Research Article

Received: 8 September 2008 Accepted: 13 May 2009 Published online in Wiley Interscience: 22 June 2009

(www.interscience.wiley.com) DOI 10.1002/jrs.2356

CARS microscopy of lipid stores in yeast: the

impact of nutritional state and genetic
background
Christian Brackmann, Joakim Norbeck, Madeleine Åkeson, Daniel Bosch,
Christer Larsson, Lena Gustafsson and Annika Enejder∗
We have developed a protocol for sub-micrometer resolved and chemically specific imaging of lipid storage in vivo employing
coherent anti-Stokes Raman scattering (CARS) microscopy of one of the most important model organisms Saccharomyces
cerevisiae – the yeast cell. By probing the carbon–hydrogen vibration using the nonlinear process of CARS, lipid droplets in the
yeast cells clearly appear, as confirmed by comparative studies on relevant labeled organelles using two-photon fluorescence
microscopy. From the images, unique quantitative data can be deduced with high three-dimensional resolution, such as
the volume, shape, number, and intracellular location of the neutral lipid stores. We exemplify the strength and usability
of the method for two cases: the impact on lipid storage of the nutritional condition (starvation and type of carbon source
available) as well as of genetic modification of two fundamental metabolic regulation pathways involving carbohydrate and
lipid storage (BCY1 and DGA1, LRO1, ARE1/2 deletions), respectively. While the impact of carbon source on the total cellular
lipid volume was minimal, long-term starvation induces a significant accumulation of lipid droplets. We also confirm that the
lipid-storage-deficient mutant is indeed unable to synthesize lipid droplets, and that the inability of the bcy1-mutant to store
carbohydrates is compensated by a two-fold increase in stored neutral lipids. We note that there is a significant cell-to-cell
variability in neutral lipid storage in general, i.e. that there is a correspondence to the noise found for gene expression also in
lipidomics. Copyright
c 2009 John Wiley & Sons, Ltd.

Keywords: yeast; Saccharomyces cerevisiae; coherent anti-Stokes Raman scattering; microscopy; neutral storage lipids

Introduction to be highly useful in understanding the biology of cells, also

of higher organisms. It offers high accessibility to the genome
Throughout evolution, organisms have been forced to cope with through the complete library of viable mutants available, is easy to
highly variable nutrient availability, and therefore evolved the
cultivate, and has a short generation time. In addition, it exhibits
capacity to store nutrients for use under starvation conditions.
high evolutionary conservation in processes as complex as the reg-
Multicellular organisms have even developed specialized cells and
ulatory mechanisms of the cell cycle[17] and, not so surprisingly,
organs which fulfill this role, such as adipocytes and hepatocytes,
in a long list of fundamental metabolic pathways.[5,18,19] Thus, the
which shows the importance of this ability. Storage nutrients
model organism S. cerevisiae has the prospect of providing, for the
involving carbon are mainly carbohydrates (e.g. glycogen) and
human well-being, important information on metabolic processes
neutral lipids (triacylglycerol (TGA) and steryl esters). Most, if not
involving storage lipids.
all, eukaryotic cells accumulate neutral lipids, mainly consisting of
Our knowledge on lipid storage and in particular droplet
TGA and acylated sterols. They usually coalesce into so-called lipid
morphology has so far relied on biochemical analysis of whole
droplets, bounded by phospholipids and specific proteins.[1 – 3]
cell contents[20] and fluorescence microscopy using lipid-specific
Defects in the nutrient storage regulation have severe implications
staining or green fluorescent protein (GFP) tagged proteins
and underlie common health problems in modern society, obesity
associated with lipid droplets.[10,21,22] These approaches have clear
and diabetes being well-known examples.[4] Understanding the
drawbacks: whole-cell biochemical analysis provides average data
pathways by which storage compounds are synthesized and
of all lipids present in the cell without any spatial or single-cell
metabolized, including the regulation thereof, is of utmost
information. On the positive side, detailed specifications on carbon
importance.
chain length, degree of saturation, etc. are readily obtained. The
For this purpose, suitable model systems have been devel-
lipid staining and protein tag approaches provide single-cell and
oped. They range from cell cultures of yeast[5] and mammalian
morphological information on the lipid droplets. However, staining
cell lines[6 – 8] to whole organisms such as zebrafish larvae,[9]
nematodes,[10] fruit flies,[11,12] and mammals,[13 – 15] each con-
tributing with different but just as important aspects of the lipid
∗ Correspondence to: Annika Enejder, Department of Chemical and Biological
metabolism. Whereas the whole organism models allow for full
systemic studies, single-cell models are useful to gain in-depth Engineering, Chalmers University of Technology, Kemigården 10, S-412 96
Göteborg, Sweden. E-mail: enejder@chalmers.se
information on specific cellular metabolic pathways. The yeast cell
(Saccharomyces cerevisiae), being the first eukaryotic organism to Department of Chemical and Biological Engineering, Chalmers University of
748

