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Thermophiles are microorganisms that thrive at high temperatures. Studying them can provide valuable information about how life adapts to extreme conditions. However, it is difficult to achieve high temperature conditions with conventional optical microscopes. Several home-made solutions based on local resistive electrical heating have been proposed, but there is no simple commercial solution. In this paper, we introduce the concept of microscale laser heating over the microscope field of view to provide high temperatures for thermophile studies while keeping the user’s environment mild. Microscale heating at moderate laser intensity can be achieved using a gold nanoparticle coated substrate as a biocompatible and efficient light absorber. Possible effects of microscale fluid convection, cell retention, and centrifugal thermophoretic motion are discussed. The method has been demonstrated in two species: (i) Geobacillus stearothermophilus, an active thermophilic bacterium that reproduces at about 65°C, which we have observed to germinate, grow and swim under microscale heating; (ii) Thiobacillus sp., an optimally hyperthermophilic archaea. at 80°C. This work paves the way for simple and safe observation of thermophilic microorganisms using modern and affordable microscopy tools.
Over billions of years, life on Earth has evolved to adapt to a wide range of environmental conditions that are sometimes considered extreme from our human perspective. In particular, some thermophilic microorganisms (bacteria, archaea, fungi) called thermophiles thrive in the temperature range from 45°C to 122°C1, 2, 3, 4. Thermophiles live in various ecosystems, such as deep sea hydrothermal vents, hot springs or volcanic areas. Their research has generated a lot of interest over the past few decades for at least two reasons. First, we can learn from them, for example, how thermophiles 5, 6, enzymes 7, 8 and membranes 9 are stable at such high temperatures, or how thermophiles can withstand extreme levels of radiation10. Second, they are the basis for many important biotechnological applications1,11,12 such as fuel production13,14,15,16, chemical synthesis (dihydro, alcohols, methane, amino acids, etc.)17, biomining18 and thermostable biocatalysts7 ,11,13. In particular, the currently well-known polymerase chain reaction (PCR)19 involves an enzyme (Taq polymerase) isolated from the thermophilic bacterium Thermus aquaticus, one of the first thermophiles to be discovered.
However, the study of thermophiles is not an easy task and cannot be improvised in any biological laboratory. In particular, living thermophiles cannot be observed in vitro with any standard light microscope, even with commercially available heating chambers, usually rated for temperatures as low as 40°C. Since the 1990s, only a few research groups have dedicated themselves to the introduction of high-temperature microscopy (HTM) systems. In 1994 Glukh et al. The heating/cooling chamber was conceived based on the use of a Peltier cell that controls the temperature of rectangular capillaries closed to maintain anaerobicity 20 . The device can be heated up to 100 °C at a rate of 2 °C/s, allowing the authors to study the motility of the hyperthermophilic bacterium Thermotoga maritima21. In 1999 Horn et al. A very similar device has been developed, still based on the use of heated capillaries suitable for commercial microscopy to study cell division/connection. After a long period of relative inactivity, the search for effective HTMs resumed in 2012, in particular in connection with a series of papers by the Wirth group that used a device invented by Horn et al. Fifteen years ago, the motility of a large number of archaea, including hyperthermophiles, was studied at temperatures up to 100°C using heated capillaries23,24. They also modified the original microscope to achieve faster heating (several minutes instead of 35 minutes to reach the set temperature) and achieve a linear temperature gradient of more than 2 cm across the medium. This temperature gradient shaping device (TGFD) has been used to study the mobility of many thermophiles within temperature gradients at biologically relevant distances 24, 25 .
Heating closed capillaries is not the only way to observe live thermophiles. In 2012, Kuwabara et al. Homemade disposable Pyrex chambers sealed with heat-resistant adhesive (Super X2; Cemedine, Japan) were used. The samples were placed on a commercially available transparent heating plate (Micro Heat Plate, Kitazato Corporation, Japan) capable of heating up to 110°C, but not originally intended for bioimaging. The authors observed efficient division of anaerobic thermophilic bacteria (Thermosipho globiformans, doubling time 24 min) at 65°C. In 2020, Pulshen et al. Efficient heating of commercial metal dishes (AttofluorTM, Thermofisher) was demonstrated using two homemade heating elements: a lid and a stage (PCR machine-inspired configuration). This association results in a uniform liquid temperature and prevents evaporation and condensation at the bottom of the lid. The use of an O-ring avoids gas exchange with the environment. This HTM, called the Sulfoscope, was used to image Sulfolobus acidocaldarius at 75°C27.
