DOI: 10.1002/cbic.201300096
In Vivo Probing of the Temperature Responses of
Intracellular Biomolecules in Yeast Cells by Label-Free
Raman Microspectroscopy
Yu-Fang Chiu, Chuan-Keng Huang, and Shinsuke Shigeto*
[a]Introduction
Temperature profoundly affects a plethora of physiological pro-cesses that take place in living cells, such as cell division, me-tabolism, protein transport, and cell death. This high sensitivity of physiological processes to temperature arises from the fact that the equilibria and kinetics of individual biochemical reac-tions involved in the physiological processes are strongly tem-perature-dependent. Along with pH and oxygen concentration, temperature has been widely used as a critical parameter in genetic and biochemical experiments. Temperature-sensitive mutation, for example, has substantially advanced understand-ing of the molecular mechanisms of many important biological processes.[1–4]We still know little, however, about how cells
re-spond to temperature variation in vivo and at the molecular level and what distinct behavior patterns intracellular compo-nents show at various temperatures.
In this study we discuss the temperature responses of major intracellular biomolecules in living fission yeast (Schizosacchar-omyces pombe) cells probed by Raman microspectroscopy. Unlike most biochemical approaches, microscopic techniques can investigate single living cells in a nondestructive manner. Fluorescence microscopy with the use of fluorescent nanother-mometers based on lanthanide nanoparticles,[5, 6] quantum
dots,[7] or polymer-embedded organic dyes[8–11] has recently
permitted accurate measurements of intracellular temperature in living cells. Of particular importance is the work done by
Okabe et al.,[9]which for the first time has extended
intracellu-lar temperature measurements at specific locations to map-ping, with a temperature resolution of < 1 K.
In contrast, the focus of this work is on the effects of envi-ronmental temperature (i.e., culture temperature in this case) rather than on intracellular temperature. Whereas intracellular temperature is closely associated with cellular thermogene-sis[12, 13](i.e., local heat generation as a consequence of cellular
activities), environmental temperature alters the rate of the metabolic activity of cells and thereby causes changes in cell proliferation rate. By analyzing the fingerprint Raman spectra (600–1800 cm 1) of fission yeast cells measured at culture
tem-peratures ranging from 26.1 to 38.3 8C, we show that the bio-synthesis of the fungal sterol ergosterol[14]—in contrast with
the less temperature-sensitive behavior of proteins and phos-pholipids—is significantly diminished at culture temperatures higher than 35 8C. This study demonstrates that Raman micro-spectroscopy can be added to the toolbox for in vivo molecu-lar-specific probing of temperature-dependent cellular activi-ties without the need to introduce fluorophores, which might perturb the physiological state of cells.
Results and Discussion
We used a laboratory-built confocal Raman microspectrome-ter[15–17] equipped with a microscope stage-top incubator to
record Raman spectra of 30 fission yeast cells under the micro-scope at ten different temperatures ranging from 26.1 to 38.3 8C. The cells were precultured in Yeast and Mold (YM) broth in a shaker incubator and then transferred to a glass-bot-Environmental temperature is an essential physical quantity
that substantially influences cell physiology by changing the equilibria and kinetics of biochemical reactions occurring in cells. Although it has been extensively used as a readily con-trollable parameter in genetic and biochemical research, much remains to be explored about the temperature responses of intracellular biomolecules in vivo and at the molecular level. Here we report in vivo probing, achieved with label-free Raman microspectroscopy, of the temperature responses of major intracellular components such as lipids and proteins in living fission yeast cells. The characteristic Raman band at 1602 cm 1, which has been attributed mainly to ergosterol,
showed a significant decrease ( 47 %) in intensity at elevated temperatures above 35 8C. In contrast to this high temperature sensitivity of the ergosterol Raman band, the phospholipid and protein Raman bands did not vary much with increasing cul-ture temperacul-ture in the 26–38 8C range. This finding agrees with a previous biochemical study that showed that the initial stages of ergosterol biosynthesis in yeast are hindered by tem-perature elevation. Moreover, our result demonstrates that Raman microspectroscopy holds promise for elucidation of temperature-dependent cellular activities in living cells, with a high molecular specificity that the commonly used fluores-cence microscopy cannot offer.