have its entire genome sequenced,[16] has for a long time proven Technology, S-412 96 Göteborg, Sweden

J. Raman Spectrosc. 2009, 40, 748–756 Copyright

c 2009 John Wiley & Sons, Ltd.
CARS microscopy of lipid stores in yeast
is an indirect and invasive method in that dyes are required to
enter the cells and bind to lipids, with potential side effects
such as altered chemical and physical properties of the lipids,
photo-induced damages, uncertainties in the labeling/expression
efficacy, and fluorescence signal generation.[23 – 27] A noninvasive
method in which lipid amounts and distributions can be studied
in vivo during an extended period of time is therefore desirable.
Coherent anti-Stokes Raman scattering (CARS) microscopy fulfils
these criteria. CARS microscopy was for the first time demonstrated
in the early 1980s.[28] Since then, the technique has benefited
greatly from advances in optics, laser, and detector technology
and was revived in later years.[29] Overviews on the development of
CARS microscopy in the 2000s are presented in review papers and
textbooks.[30 – 32] CARS is a nonlinear, four-wave mixing, optical
process, where the signal is generated through the interaction
of three electric fields with a target sample, via the third-order
nonlinear susceptibility. An enhanced signal is achieved as the
frequencies of the applied laser fields are tuned into resonance
with a specific molecular vibration, and thereby target specific
species or molecular groups. For studies of lipids, vibrations
between carbon and hydrogen are suitable for excitation due
to the high content of carbon–hydrogen bonds and their high
vibrational Raman cross section. CARS microscopy has successfully
been applied for studies of lipids in vesicles and lipid layers,[33 – 35]
in single cells,[36] as well as in multicellular organisms.[27] From
these studies it can be concluded that the symmetric vibration of
the CH2 groups at wavenumber 2845 cm−1 should be probed for
chemically specific imaging of lipids.
Contrary to the established fluorescence microscopy technique,
CARS microscopy requires no labeling of the sample, thereby
avoiding all uncertainties associated with uptake and distribution
of the fluorescent marker in the lipids. This important quality
is shared by the conventional (incoherent) Raman microscopy
technique (see for instance Ref. [37]), which is also based on
the probing of molecular vibrations. However, this method relies
on spontaneous Raman scattering, a process significantly weaker
than CARS, resulting in lower signal strengths and, for living
systems, unacceptably long integration times. Third-harmonic
generation (THG) microscopy has also been applied for imaging
of lipid droplets in biological material,[38] and the method is
able to distinguish lipid droplets from the surrounding structure
primarily due to their higher density. However, THG signals are
also generated by other dense structures in the sample[39,40] and
there are no means to distinguish them despite different molecular
composition.
In the present study, CARS microscopy has been evaluated
as a tool for studying lipid storage in a single-cell model
organism – yeast. CARS microscopy here serves as an excellent
method for monitoring neutral lipid storage in vivo, with the
sub-micrometer spatial resolution required for this small-sized cell
model. The effect of nutrient availability was visualized by studying
starved cells, as well as cells grown on a fermentative carbon source
(glucose) or a respiratory carbon source (ethanol/glycerol). As first
examples of how the genetic impact on the lipid metabolism can
be screened by means of CARS microscopy, two mutants lacking
genes central for the regulation of the cell metabolism were
studied. For verification, a completely lipid-free strain (H1246)[41]
was monitored, lacking four genes essential for the TGA and steryl
ester synthesis. In addition, a bcy1-deletion strain was studied,
which is defective in the alternative way of storing energy, i.e. as
carbohydrates.[42] If and to which extent this has any implications
for the lipid storage pathways was previously not known, a
J. Raman Spectrosc. 2009, 40, 748–756 Copyright