A recognized limitation of all these systems was the restriction to the use of air objectives, any oil immersion being unsuited for such high temperature and for imaging through >1-mm thick transparent samples. A recognized limitation of all these systems was the restriction to the use of air objectives, any oil immersion being unsuited for such high temperature and for imaging through >1-mm thick transparent samples. Общепризнанным недостатком всех этих систем было ограничение на использование воздушных объективов, поскольку любое иммерсионное погружение в масло не подходило для такой высокой температуры и для визуализации через прозрачные образцы толщиной > 1 мм. A recognized shortcoming of all these systems was the limitation to the use of air objectives, since any oil immersion was not suitable for such high temperature and for visualization through transparent samples > 1 mm thick.所有这些系统的一个公认限制是限制使用空气物镜，任何油浸都不适合这样的高温和通过> 1 毫米厚的透明样品成像。 A recognized limitation of all these systems is the limitation of using an air-entrained mirror, as any oil immersion is unsuitable for imaging transparent samples >1 mm thick at such high temperatures. Общепризнанным недостатком всех этих систем является ограниченное использование воздушных объективов, любое иммерсионное погружение в масло непригодно для таких высоких температур и визуализации через прозрачные образцы толщиной >1 мм. A recognized drawback of all these systems is the limited use of air lenses, any oil immersion is unsuitable for such high temperatures and visualization through transparent samples >1 mm thick. More recently, this limitation was lifted by Charles-Orzag et al. 28, who developed a device that no longer provides heat around the system of interest, but rather inside the cover glass itself, covered with a thin transparent layer of a resistor made of ITO (indium-tin oxide). The lid can be heated up to 75 °C by passing an electric current through the transparent layer. However, the author must also heat the lens to the objective, but not more than 65 °C, so as not to damage it.
These works show that the development of efficient high-temperature optical microscopy has not been widely adopted, often requires homemade equipment, and is often achieved at the cost of spatial resolution, which is a serious disadvantage given that thermophilic microorganisms are no larger than a few micrometers. Reduced heating volume is the key to solving three inherent problems of HTM: poor spatial resolution, high thermal inertia when the system heats up, and harmful heating of surrounding elements (immersion oil, objective lens… or user’s hands) at extreme temperatures. ).
In this paper, we introduce an HTM for thermophile observation that is not based on resistive heating. Instead, we achieved localized heating within a limited region of the microscope’s field of view by laser irradiation of a light-absorbing substrate. The temperature distribution was visualized using quantitative phase microscopy (QPM). The effectiveness of this method is demonstrated by Geobacillus stearothermophilus, a motile thermophilic bacterium that reproduces at about 65°C and has a short doubling time (about 20 minutes), and Sulfolobus shibatae, a hyperthermophile that grows optimally at 80°C (archaea) to illustrate. Normal replication rate and swimming were observed as a function of temperature. This laser HTM (LA-HTM) is not limited by the thickness of the coverslip or by the nature of the objective (air or oil immersion). This allows any high resolution lens on the market to be used. It also does not suffer from slow heating due to thermal inertia (achieves instant heating on a millisecond scale) and uses only commercially available components. The only new safety concerns are related to the presence of powerful laser beams (typically up to 100 mW) inside the device and possibly through the eyes, which require protective goggles.