[a] Y.-F. Chiu, C.-K. Huang, Prof. Dr. S. Shigeto
Department of Applied Chemistry and Institute of Molecular Science National Chiao Tung University
1001 Ta-Hsueh Road, Hsinchu 30010 (Taiwan) E-mail: shigeto@mail.nctu.edu.tw
tomed dish for Raman measurements (see the Experimental Section for details). The growth curves of fission yeast cells at optimum temperature[18] (30 8C) and at 38 8C are compared in
Figure 1 A. After 40 h, the cells had reached stationary phase
even at 38 8C, but the number of yeast cells cultured at this ad-verse temperature was not as large as that at 30 8C. No notice-able difference between the bright-field optical images of typi-cal living yeast cells grown at 30 and 38 8C was found (Fig-ure 1 B). With 632.8 nm excitation at 2.6 mW excitation power (see the Experimental Section), the target yeast cell tended to be optically trapped. Optical trapping (also known as optical tweezers) is a powerful technique for capturing and manipulat-ing biological particles at the laser focus.[19] It facilitates both
efficient Raman excitation and collection of Raman scattered light in a confocal configuration.[20–22]
Figure 2 displays a series of averaged Raman spectra of opti-cally trapped yeast cells at 10 different culture temperatures. Each spectrum is the average of 30 background-subtracted spectra and has been normalized to the peak area of the CH bending band at 1440 cm 1. The use of the peak area of the
1440 cm 1 band as a normalization standard is justified
be-cause it has been found to remain almost constant over the whole temperature range studied.
A number of characteristic Raman bands can be seen in Figure 2. Vibrational assignments of those bands, except for the band at 1602 cm 1, have been extensively discussed in the
literature,[23–25] as summarized below. The weak band at
1744 cm 1is assigned to the C=O stretch of the ester moieties
of phospholipids. The band at 1655 cm 1 is attributed to the
C=C stretch of the cis CH=CH linkages of unsaturated lipid
chains. As described above, the prominent 1440 cm 1band is
assigned to the CH bending modes, including CH2scissors and
CH3 degenerate deformation. The band at 1300 cm 1 is
as-signed to the in-plane CH2 twisting mode. All these Raman
bands are thought to be predominantly due to lipids, because the yeast cells are likely to be trapped at lipid droplets (dark spots indicated by arrowheads in Figure 1 B), extremely lipid-rich organelles. Possible contributions from overlapping pro-tein bands (e.g., amide I) are therefore considered minor. The sharp peak at 1003 cm 1is a protein Raman band arising from
the ring-breathing mode of the phenylalanine residues. The band at 1602 cm 1is an interesting Raman signature of
yeast and has been intensively studied by Hamaguchi and co-workers. They examined the behavior of the 1602 cm 1 band
on treatment of the cells with respiration inhibitors such as KCN[24] and NaN
3,[26] under different nutrition and atmospheric
conditions[27] and in the presence of oxidative stress induced
by addition of H2O2.[27]Despite these thorough investigations,
the origin of the band had long been unclear. Very recently, however, it has been shown that ergosterol is the main con-tributor to the 1602 cm 1band.[28]
The spectral features shown in Figure 2 appear to be quite similar at all measurement temperatures. Note that the shapes of the phospholipid Raman bands did not change from well-formed peaks to diffuse, broadened profiles at high tempera-ture; this suggests that the double-membrane structure re-mained intact[24] and the cells were alive, albeit presumably
not very active.
Intriguingly, though, the 1602 cm 1 band alone exhibits
a marked decrease in intensity at elevated temperatures of 35.5, 36.8, and 38.3 8C. To illustrate this unique temperature
de-Figure 1. A) Growth curves of fission yeast cells cultured in YM broth at 30 (*) and 38 8C (*). Solid lines are sigmoid fits provided as a guide for the eye. B) Bright-field optical images of typical living yeast cells cultured at 30 (left) and 38 8C (right). White arrowheads indicate the intracellular locations of several lipid droplets (dark spots). Scale bars : 5mm.
