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Precise and millidegree stable temperature control for fluorescence imaging: Application to phase transitions in lipid membranes
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View: Figures


Image of FIG. 1.
FIG. 1.

(a) Block diagram of the imaging apparatus. 780 nm excitation light (red arrow) from a Ti:sapphire laser is routed to a Bio-Rad MRC600 scanbox, which raster scans the beam through the objective lens and across the sample. The sample consists of an aqueous suspension of GUVs (red circles) between two glass coverslips (dark blue) and sealed from the bath water by clear nail polish and Fomblin perfluorinated vacuum grease. The entire sample and part of the objective lens are contained within the sample bath and submerged in water as shown. Two heaters (orange) and two RTD thermometers (one for each heater) are connected to the CryCon model 32B temperature controller, which is remotely operated by an RS232 connection to a PC. The excitation light is separated from the fluorescence light (orange and green arrows, denoting two different fluorophores) by dichroic mirror 1, and the fluorescence emissions are then separated by color by dichroic mirror 2 before being collected by the PMTs. (b) Close-up of the sample bath. The components are labeled i–xi: (i) Pt100 RTD, (ii) copper sample holder, (iii) small (loop 2) cable immersion heater, (iv) Teflon cup (purple), (v) large (loop 1) bath water heater, (vi) Teflon rod for the sample holder, (vii) o-ring seal (black) between objective lens and Teflon cup, (viii) foam insulation (blue), (ix) objective lens (UPlan Apochromat 60×, Olympus), (x) thermal isolator for objective lens, and (xi) GUV (red circles) samples between two round coverslips (dark blue).

Image of FIG. 2.
FIG. 2.

Dye partitioning and phase transition in a GUV. For this sample, SSM/DOPC/. The black and white images depict the two different fluorescence channels at prior to merging and false color assignment. The upper black-and-white image shows the LR-DPPE fluorescence ( phase) and the lower image shows naphthopyrene fluorescence ( phase). The middle image shows an equatorial section of a GUV with phase coexistence at and the right image shows the GUV at , after it has undergone a miscibility transition. The red false color denotes the phase probe LR-DPPE and the blue false color denotes the phase probe naphthopyrene.

Image of FIG. 3.
FIG. 3.

Temporal stability of the temperature for a fixed location in the sample bath. The sample temperature at a fixed location is stable to within milli-Kelvins of the set-point temperature for periods of hours. The temperatures displayed in the insets are the recorded temperatures; the set point temperatures were (a) and (b) . Each graph depicts one run only; thus, the error bars depicted on the graphs for the actual temperature (vs setpoint temperature) refer to the standard deviations of time averages of the temperature readings. Therefore, all are the same in length for a given data set.

Image of FIG. 4.
FIG. 4.

Spatial temperature gradients. The open circles and black circles refer to the two different locations. (a) Measurement of the temperature vs time at positions 1 cm apart, at a constant depth in the sample chamber (set point of ). (b) Measurement of the temperature at different depths in the sample chamber, 3 cm apart. Each open circle or black circle data point is an average of two measurements. For a given plot, the error bars represent a combination of the standard deviation of two data sets used to produce the depicted averaged plot and the time average of the temperature readings for the averaged plot. It should be noted that the data were recorded at 5 s intervals, but those displayed on the graphs have been made sparse (30 s intervals instead of 5 s intervals) for the sake of clarity.

Image of FIG. 5.
FIG. 5.

Temperature-dependent ternary diagram for cholesterol/SSM/DOPC. (a) Ternary diagram. Color denotes miscibility transition temperatures as indicated by the scale to the right, and the coexistence boundaries for the colored regions are for . Blue points refer to one-phase samples at , black points to coexistence at , red points to gel- coexistence at , and green points to three-phase samples at , as observed using two-photon microscopy. The red star denotes the critical point at and the blue star denotes the overall critical point at . (b) A schematic of the coexistence region with the line of critical points (red) extending from the point at to the upper critical point at .

Image of FIG. 6.
FIG. 6.

Quench data. (a) An example of the temperature reading near the sample vs time (second) for a quench experiment. The inset shows the error bars up close. (b) Average domain radius (pixels) vs time (second) for four different compositions quenched below the miscibility gap. The error bars in (a) reflect the uncertainty in the thermometer only; thus, they are of constant length for all data points. The error bars depicted in (b) reflect the standard deviations of the sample averages.


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Precise and millidegree stable temperature control for fluorescence imaging: Application to phase transitions in lipid membranes