The principle of LA-HTM is to use a laser to heat the sample locally within the field of view of the microscope (Fig. 1a). To do this, the sample must be light-absorbing. To use a reasonable laser power (less than 100 mW), we did not rely on the absorption of light by the liquid medium, but artificially increased the absorption of the sample by coating the substrate with gold nanoparticles (Fig. 1c). Heating gold nanoparticles with light is of fundamental importance to the field of thermal plasmonics, with expected applications in biomedicine, nanochemistry or sunlight harvesting29,30,31. Over the past few years, we have used this LA-HTM in several studies related to thermal plasma applications in physics, chemistry and biology. The main difficulty with this method is in displaying the final temperature profile, since the elevated temperature is limited to a microscale region within the sample. We have shown that temperature mapping can be achieved with the four-wavelength transverse shear interferometer, a simple, high-resolution, and very sensitive method of quantitative phase microscopy based on the use of two-dimensional diffraction gratings (also known as cross gratings) 33,34,35,36. The reliability of this thermal microscopy technique, based on crossed grating wavefront microscopy (CGM), has been demonstrated in a dozen papers published over the past decade37,38,39,40,41,42,43.
Scheme of installation of parallel laser heating, shaping and temperature microscope. b Sample geometry consisting of an AttofluorTM chamber containing a coverslip coated with gold nanoparticles. c Look closely at the sample (not to scale). d represents the uniform laser beam profile and (e) the simulated subsequent temperature distribution on the sample plane of the gold nanoparticles. f is an annular laser beam profile suitable for generating a uniform temperature as shown in the simulation of the resulting temperature distribution shown in (g). Scale bar: 30 µm.
In particular, we recently achieved heating of mammalian cells with LA-HTM and CGM and tracked cellular heat shock responses in the range of 37-42°C, demonstrating the applicability of this technique to single living cell imaging. However, the application of LA-HTM to the study of microorganisms at high temperatures is not unambiguous, as it requires more caution compared to mammalian cells: firstly, heating the bottom of the medium by tens of degrees (rather than a few degrees) leads to a strong vertical temperature gradient. can create fluid convection 44 which, if not firmly attached to the substrate, can cause undesirable movement and mixing of bacteria. This convection can be eliminated by reducing the thickness of the liquid layer. For this purpose, in all the experiments presented below, bacterial suspensions were placed between two coverslips approximately 15 µm thick placed inside a metal cup (AttofluorTM, Thermofisher, Fig. 1b,c). In principle, convection can be avoided if the thickness of the liquid is smaller than the beam size of the heating laser. Secondly, working in such a limited geometry can suffocate aerobic organisms (see Fig. S2). This problem can be avoided by using a substrate that is permeable to oxygen (or any other vital gas), by leaving trapped air bubbles inside the coverslip, or by drilling holes in the top coverslip (see Fig. S1) 45 . In this study, we chose the latter solution (Figures 1b and S1). Finally, laser heating does not provide uniform temperature distribution. Even at the same intensity of the laser beam (Fig. 1d), the temperature distribution is not uniform, but rather resembles the Gaussian distribution due to thermal diffusion (Fig. 1e). When the goal is to establish precise temperatures in the field of view for studying biological systems, uneven profiles are not ideal and can also lead to thermophoretic movement of bacteria if they do not adhere to the substrate (see Fig. S3, S4)39. To this end, we used a spatial light modulator (SLM) to shape the infrared laser beam according to the shape of the ring (Fig. 1f) in the plane of the sample to achieve a perfectly uniform temperature distribution within a given geometric area, despite thermal diffusion (Fig. 1d) 39 , 42, 46. Place a top coverslip over a metal dish (Figure 1b) to avoid evaporation of the medium and observe for at least a few days. Because this top coverslip is not sealed, additional medium can be easily added at any time if necessary.
To illustrate how LA-HTM works and demonstrate its applicability in thermophilic research, we studied the aerobic bacteria Geobacillus stearothermophilus, which have an optimum growth temperature of around 60-65°C. The bacterium also has flagella and the ability to swim, providing another indicator of normal cellular activity.
Samples (Fig. 1b) were pre-incubated at 60°C for one hour and then placed in an LA-HTM sample holder. This pre-incubation is optional, but still useful, for two reasons: First, when the laser is turned on, it causes the cells to immediately grow and divide (see movie M1 in Supplementary Materials). Without pre-incubation, bacterial growth is typically delayed by about 40 minutes each time a new viewing area is heated on the sample. Second, the 1 hour pre-incubation promoted adhesion of the bacteria to the coverslip, preventing cells from drifting out of the field of view due to thermophoresis when the laser was turned on (see film M2 in Supplementary Materials). Thermophoresis is the movement of particles or molecules along a temperature gradient, usually from hot to cold, and bacteria are no exception43,47. This undesirable effect is eliminated over a given area by using SLM to shape the laser beam and achieve a flat temperature distribution.