Figure 2. Averaged Raman spectra (fingerprint region), of fission yeast cells cultured at 10 different temperatures. Each spectrum is the average of 30 Raman spectra obtained from 30 different randomly chosen yeast cells. All the spectra have been normalized to the peak area of the CH bending band at 1440 cm1
pendence better, we plot the ratios of the averaged peak areas of the four Raman bands at 1602, 1655, 1300, and 1003 cm 1
to that of the 1440 cm 1band as a function of measurement
temperature (Figure 3). A detailed description of peak area
de-termination including baseline correction can be found in the Experimental Section. For the 1602 cm 1 band (mainly
ergo-sterol), the peak area ratio remains unchanged between 26.1 and 34.5 8C, but at 35.5 8C it suddenly drops by as much as 47 % and subsequently becomes constant again up to 38.3 8C (Figure 3 A). In sharp contrast, the ratios for the bands at 1655 (mainly lipids, Figure 3 B), 1300 (lipids, Figure 3 C), and 1003 cm 1 (proteins, Figure 3 D) show much less pronounced
temperature dependence; they appear to be nearly independ-ent of temperature with some fluctuation around the mean value. One might argue that the ratio for the 1655 cm 1band
also decreases slightly as the temperature is elevated. This de-crease is estimated to be at most 19 %, however, and is much smaller than the temperature response that we observed for the 1602 cm 1band (19 vs. 47 %). We can therefore conclude
that the ergosterol band at 1602 cm 1represents a highly
sen-sitive indicator of a cell’s surrounding temperature.
The data presented in Figure 2 and 3 are averages over 30 cells, and as such they lack information on the statistical distributions of the band areas for individual cells. To examine whether the unique temperature response of the 1602 cm 1
band is also evident without averaging and normalization, the distributions of the Raman peak areas of the 1602 and 1440 cm 1 bands of all 30 cells are compared at 26.1 and
38.3 8C (Figure 4). The distribution of the 1602 cm 1band area
at 38.3 8C is clearly shifted (by 2515 arbitrary units) toward lower value relative to that at 26.1 8C, whereas the area distri-bution of the 1440 cm 1band does not change much between
the two temperatures. Similar results were obtained from com-parison of the 1602 cm 1 band with the other Raman bands ;
this confirms that the temperature response of the 1602 cm 1
band is significantly different from those of the other phospho-lipid/protein Raman bands at 1440, 1655, 1300, or 1003 cm 1.
In addition, it supports the assignment of the 1602 cm 1band
to ergosterol,[28]which is neither a phospholipid nor a protein.
This finding is consistent with a previous study by Shimizu and Katsuki[29]that showed that ergosterol biosynthesis in
aer-ated budding yeast at 40 8C is lowered to only 32–35 % relative to that at lower temperature (20 or 30 8C). This decrease was interpreted as a consequence of repression of the enzymes in-volved in the synthesis of mevalonate from acetyl-CoA, the ini-tial stages of ergosterol biosynthesis.[14]We note, however, that
Shimizu’s and Katsuki’s results were obtained by biochemical methods including extraction. Our study shows that the tem-perature sensitivity of the biosynthesis and metabolic activity in cells can now be probed in a nondestructive manner by Raman microspectroscopy.
Conclusions
We have revealed that the Raman band of ergosterol appear-ing at 1602 cm 1in fission yeast cells responds to
environmen-tal temperature in such a sensitive way that the peak area abruptly decreases by 47 % above 35 8C. This high sensitivity to temperature contrasts with less characteristic temperature dependence exhibited by other lipid and protein bands. It should be noted that this study, unlike the previous fluores-cence studies,[5–11]is not an attempt to perform real-time
moni-Figure 3. Temperature dependence of the peak area ratio of the A) 1602, B) 1655, C) 1300, and D) 1003 cm 1
bands to the 1440 cm 1
band. The peak area of each Raman band was derived by calculating the area between the band contour and a straight line connecting the two edges of the band (see the Experimental Section). Data represent mean values standard devia-tions of triplicate experiments.
Figure 4. Peak area distributions of A) the 1602 cm 1
, and B) the 1440 cm 1
bands for yeast cells cultured at 26.1 8C (top) and 38.3 8C (bottom). Dashed lines indicate the mean values (n = 30).
toring of intracellular temperature. Rather it has shed new light on molecular-level understanding of the responses of in-tracellular components in living cells to environmental temper-ature, which is as central to chemical biology as intracellular thermometry.
Ergosterol represents an important distinction between fungi (including yeast) and animals (which use cholesterol in-stead of ergosterol). Indeed, many antifungal agents function by interacting with ergosterol. Amphotericin B, a well-known antifungal drug, for example, has been shown to kill yeast by directly binding ergosterol.[30] Our Raman microspectroscopic
approach to monitoring ergosterol concentrations through the 1602 cm 1band could help understanding, both in vivo and at
the molecular level, of how these drugs work inside the cell and whether their antifungal ability is enhanced or impaired by changing environmental temperature.
