093 Ultra rapid warming of cryo samples using an IR laser pulse

Abstract

Our laboratory has been investigating the effect of warming rate on survival of mouse oocytes after very rapid cooling. Cooling and warming rates are affected by sample thermal mass, surface area, thermal impedance, and delta T. To maximize these rates we use 0.1 ul samples placed on Cryotops and plunged directly into LN2 for cooling and into room temperature buffer for warming. The maximum warming rate obtained is ∼ 100,000 C/min and it yields the highest survivals. However, extrapolation of the experimental data suggests faster warming would yield higher survivals. Here we report on the use of an IR laser pulse to achieve warming rates 3.5 to 100 times faster. Our objective was to choose a laser wavelength which would warm the cryo buffer while leaving the cells ‘untouched’, thus leading to indirect warming of the cells. Optical tweezer studies show that a wide variety of cell types can tolerate very high intensities of 1064 nm IR energy emitted by Nd:YAG lasers and so this was the type chosen. Calculations indicate the laser intensity for cryo melting to be significantly less than that used for optical tweezers. Most cryo buffers, including the EAFS we use are relatively transparent in the IR. The buffer IR absorption is easily increased by adding a little India ink. India ink consists primarily of carbon particles from 0.1 to 1 um in diameter, and at the concentration we use, 0.25% (V/V), is harmless to the oocytes. The concentration is carefully chosen to allow most of the IR energy to pass completely through the sample. This yields relatively even sample heating from front to back. We estimate ink-free samples absorb ∼ 9% of the incident energy, and the added ink absorbs ∼ 25% of the remaining energy, yielding a total absorption of ∼ 30%. We have developed a small cryo ‘jig’ which lifts the sample out of an LN2 bath, covers the bath, and fires a laser pulse, all in less than one sec. This assures that the sample is rapidly warmed by the laser pulse rather than by slower air warming. Thus, the samples are warmed from ∼ −180°C to ∼ 0°C by the laser pulse. We have computed the required laser energy for melting, but there are so many variables that we have found it preferable to empirically determine the required laser energy to just melt the samples for a given experiment. This is done by direct visual observation with a 15 x stereo microscope built into the laser system. We use a 40 joule welding laser (for long pulse durations!) with a beam diameter of 2 mm and a pulse length of 0.5 to 30 msec. This yields warming rates from 3.5 x 10 5 to 2 x 10 7 C/min. However, modeling indicates that rates higher than 1 x 10 7 C/min yield unacceptably large thermal gradients across the 70 um diameter oocytes and are not used. Preliminary experiments at a warming rate of 3.5 x 10 5 C/min yield survivals comparable to our traditional methods. Source of Funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. E. Paredes FPU Research Stays Fellowship from the Spanish Government, 1 November 2012-31 January 2013. Conflict of Interest: None declared. fkleinha@iupui.edu