Thermophiles, heat-loving microorganisms including bacteria and archaea, thrive at temperatures ranging from 45°C to 122°C, inhabiting environments such as hydrothermal vents and hot springs. Their study is crucial for understanding adaptation to extreme conditions and for biotechnological applications, including fuel generation, chemical synthesis, biomining, and the production of thermostable biocatalysts. A prime example is Taq polymerase from *Thermus aquaticus*, essential for PCR. However, studying thermophiles *in vitro* presents challenges, as standard optical microscopes are not equipped for high-temperature observation. Existing high-temperature microscopy (HTM) systems, often custom-built, suffer from limitations such as poor spatial resolution, slow heating, and the risk of damaging the microscope's components. Previous approaches involved resistive heating of capillaries or chambers, sometimes requiring heating the objective lens as well. These limitations highlight the need for a more efficient and accessible HTM system that overcomes these challenges.

Several research groups have attempted to address the limitations of high-temperature microscopy since the 1990s. Gluch et al. (1994) developed a heating/cooling chamber using Peltier cells to control the temperature of a sealed capillary, enabling the study of *Thermotoga maritima* motility. Horn et al. (1999) used a similar heated capillary system for investigating cell division. Wirth's group, starting in 2012, further advanced this approach, enabling faster heating and creating temperature gradients. Other methods included using disposable Pyrex chambers on a hot plate (Kuwabara et al., 2012) and metallic dishes with custom-made heating elements (Pulschen et al., 2020). Charles-Orszag et al. (2021) improved upon this by heating the coverslip itself with a resistive layer, but still required objective lens heating. While these methods achieved high-temperature observation, they often compromised spatial resolution, featured slow heating, and required extensive modifications or custom-built equipment.

This study introduces a novel laser-assisted high-temperature microscopy (LA-HTM) technique. Instead of resistive heating, LA-HTM uses a laser to locally heat a sample within the microscope's field of view. To achieve efficient heating with moderate laser power (less than 100 mW), the substrate is coated with gold nanoparticles, which act as efficient light absorbers. The resulting temperature distribution is precisely mapped using quantitative phase microscopy (QPM), specifically cross-grating wavefront microscopy (CGM). To address potential issues such as microscale fluid convection, cell confinement, and thermophoretic motion, the bacterial suspensions are sandwiched between coverslips (approximately 15 µm thick), with a hole drilled in the top coverslip to allow gas exchange. A spatial light modulator (SLM) shapes the infrared laser beam to create a uniform temperature profile within the target area, eliminating undesired temperature gradients and bacterial movement due to thermophoresis. The technique was tested on *Geobacillus stearothermophilus* and *Sulfolobus shibatae*. *G. stearothermophilus* samples were pre-incubated at 60°C for an hour to facilitate immediate growth upon laser activation. The growth and motility of *G. stearothermophilus* and *S. shibatae* were monitored over several hours using CGM, which simultaneously measures temperature and biomass. The Cardinal Model was used to fit the growth rate data of *G. stearothermophilus*, while the growth rate of *S. shibatae* was observed to be linear.

LA-HTM successfully achieved microscale temperature control and allowed for the observation of various aspects of thermophilic microbial life. Using *G. stearothermophilus*, the study demonstrated that the LA-HTM enabled observation of active bacterial growth and motility at the optimal growth temperature of 65°C. A flat temperature profile of 65°C was achieved using a ring-like laser beam shaped by the SLM. Growth at different temperatures followed an exponential increase, with an optimal growth rate around 60-65°C, consistent with literature values. The LA-HTM also enabled the observation of bacterial swimming behavior, distinguishing it from Brownian motion, convection, and thermophoresis. Furthermore, the researchers observed for the first time the real-time germination of *G. stearothermophilus* spores, which germinated independently at different times. The growth rate of *G. stearothermophilus* from spores was higher than that of normally growing cells. Applying LA-HTM to *S. shibatae*, a hyperthermophile, revealed a surprising linear growth pattern at 73°C, which might be due to cell inhibition resulting from close packing. The CGM technique’s high precision allowed for a reliable distinction between exponential and linear growth.

The LA-HTM offers several advantages over previous HTM techniques. The microscale heating reduces thermal inertia, enabling rapid temperature changes and precise time control. The confinement of heating to a small volume allows for localized observation of microorganisms while leaving surrounding cells dormant for further investigation. The use of gold nanoparticles as light absorbers ensures biocompatibility, chemical inertness, and tunable absorbance. CGM provides simultaneous temperature and biomass measurements, avoiding the limitations and potential artifacts associated with fluorescence-based thermometry. The ability to study *S. shibatae*, a hyperthermophile, demonstrates LA-HTM's potential for exploring a wider range of thermophiles and potentially exceeding 100°C. The observed linear growth of *S. shibatae* highlights the technique's ability to uncover unexpected biological phenomena.

This study demonstrates the potential of LA-HTM as a powerful and versatile tool for studying thermophiles. LA-HTM provides a significant improvement over existing techniques due to its simplicity, speed, and high spatial resolution. The ability to observe various biological processes, including growth, motility, and germination, under precisely controlled temperatures opens up new opportunities for investigating thermophile biology and uncovering novel biological phenomena. Future research directions may include exploring the high-temperature limits of LA-HTM, investigating different thermophilic species, and studying the impact of various environmental factors on thermophile biology.

The main limitation of LA-HTM is the potential for non-adherent or motile cells to move out of the field of view. However, this can be partially mitigated using lower magnification objectives, though this would reduce spatial resolution. The use of pre-incubation for *G. stearothermophilus* might slightly influence the initial growth kinetics. Further, the observed linear growth in *S. shibatae* needs further investigation to fully understand the underlying mechanisms. Future studies will need to focus on minimizing these limitations to improve the robustness of LA-HTM experiments.