Experimental Section
Cell culture: Fission yeast cells were cultured in YM broth (Acume-dia, 7363) in a shaking incubator at 150 rpm at 10 different tem-peratures (26.1, 28.4, 30.3, 31.7, 32.5, 33.8, 34.5, 35.5, 36.8, and 38.3 8C) for 40 h so that cells reached stationary phase (see Fig-ure 1 A). About 200mL of the culture medium containing yeast cells was transferred to a glass-bottomed dish coated with poly-d-lysine (MatTek, 35G-1.5-14-C). Medium taken from the YM broth (1 mL) was centrifuged at 1200 rpm for 1 min (Beckman Coulter, Microfuge 16), and the supernatant fluid was then used to dilute the sample put into the poly-d-lysine-coated dish in order to pre-vent possible phase change and laser trapping of multiple cells. The diluted sample was used for Raman measurements. The growth curves of yeast cells (Figure 1 A) were determined by using a cell counting chamber (Marienfeld, 0640130) under the micro-scope.
Raman microspectroscopy: Raman spectroscopic measurements were performed with a laboratory-built confocal Raman microspec-trometer, which has been described in detail elsewhere.[15–17]
The 632.8 nm output of a He-Ne laser was used as the excitation light. The beam was magnified by a factor of 2.7 to effectively cover the exit pupil of the objective used. The expanded beam was intro-duced into an inverted microscope (Nikon, TE-2000 modified) and was focused onto the sample with an oil-immersion objective (CFI Plan Fluor, 100 , NA 1.3). The focus spot size was 1 mm. Back-scattered Raman light was analyzed with an imaging spectrometer (HORIBA Scientific, iHR320) and detected with a back-illuminated, deep-depletion, liquid-N2-cooled charge-coupled device (CCD)
de-tector (Princeton Instruments, Spec-10:100) with 100 1340 pixels operating at 120 8C. A grating with 600 grooves per mm was used to cover a wide spectral range (> 2000 cm 1
) with an effective spectral resolution of 7 cm 1
. For bright-field observation, the sample was illuminated with a halogen lamp, and optical micro-graphs were acquired with a digital camera (Nikon, DS-Ri1) mount-ed on the microscope.
During the measurement, the sample temperature was kept the same as the culture temperature by use of an incubator (Tokai Hit, INU-ONICS-F1) mounted on the inverted-microscope stage. The target temperature was achieved with an accuracy of 0.1 K by in-dependently controlling the temperatures of four heaters available in the incubator and was monitored by measuring the temperature of water in the glass-bottomed dish with a thermometer. Fresh air
( 0.2 bar) was continuously passed into the stage-top incubator. At every temperature, Raman spectra of 30 (or more) randomly chosen yeast cells were obtained with 2.6 mW laser power at the sample point and with a 60 s exposure time. The relatively long exposure time yielded decent Raman spectra (see Figure 2), so no noise reduction of the recorded spectra, such as use of singular value decomposition,[15]
was performed. Estimated spatial resolu-tion was 0.3mm in the lateral (XY) direction and 2.4 mm in the axial (Z) direction. Because the thickness of a fission yeast cell is typically 2 mm, the focal volume contained the entire cell along the Z di-rection.
Data analysis: Raman peak area was determined by integrating the area between the band contour and a baseline connecting the two edges of the band. The spectral interval used for the peak area determination was 1631–1686 cm 1
for the 1655 cm 1 band, 1587–1620 cm 1 for the 1602 cm1 band, 1411–1495 cm 1 for the 1440 cm 1 band, 1279–1332 cm 1 for the 1300 cm 1 band, and 997–1008 cm 1 for the 1003 cm 1
band. Small variations in these intervals ( a few wavenumbers) had little effect on the peak area ratio shown in Figure 3. All spectral data analysis was performed with IGOR Pro 6 (Wave Metrics).
Acknowledgements
We thank Kung-Hui He for help in data analysis. This work was supported by the National Science Council of Taiwan (Grant NSC100-2113M-009-009-MY2).
Keywords: biophysics · ergosterol · fungi · lipids · Raman spectroscopy · temperature sensitivity
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Received: February 25, 2013 Published online on April 29, 2013
