) (Fig. 1(c)), two-axis microelectromechanical systems (MEMS) scanning mirror (Fig. 1(d)), miniature relay lens system, and gradient index (GRIN) objective lens. These components provide the advantages of compact size and the ability to handle high peak intensity laser pulses to enable FLMS and two-photon microscopy (TPM) in a miniaturized system
This design uses the same optical pathways for both microsurgery and fluorescence excitation, providing visualization and guidance at the exact location of ablation. Though several of these optical components have been utilized in previous miniature two-photon microscope designs, the incorporation of amplified high peak-intensity pulses for microsurgery led to a design that enables FLMS, but it improves imaging capabilities as well
Summary
Femtosecond laser microsurgery (FLMS) has emerged as a superior technique for ablation of cells and subcellular structures and offers the highest precision for microsurgery in three-dimensional (3D) tissue [1,2,3,4]. Femtosecond lasers require much less energy for ablation and lead to significantly less heating of surrounding tissue (especially for repetition rates < 1 MHz) when compared to ablation with nanosecond or longer duration laser pulses [3, 5] Owing to these advantages, FLMS has been gradually moving from the laboratory to the physician’s office, most notably in ophthalmology, where femtosecond laser systems produced by IntraLase Corp. The use of femtosecond lasers for both imaging and manipulation of biological samples has been demonstrated in laboratory settings using large table-top systems [12,13,14,15,16] This combined tool can be used for diagnosis and treatment of various diseases as well as for in vivo monitoring of disease progression
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