c 2009 John Wiley & Sons, Ltd.
question which we are able to respond to by means of CARS
microscopy.
Experimental
The CARS microscope
An outline of the CARS microscopy setup is shown in Fig. 1,
the main components of which are a laser system, consisting
of a Nd : Van laser (Picotrain, HighQ Lasers GmbH) pumping two
optical parametric oscillators (Levante OPO, APE GmbH), and
an inverted research microscope (Eclipse TE-2000-E, Nikon). The
Nd : Van laser generates pulse trains with a pulse duration of
7 ps at a repetition frequency of 76 MHz, the laser wavelength
is 1064 nm, and the linewidth is approximately 3 cm−1 . The
average output power of the pump laser is 10 W and the beam
is split and directed into the two optical parametric oscillators
(OPOs) as well as into the microscope using an arrangement
of dichroic mirrors. A telescope is used in the common beam
path to adjust the beam diameters prior to entering the mirror-
scanning unit of the microscope. Temporal overlap between the
laser pulses at the sample is ensured by having adjustable delay
lines with retroreflectors for the OPO beams. The laser beams
are focused on the sample using a microscope objective of high
numerical aperture (60 × N.A. 1.2 water immersion or 100 ×
N.A. 1.49 oil immersion). The experimental setup enables different
combinations of beams in the near-infrared regime to be coupled
into the microscope, and thereby the excitation of different
molecular vibrations as well as fluorescent marker molecules
(two-photon fluorescence). The symmetric vibration of the CH2
group in the acyl chains of lipids at 2845 cm−1 is excited using
a combination of one OPO tuned to 817 nm together with the
pump beam (1064 nm). The CARS and two-photon fluorescence
excitation schemes for this beam combination are shown in the
insert of Fig. 1. The emitted CARS signal, co-propagating in the
forward direction with the laser beams, is focused onto a single-
photon counting photomultiplier tube (PMC 100-1, Hamamatsu)
by an aspherical lens of N.A. 0.8 mounted above the sample.
An additional photomultiplier tube is mounted on a side port
of the microscope for the simultaneous detection of two-photon
fluorescence in epi-geometry as illustrated in Fig. 1. Bandpass
filters suppress the radiation at the laser wavelengths and merely
transmits the CARS signal generated at 663 nm in the forward
direction and two-photon fluorescence in the wavelength range
500–550 nm in the epi-direction. Three-dimensional imaging
is achieved by scanning a sequence of horizontal planes at
different vertical positions, by translating the objective using a
motorized stage.
Cell preparation
For the identification of the organelles appearing in the CH2
stretch CARS microscopy images, cells carrying a C-terminal GFP
tag on genes associated with the mitochondrion, peroxisome,
vacuole, and the lipid droplet were acquired and cultured in
a defined medium (5 ml medium in 50 ml tubes) to reach the
mid-log phase. In this investigation, mitochondria were marked
by ATP2-GFP, peroxisomes by PEX11-GFP, the vacuole by VMA1-
GFP, and lipid droplets by ERG6-GFP, identical to the strains
used in a previous study on global protein localization.[43] In the
subsequent lipid storage studies, four different strains of yeast
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cells were monitored by means of CARS microscopy: commercial
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 C. Brackmann et al.