On fig. Figure 2 shows the temperature distribution measured by CGM obtained by irradiating a glass substrate coated with gold nanoparticles with an annular laser beam (Fig. 1f). A flat temperature distribution was observed over the entire area covered by the laser beam. This zone was set to 65°C, the optimal growth temperature. Outside this region, the temperature curve naturally falls to \(1/r\) (where \(r\) is the radial coordinate).
a Temperature map of CGM measurements obtained by using an annular laser beam to irradiate a layer of gold nanoparticles to obtain a flat temperature profile over a circular area. b Isotherm of the temperature map (a). The contour of the laser beam is represented by a gray dotted circle. The experiment was repeated twice (see Supplementary Materials, Figure S4).
The viability of bacterial cells was monitored for several hours using LA-HTM. On fig. 3 shows the time interval for four images taken from a 3 hour 20 minute movie (Movie M3, Supplementary Information). Bacteria were observed to actively proliferate within the circular area defined by the laser where the temperature was optimal, approaching 65°C. In contrast, cell growth was significantly reduced when the temperature fell below 50°C for 10 s.
Optical depth images of G. stearothermophilus bacteria growing after laser heating at different times, (a) t = 0 min, (b) 1 h 10 min, (c) 2 h 20 min, (d) 3 h 20 min, out of 200 Extracted from a one-minute film (M3 film provided in Supplementary Information) superimposed on the corresponding temperature map. The laser turns on at time \(t=0\). Isotherms have been added to the intensity image.
To further quantify cell growth and its dependence on temperature, we measured the increase in biomass of various colonies of initially isolated bacteria in the Movie M3 field of view (Fig. 4). The parent bacteria selected at the start of mini colony forming unit (mCFU) formation are shown in Figure S6. Dry mass measurements were taken with a CGM 48 camera which was used to map the temperature distribution. The ability of the CGM to measure dry weight and temperature is the strength of the LA-HTM. As expected, high temperature caused faster bacterial growth (Fig. 4a). As shown in the semi-log plot in Fig. 4b, growth at all temperatures follows exponential growth, where the data uses the exponential function \(m={m}_{0}{10}^{t/\ tau }+{{ \mbox{cst}}}\), where \(\tau {{{{{\rm{log }}}}}}2\) – generation time (or doubling time), \( g =1/\tau\) – growth rate (number of divisions per unit time ). On fig. 4c shows the respective growth rate and generation time as a function of temperature. Fast growing mCFUs are characterized by saturation of growth after two hours, an expected behavior due to high bacterial density (similar to the stationary phase in classical liquid cultures). The general shape \(g\left(T\right)\) (Fig. 4c) corresponds to the expected two-phase curve for G. stearothermophilus with an optimal growth rate around 60-65°C. Match the data using a cardinal model (Figure S5)49 where \(\left({{G}_{0}{;\;T}}_{{\min }};{T}_{{opt} } ;{T}_{{\max}}\right)\) = (0.70 ± 0.2; 40 ± 4; 65 ± 1.6; 67 ± 3) °C, which agrees well with other values cited in the literature49. Although the temperature dependent parameters are reproducible, the maximum growth rate of \({G}_{0}\) may vary from one experiment to another (see figures S7-S9 and movie M4). In contrast to temperature fitting parameters, which should be universal, the maximum growth rate depends on the properties of the medium (availability of nutrients, oxygen concentration) within the observed microscale geometry.
a Microbial growth at various temperatures. mCFU: Miniature Colony Forming Units. Data obtained from a video of a single bacterium growing in a temperature gradient (movie M3). b Same as (a), semi-logarithmic scale. c Growth rate\(\tau\) and generation time\(g\) calculated from linear regression (b). Horizontal error bars: temperature range over which mCFUs expanded into the field of view during growth. Vertical error bars: linear regression standard error.
