Page 88 - IJB-9-4

International Journal of Bioprinting Laser transfer for CTC isolation

Figure 3. Optical set-up for laser transfer and cell identification and selection. Our arrangement combines laser irradiation for BA-LIFT process, in situ
video recording and analysis of the sample fluorescence. The focusing optics are common to the different optical systems.
Focusing optics were fixed and the beam was scanned path, bright field microscopy and fluorescence microscopy
along the sample by moving the sample using lineal servo are combined by means of appropriate dichroic mirrors.
XY axes (Parker) with an accuracy of 1 µm. The distance As it has been previously detailed, the laser source used for
2
between the sample and the laser beam focal point was BA-LIFT is a UV, ns-pulsed laser, delivering 3.5 J/cm laser
controlled using a manual micrometric Z stage with and pulses, in terms of fluence, at the sample.
accuracy of 10 µm. The laser was fired directly to the The microscope objective used for focusing the laser
location of the desired cell, which was centered in the beam onto the samples was also used for obtaining in situ,
vision system; in that way, the possible mechanical damage real-time microscope images of the processed sample with
induced by the shear stress in the edges of the affected two independent CMOS cameras, with one recording
volume of liquid during jet formation can be minimized.
bright field B/W images (Thorlabs DCC1545M) and the
Laser pulse energy of 5 µJ is typically used for other recording fluorescence images (Andor Zyla 5.5). UV
transferring cells with single laser pulses, resulting in a laser and visible microscopy optical paths are combined
mean laser fluence of 3.5 J/cm . With these parameters, by means of a UV dichroic mirror just before the focusing
2
the volume of the transferred droplet containing the cell optics.
is around 50 pL.
A LED source (Thorlabs M565L3) emitting at a
wavelength range from 475 to 750 nm with peak intensity
3.7. Fluorescence system at 550 nm was used for illuminating the sample. LED
In our experiments, the optical set-up used combines laser light was initially filtered using a single band exciter
irradiation for LIFT process, in situ video recording and filter (Semrock FF01-531/40) with high transmission
analysis of the sample fluorescence (Figure 3). The focusing from 500 to 550 nm and a fluorescence dichroic mirror
optics are common to the different optical systems and (Semrock FF562-Di03) cutting at 562 nm. The reflected
consist of a 10× microscope objective, while the laser beam light returning from the sample was divided in two

Volume 9 Issue 4 (2023) 80 https://doi.org/10.18063/ijb.720