Figure 1. Schematic illustration of the combined CARS and two-photon fluorescence microscopy setup consisting of a tunable laser system, operating in
the near infrared regime, and an inverted research microscope. While the CARS signal is detected in the forward direction, the two-photon fluorescence
is collected in the epi-direction. The inserts in the figure shows the excitation scheme for the CARS and the two-photon fluorescence processes when
probing the symmetric CH2 vibration at 2845 cm−1 and the GFP fluorophore between 500 and 550 nm, respectively.

grade Baker’s yeast, BY4742 wild type, BY4742 bcy1::KanMX, and
a lipid-free strain (H1246) in the w303-background.[41] All cells
were grown in SC medium with 2% glucose and ammonium
sulfate as carbon and nitrogen sources, with the exception of
a subset of the wild type which was grown on 3% ethanol/2%
glycerol as carbon source. A small volume (∼3 µl) of medium and
cells at log-phase was deposited in a microcuvette made of two
cover slides attached with a thin gasket (Invitrogen Secure-Seal
Spacer). This resulted in a small chamber (diameter: 9 mm, height:
0.12 mm) between the cover slides, which is suitable for CARS
microscopy.
Biochemical analysis of TGA
The total amounts of TGA in full populations (∼5×106 cells) of wild-
type yeast cells grown on fermentative (glucose) and respiratory
(ethanol/glycerol) medium, respectively, were quantified by
means of biochemical analysis. Lipids were extracted by using
KOH/ethanol according to Andlid et al.[44] and the liberated
glycerol was quantified by using an enzyme combination kit
(Boehringer Mannheim GmbH, Mannheim Germany).
Data acquisition and image evaluation
The data were acquired as z-stacks covering fields of view of
20 × 20 µm2 and 30 × 30 µm2 in the horizontal plane, and
images were collected over a vertical distance of 10–18 µm,
750
the initial co-localization study, pairs of simultaneously recorded
CARS and two-photon fluorescence z-stacks were acquired for
the organelle-labeled yeast strains. Co-localization images were
formed by means of ImageJ,[45] and the degree of overlap between
the highlighted organelles in the fluorescence and CARS images
was estimated. In the studies involving nutrient availability and
different mutants, 13 z-stacks were collected on the starved Baker’s
yeast cells; 16 and 19 z-stacks on the glucose and ethanol/glycerol
metabolizing cells, respectively; and finally 18 z-stacks on the
bcy1-mutant. Each scanned image plane of a stack was acquired
during an integration time of 20 s. The measurements on cells
metabolizing glucose, ethanol/glycerol, and the co-localization
studies were carried out using the 60 × objective, whereas the
100 × objective was used for the bcy1-mutant, the Baker’s yeast,
the lipid-free mutant, and the images of Fig. 2. The number of
cells and stationary lipid droplets evaluated from the z-stacks for
each category is specified in Table 1. A potential movement of a
lipid droplet during the acquisition would introduce a smearing
of the signal over the cell area and not result in a sharp image
of the droplet. Therefore, the evaluation is limited to stationary
droplets as those distinguishable in the images of Fig. 5. In order
to obtain quantitative data from the large set of CARS microscopy
images, a segmentation algorithm of Local thresholding, which
has been proven particularly useful for nonlinear microscopy,[46]
was employed for converting the z-stacks to binary images (areas
identified as organelles −1, background −0), segmenting the

chosen to include the entire volume of the imaged yeast cells. In lipid droplets from the background signal generated by the yeast

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c 2009 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2009, 40, 748–756
CARS microscopy of lipid stores in yeast

(a) Table 1. Summary of the results obtained from the quantitative

analysis of the CARS microscopy images presenting the amounts of
lipids for the different types of yeast cells investigated. The results
indicated by (∗ ) were evaluated neglecting the smallest lipid droplets
present in only one z-stack plane
Baker’s
Glucose Ethanol BCY1 yeast
1200

800 Average droplet volume/µm3 0.21 0.18 0.23 0.31

400
Standard deviation/µm3 0.17 0.14 0.22 0.29
Average droplet volume/µm3 (∗ ) 0.29 0.25 0.31 0.39
0
Standard deviation/µm3 (∗ ) 0.16 0.14 0.23 0.30
Average droplets per cell 1.1 1.6 2.5 2.6
(b)
Maximum droplets per cell 4 4 5 19
Minimum droplets per cell 0 0 0 0
Lipid volume per cell/µm3 0.23 0.28 0.58 1.06