In addition to normal growth, some bacteria sometimes floated into view during laser heating, which is an expected behavior for bacteria with flagella. The movie M5 in additional information shows such swimming activities. In this experiment, uniform laser radiation was used to create a temperature gradient, as shown in Figures 1d, e and S3. Figure 5 shows two image sequences selected from the M5 movie showing that one bacterium exhibits directional movement while all other bacteria remain motionless.
The two time frames (a) and (b) show the swimming of two different bacteria marked with dotted circles. The images were extracted from the M5 movie (provided as supplementary material).
In the case of G. stearothermophilus, the active movement of bacteria (Fig. 5) began a few seconds after the laser beam was turned on. This observation emphasizes the temporal response of this thermophilic microorganism to an increase in temperature, as already observed by Mora et al. 24 . The topic of bacterial motility and even thermotaxis can be further explored using LA-HTM.
Microbial swimming should not be confused with other types of physical motion, namely (i) Brownian motion, which appears to be chaotic motion with no definite direction, (ii) convection 50 and thermophoresis 43, consisting in a regular drift of motion along a temperature gradient.
G. stearothermophilus is known for its ability to produce highly resistant spores (spore formation) when exposed to adverse environmental conditions as a defense. When environmental conditions become favorable again, the spores germinate, forming living cells and resuming growth. Although this sporulation/germination process is well known, it has never been observed in real time. Using LA-HTM, we report here the first observation of germination events in G. stearothermophilus.
On fig. 6a shows time-lapse images of optical depth (OT) obtained using a CGM set of 13 spores. For the entire collection time (15 h 6 min, \(t=0\) – the beginning of laser heating), 4 out of 13 spores germinated, at successive time points \(t=2\) h, \( 3\ ) h \(10 \)’, \(9\) h \(40\)’ and \(11\) h \(30\)’. Although only one of these events is shown in Figure 6, 4 germination events can be observed in the M6 movie in the supplementary material. Interestingly, germination appears to be random: not all spores germinate and do not germinate at the same time, despite the same changes in environmental conditions.
a Time-lapse consisting of 8 OT images (oil immersion, 60x, 1.25 NA objective) and (b) biomass evolution of G. stearothermophilus aggregates. c (b) Drawn on a semi-log scale to highlight the linearity of the growth rate (dashed line).
On fig. 6b,c shows the biomass of cell populations in the field of view as a function of time over the entire period of data collection. The fast decay of the dry mass observed at \(t=5\)h in fig. 6b, c, due to the exit of some cells from the field of view. The growth rate of these four events is \(0.77\pm 0.1\) h-1. This value is higher than the growth rate associated with Figure 3. 3 and 4, where cells grow normally. The reason for the increased growth rate of G. stearothermophilus from spores is unclear, but these measurements highlight the interest of LA-HTM and work at the single cell level (or at the single mCFU level) to learn more about the dynamics of cell life.
To further demonstrate the versatility of LA-HTM and its performance at high temperatures, we examined the growth of Sulfolobus shibatae, a hyperthermophilic acidophilic archaea with an optimum growth temperature of 80°C51. Compared to G. stearothermophilus, these archaea also have a very different morphology, resembling 1 micron spheres (cocci) rather than elongated rods (bacilli).
Figure 7a consists of sequential optical depth images of S. shibatae mCFU obtained using CGM (see M7 feature film in Supplementary Materials). This mCFU grows at around 73°C, below the optimum temperature of 80°C, but within the temperature range for active growth. We observed multiple fission events that made mCFUs look like micrograpes of archaea after a few hours. From these OT images, mCFU biomass was measured over time and presented in Figure 7b. Interestingly, S. shibatae mCFUs showed linear growth rather than the exponential growth seen with G. stearothermophilus mCFUs. There has been a long-standing discussion 52 about the nature of cell growth rates: while some studies report growth rates of microbes that are proportional to their size (exponential growth), others show a constant rate (linear or bilinear growth). As explained by Tzur et al.53, distinguishing between exponential and (bi)linear growth requires a precision of <6% in biomass measurements, which is out of reach for most QPM techniques, even involving interferometry. As explained by Tzur et al.53, distinguishing between exponential and (bi)linear growth requires a precision of <6% in biomass measurements, which is out of reach for most QPM techniques, even involving interferometry. Как объяснили Цур и др.53, различение экспоненциального и (би)линейного роста требует точности <6% в измерениях биомассы, что недостижимо для большинства методов QPM, даже с использованием интерферометрии. As explained by Zur et al.53, distinguishing between exponential and (bi)linear growth requires <6% accuracy in biomass measurements, which is unattainable for most QPM methods, even using interferometry. As explained by Zur et al. 53, distinguishing between exponential and (bi)linear growth requires less than 6% accuracy in biomass measurements, which is unattainable for most QPM methods, even when interferometry is used. CGM achieves this accuracy with sub-pg accuracy in biomass measurements36,48.
