INFORMATION TECHNOLOGY

Biophotofluidics in quasi-thermostatic environments

Recently, Professor Moritz Kreysing from the Max Planck Institute of Molecular Cell Biology and Genetics and the Karlsruhe Institute of Technology, and Professor Eric Lauga of the University of Cambridge, collaborated to achieve the directional flow of nematode embryos in the system under a quasi-thermostatic distribution field by using symmetry relationships to decouple the relationship between laser heating and heat flow guidance during laser scanning. The ISO-FLUCS technology is expected to become the new standard for optical manipulation in temperature-sensitive systems in biology and materials science.

The article was published in the eLight issue of the Excellence Program under the title “ISO-FLUCS: Symmetrization of optofluidic manipulations in quasi-isothermal micro-environments.”

Micro-nano manipulation technology is widely used in materials science, colloidal physics and life sciences. Optically guided thermoviscous flow, also known as focused light-guided cytoplasmic flow (FLUCS), is one of the micro-nano manipulation technologies that has attracted much attention in recent years. Its principle is based on the thermoviscous flow generated by the complex interaction between thermal expansion and temperature-induced viscosity changes, resulting in a change in fluid density and viscosity through local heating generated by infrared laser focusing that irradiates the sample, thereby triggering a local liquid flow as well as a transport process for manipulating the cytoplasm of cells and developing embryos. FLUCS has important applications for biomedical research because it has the advantage of generating directed flow and reducing invasive and direct contact control. However, in FLUCS, local laser scanning can cause temperature perturbations of biological samples (Figure 1b), which may have a large impact on highly thermally sensitive systems such as heat-sensitive mammalian cells, thermally degradable oocytes, thermally regulated gill tissue, etc., thus greatly limiting the application of FLUCS.

In this paper, the authors investigate the use of step-by-step scanning to separate the flow and heat generated by laser guidance. Additional opposing paths are introduced in the symmetrical distribution around the desired trajectory, resulting in a more uniform temperature distribution within the region of interest (ROI, i.e., the region of interest). Further, the authors also scan at as many as three different scales (i.e., three laser scan lines) based on the symmetry relationship described above, which can result in a more uniform local temperature distribution while also producing directional flows with essentially no change in the path of these flows (Figure 1c). Peltier is also used externally to cool the sample to its desired temperature. The authors experimentally demonstrated that this technique, called isothermal FLUCS (ISO-FLUCS), significantly reduces the effect of laser heating, and at the same time can achieve the thermal viscosity directional flow of nematode embryos, and far exceed the speed of their endogenous flow, verifying the effect of the system. Given that it not only greatly reduces the effects of uneven temperature distribution due to heating, but also retains the main characteristics of FLUCS (directional, versatile, non-invasive, tactile), ISO-FLUCS is expected to become the new standard for optical manipulation in high-temperature sensitive systems in biology and materials science.

Figure 1: a. The optical path of the FLUCS device enables simultaneous imaging and directing the flow of cells through the liquid. The infrared laser beam that directs the flow of cells is X/Y scanned by an acousto-optic deflector (AOD) and irradiated onto a dichroic mirror, thus binding to the imaging optical path. Both are coupled to the back focal plane of a high numerical aperture, custom-coated microscope objective. Laser irradiation within the ROI causes the temperature to rise and produce directional flow. At the same time, the ROI heats or cools the entire sample through a Peltier element attached to a highly conductive sapphire slide. b. Simulated temperature distribution generated by repeated scanning of infrared laser along a single line. The two-step scanning mode uniformly heats the simulated temperature distribution of the entire ROI.

In order to achieve the performance of conventional FLUCS without creating large temperature gradients, it is necessary to decouple temperature from the introduction of thermovisus streams. One way to achieve this is to create additional scanning paths to homogenize the temperature over a larger area. The speed of the thermoviscous flow depends primarily on how often the laser scans along a particular path, not on how quickly the laser heats up. Thus, the main scan mode can be accelerated (i.e. in the direction of the directional flow rate) and the scan signal can be compressed to occupy only a small part of the original area. This effective reduction in duty cycle will provide space for the introduction of secondary scan points, allowing heating to spread over a larger area within a given duty cycle, effectively homogenizing the temperature.

Guided by the above basic protocol, the authors first explored the mid-infrared fluorescence excitation in single-laser hotspot scanning mode and the intensity and temperature distribution due to the spiral pattern (Figure 2a-d). The authors then focused on the effect between the resolution of multiple hot spots and the resulting temperature distribution in the case of multiple laser heating points, and when the specific parameters of the light spot were not taken into account, it was finally concluded that the optimal distance between the two heating points was 5.6 microns (Figure 2e-f).

The next step is to design a temporal scan sequence that requires (1) all the points required to be uniformly heated at the same frequency during a scan cycle, and (2) still be able to direct the desired directional flow field. It should also be noted that the frequency of the scan needs to be high enough that the time delay between visits to the same point in two consecutive periods is significantly lower than the characteristic timescale of the thermoviscosity phenomenon. Therefore, the authors propose a simple ISO-FLUCS pattern consisting of seven parallel equidistant lines (Figure 3) and guide the directional flow field only on the middle line.

Figure 2: a. Time-average fluorescence image of rhodamine B when exposed to infrared laser. Temperature profile measured by fluorescence of rhodamine B in glycerol/aqueous solution. c. Simulated temperature distribution caused by the spiral pattern and d corresponding temperature histogram. e. Simulated temperature distribution resulting from two hot spots placed at different separation distances. The corresponding intensity cross-section shows a relatively uniform temperature distribution within the ROl at a separation distance of approximately 5.6 microns. The standard deviation of the temperature distribution measured within ROl in f panel e as a function of the separation distance between hot spots, indicates that the optimal distance is 5.6 μm. Simulated temperature distribution caused by spiral pattern in multiple hot spot cases and h-corresponding temperature histogram.

Figure 3: Diagram of the time scanning unit used to compose the ISO-FLUCS pattern.

To determine whether the performance of ISO-FLUCS can match that of conventional FLUCS, the authors performed experimental verification (Figure 4). Based on the distribution of particle trajectories and fields in the experiment (Figure 4a, e), it can be found that the heating effect of the outer quadrupole (four scan lines) in ISO-FLUCS is almost completely suppressed, and a field symmetry similar to unipolar heating is generated. The velocity of the particles in the field was also measured (Figure 4b,f), and it was found that the velocity of the particles in ISO-FLUCS was lower because the energy on the scan line after averaging was low, but the speed could be easily adjusted by the power of the laser (Figure 4i, j). Finally, to illustrate the superiority of ISO-FLUCS, a significant homogenization of the temperature range within the ROI can be found compared to conventional FLUCS (Figure 4c, d, g, h, k, l).

In addition, the authors explore more complex scanning modes while retaining the characteristics of ISO-FLUCS. And experimentally manipulated the cytoplasm of nematodes in vivo. This further verifies the superiority of ISO-FLUCS and provides a new way to manipulate temperature-sensitive samples. (Source: China Optics WeChat public account)

Figure 4: ISO-FLUCS reduces the effects of temperature while still directing thermoviscous flow.

Related paper information:https://doi.org/10.1186/s43593-023-00049-z

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