a Time-lapse consisting of 6 OT images (oil immersion, 60x, NA objective 1.25) and (b) micro-CFU biomass evolution measured with CGM. See movie M7 for more information.
The perfectly linear growth of S. shibatae was unexpected and has not yet been reported. However, exponential growth is expected, at least because over time, multiple divisions of 2, 4, 8, 16 … cells must occur. We hypothesized that linear growth might be due to cell inhibition due to dense cell packing, just as cell growth slows down and eventually reaches a dormant state when cell density is too high.
We conclude by discussing the following five points of interest in turn: reduction in heating volume, reduction in thermal inertia, interest in gold nanoparticles, interest in quantitative phase microscopy, and a possible temperature range in which LA-HTM can be used.
Compared to resistive heating, laser heating used for HTM development offers several advantages, which we illustrate in this study. In particular, in liquid media in the field of view of the microscope, the heating volume is kept within a few (10 μm) 3 volumes. In this way, only the observed microbes are active, while other bacteria are dormant and can be used to further study the sample – there is no need to change the sample every time a new temperature needs to be checked. In addition, microscale heating allows direct examination of a large range of temperatures: Figure 4c was obtained from a 3-hour movie (Movie M3), which usually requires the preparation and examination of several samples – one for each of the samples under study. y is the temperature representing the number of days in the experiment. Reducing the heated volume also keeps all the surrounding optical components of the microscope, especially the objective lens, at room temperature, which has been a major problem faced by the community so far. LA-HTM can be used with any lens, including oil immersion lenses, and will stay at room temperature even with extreme temperatures in the field of view. The main limitation of the laser heating method that we report in this study is that cells that do not adhere or float may be far from the field of view and difficult to study. A workaround could be to use low magnification lenses to achieve a larger temperature rise in excess of a few hundred microns. This caution is accompanied by a decrease in spatial resolution, but if the goal is to study the movement of microorganisms, high spatial resolution is not required.
The time scale for heating (and cooling) the system \({{{{{\rm{\tau }}}}}}}}_{{{\mbox{D}}}}\) depends on its size , according to the law \({{{({\rm{\tau }}}}}}}_{{{\mbox{D}}}}={L}^{2}/D\), where \(L\ ) is the characteristic size of the heat source (the diameter of the laser beam in our study is \(L\ about 100\) μm), \(D\) is the thermal diffusivity of the environment (average in our case, glass and water Diffusion rate\(D\ about 2\fold {10}^{-7}\) m2/s). Therefore, in this study, time responses of the order of 50 ms, i.e., quasi-instantaneous temperature changes, can be expected. This instantaneous establishment of temperature rise not only shortens the duration of the experiment, but also allows precise timing \(t=0\) for any dynamic study of temperature effects.
Our proposed method is applicable to any light-absorbing substrate (for example, commercial samples with ITO coating). However, gold nanoparticles are able to provide high absorption in the infrared and low absorption in the visible range, the latter characteristics of which are of interest for effective optical observation in the visible range, especially when using fluorescence. In addition, gold is biocompatible, chemically inert, optical density can be adjusted from 530 nm to near infrared, and sample preparation is simple and economical29.
Transverse grating wavefront microscopy (CGM) allows not only temperature mapping at the microscale, but also biomass monitoring, making it particularly useful (if not necessary) in combination with LA-HTM. Over the past decade, other temperature microscopy techniques have been developed, especially in the field of bioimaging, and most of them require the use of temperature-sensitive fluorescent probes54,55. However, these methods have been criticized and some reports have measured unrealistic temperature changes within cells, possibly due to the fact that fluorescence depends on many factors other than temperature. In addition, most fluorescent probes are unstable at high temperatures. Therefore, QPM and in particular CGM represent an ideal temperature microscopy technique for studying life at high temperatures using optical microscopy.
Studies of S. shibatae, which live optimally at 80°C, show that LA-HTM can be applied to study hyperthermophiles, not just simple thermophiles. In principle, there is no limit to the range of temperatures that can be reached using LA-HTM, and even temperatures above 100°C can be reached at atmospheric pressure without boiling, as demonstrated by our group of 38 in hydrothermal chemistry applications at atmospheric pressure A. A laser is used for heating gold nanoparticles 40 in the same way. Thus, LA-HTM has the potential to be used to observe unprecedented hyperthermophiles with standard high resolution optical microscopy under standard conditions (i.e. under environmental stress).
All experiments were performed using a homemade microscope, including Köhler illumination (with LED, M625L3, Thorlabs, 700 mW), specimen holder with manual xy movement, objectives (Olympus, 60x, 0.7 NA, air, LUCPlanFLN60X or 60x, 1.25 NA, Oil, UPLFLN60XOI), CGM camera (QLSI cross grating, 39 µm pitch, 0.87 mm from Andor Zyla camera sensor) to provide intensity and wavefront imaging, and sCMOS camera (ORCA Flash 4.0 V3, 16-bit mode , from Hamamatsu) to record the data shown in Figure 5 (bacterial swimming). The dichroic beam splitter is a 749 nm BrightLine edge (Semrock, FF749-SDi01). The filter on the front of the camera is a 694 short pass filter (FF02-694/SP-25, Semrock). Titanium sapphire laser (Laser Verdi G10, 532 nm, 10 W, pumped tsunami laser cavity, Spectra-Physics in Fig. 2-5, further replaced by Millenia laser, Spectraphysics 10 W, pumped Mira laser cavity, Coherent, for Fig. 2-5). 6 and 7) are set to the wavelength \({{{({\rm{\lambda }}}}}}=800\) nm, which corresponds to the plasmon resonance spectrum of gold nanoparticles. Spatial light modulators (1920 × 1152 pixels) were purchased from Meadowlark Optics. The holograms were calculated using the Gerchberg-Saxton algorithm as described in the link. 39.
Cross grating wavefront microscopy (CGM) is an optical microscopy technique based on combining a two-dimensional diffraction grating (also known as cross grating) at a distance of one millimeter from a conventional camera’s sensor. The most common example of a CGM that we have used in this study is called a four-wavelength transverse shift interferometer (QLSI), where the cross-grating consists of an intensity/phase checkerboard pattern introduced and patented by Primot et al. in 200034. The vertical and horizontal grating lines create grid-like shadows on the sensor, the distortion of which can be numerically processed in real time to obtain the optical wavefront distortion (or equivalent phase profile) of the incident light. When used on a microscope, a CGM camera can display the optical path difference of an imaged object, also known as optical depth (OT), with a sensitivity on the order of nanometers36. In any CGM measurement, in order to eliminate any defects in the optical components or beams, a primary reference OT image must be taken and subtracted from any subsequent images.
Temperature microscopy was performed using a CGM camera as described in the reference. 32. In short, heating a liquid changes its refractive index, creating a thermal lens effect that distorts the incident beam. This wavefront distortion is measured by the CGM and processed using a deconvolution algorithm to obtain a three-dimensional temperature distribution in the liquid medium. If the gold nanoparticles are evenly distributed throughout the sample, temperature mapping can be done in bacteria-free areas to produce better images, which is what we sometimes do. The reference CGM image was acquired without heating (with the laser off) and subsequently captured at the same location in the image with the laser on.
Dry mass measurement is achieved using the same CGM camera used for temperature imaging. CGM reference images were obtained by rapidly moving the sample in x and y during exposure as a means of averaging any inhomogeneity in the OT due to the presence of bacteria. From OT images of bacteria, their biomass was obtained using an ensemble of images over areas selected using Matlab’s homemade segmentation algorithm (see subsection “Numerical code”), following the procedure described in ref. 48. In short, we use the relation \(m={\alpha}^{-1}\iint {{\mbox{OT}}}\left(x,y\right){{\mbox{d}} } x{{\mbox{d}}}y\), where \({{\mbox{OT}}}\left(x,y\right)\) is the optical depth image, \(m\) is the dry weight and \({{{{{\rm{\alpha }}}}}}\) is a constant. We chose \({{{{\rm{\alpha))))))=0.18\) µm3/pg, which is a typical constant for living cells.
A cover slip 25 mm in diameter and 150 µm thick coated with gold nanoparticles was placed into an AttofluorTM chamber (Thermofisher) with the gold nanoparticles facing up. Geobacillus stearothermophilus was precultured overnight in LB medium (200 rpm, 60°C) before each day of experiments. A drop of 5 µl of a suspension of G. stearothermophilus with an optical density (OD) of 0.3 to 0.5 was placed on a cover slip with gold nanoparticles. Then, a round cover slip 18 mm in diameter with a hole 5 mm in diameter in the center was dropped onto the drop, and 5 μl of bacterial suspension with the same optical density was repeatedly applied to the center of the hole. The wells on coverslips were prepared in accordance with the procedure described in ref. 45 (see Supplementary Information for more information). Then add 1 ml of LB medium to the coverslip to prevent the liquid layer from drying out. The last coverslip is placed over the closed lid of the Attofluor™ chamber to prevent evaporation of the medium during incubation. For germination experiments, we used spores, which, after conventional experiments, sometimes covered the top coverslip. A similar method was used to obtain Sulfolobus shibatae. Three days (200 rpm, 75°C) of preliminary cultivation of Thiobacillus serrata were carried out in medium 182 (DSMZ).
Samples of gold nanoparticles were prepared by micellar block copolymer lithography. This process is described in detail in Chap. 60. Briefly, micelles encapsulating gold ions were synthesized by mixing the copolymer with HAuCl4 in toluene. The cleaned coverslips were then immersed in the solution and treated with UV irradiation in the presence of a reducing agent to obtain gold seeds. Finally, gold seeds were grown by contacting a coverslip with an aqueous solution of KAuCl4 and ethanolamine for 16 minutes, which resulted in a quasi-periodic and very uniform arrangement of non-spherical gold nanoparticles in the near infrared.
To convert the interferograms to OT images, we used a homemade algorithm, as detailed in the link. 33 and is available as a Matlab package in the following public repository: https://github.com/baffou/CGMprocess. The package can calculate intensity and OT images based on recorded interferograms (including reference images) and camera array distances.
To calculate the phase pattern applied to SLM to obtain a given temperature profile, we used a previously developed homemade algorithm39,42 which is available in the following public repository: https://github.com/baffou/SLM_temperatureShaping. The input is the desired temperature field, which can be set digitally or via a monochrome bmp image.
To segment the cells and measure their dry weight, we used our Matlab algorithm published in the following public repository: https://github.com/baffou/CGM_magicWandSegmentation. On each image, the user must click on the bacteria or mCFU of interest, adjust the wand sensitivity, and confirm the selection.
For more information on study design, see the Nature Research Report abstract linked to this article.
Data supporting the results of this study are available from the respective authors upon reasonable request.
The source code used in this study is detailed in the Methods section, and debug versions can be downloaded from https://github.com/baffou/ in the following repositories: SLM_temperatureShaping, CGMprocess, and CGM_magicWandSegmentation.
Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight into thermophiles and their wide-spectrum applications. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK Insight into thermophiles and their wide-spectrum applications. Mehta, R., Singhal, P., Singh, H., Damle, D. and Sharma, A.K. Overview of thermophiles and their wide application. Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK 深入了解嗜热菌及其广谱应用。 Mehta, R., Singhal, P., Singh, H., Damle, D. & Sharma, AK. Mehta R., Singhal P., Singh H., Damle D. and Sharma A. K. A deep understanding of thermophiles and a wide range of applications. 3 Biotechnology 6, 81 (2016).
Post time: Sep-26-2022