1200 810 nm. While organelles with high density of CH2 bonds clearly
800 appear at resonance (Fig. 2a), they appear with reduced contrast
400 at 2952 cm−1 (Fig. 2b), which is also shown in the intensity profile
0
of Fig. 2c, where a signal decrease by a factor of 2.5 can be seen for
the larger structures. In addition, the smaller organelles in the yeast
(c) 1000 cell located in the upper region of the images show a further signal
decrease by a factor of 5–6. An identification of the most prominent
CARS signal/ Arbitr. Units

2845 cm-1 organelles was carried out by combining CARS with two-photon
800 2952 cm-1
fluorescence microscopy on GFP-tagged strains of selected marker
proteins associated with the mitochondrion, peroxisome, vacuole,
600
and the lipid droplet.[43] Simultaneous measurements allowed co-
localization between CARS and fluorescence images the degree of
400
which allowed identification of the organelle selectively probed by
CARS microscopy. Examples of CARS and two-photon fluorescence
200
image pairs of all organelle categories investigated are shown in
the first two columns in Fig. 3. In the co-localization images in the
0
0 5 10 15 20 third column, red color represents the CARS signal, green color
the GFP fluorescence, and blue color indicates co-localization
x/µm
between the CARS and fluorescence signals. Figure 3a shows
Figure 2. CARS microscopy images of yeast cells collected (a) at 2845 cm−1 , images of yeast cells having the mitochondria labeled. In the
the resonance of the CH2 vibration and (b) at 2952 cm−1 , illustrating the overlay image, it can be seen that the red and green areas are
molecular specificity with which organelles with high density of CH2 groups separated without any blue color appearing, indicating no signal
appear in CARS microscopy. Intensity profiles along the white dashed lines
overlap. The same observation can be made for the peroxisome
are shown in the diagram (c), where a reduction in CARS signal relative
to the background by a factor of 2.5 can be seen in the organelles when (Fig. 3b) and the vacuole (Fig. 3c). The fluorescence image in Fig. 3d
tuning to 2952 cm−1 . shows lipid droplets of the yeast cells tagged by GFP (via ERG6).
Here, the CARS signal co-localizes well with the fluorescence,
which is observed as blue color in the droplets. The analysis of
cell bodies and the surrounding medium. After segmentation, GFP ERG6 co-localization measurements for ∼140 cells resulted
the particles in the binary image stacks were analyzed using in 169 lipid droplets identified in the CARS images, with 123 of
a 3D objects counter developed for ImageJ. Adding the signal them having a corresponding droplet in the fluorescence image,
contributions from a droplet distributed in depth over several giving an average degree of co-localization of 73%. The lower
two-dimensional images in a z-stack provided a measure of the number of lipid droplets identified in the fluorescence images
total lipid droplet volume. could possibly be explained by variations in protein expression.
Hence, the objects visualized with a strong signal in the CARS
images can be identified as lipid droplets. The relatively strong
Results CARS signal from these droplets, which is obtained when probing
the CH2 stretch vibration, is in agreement with the long chains
CARS microscopy allows specific imaging of lipid droplets
of CH2 groups of lipids found in the droplets. As an illustration
in yeast
of the detailed information provided by CARS microscopy on the
To illustrate the molecular specificity with which organelles appear distribution and morphology of lipid droplets in a single yeast cell
in CARS microscopy, an image of yeast cells was collected at in vivo, a full high-resolution three-dimensional image is shown
the resonance of the CH2 stretch vibration (2845 cm−1 ) and at in Fig. 4. It is reconstructed from an z-stack of CARS microscopy
751

2952 cm−1 by tuning the wavelength of the OPO from 817 to images consisting of 30 two-dimensional images. This promises

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c 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jrs
 C. Brackmann et al.

(a)

(b)

(c)

(d)

Figure 3. CARS (left column) and simultaneously collected two-photon fluorescence (middle column) microscopy images of labeled yeast cells. The
right column shows corresponding co-localization images with the CARS signal indicated by red color, the fluorescence by green color, and co-localized
regions by blue color. The cells had different organelles tagged by green fluorescent protein (GFP); (a) mitochondria, (b) peroxysomes (c) vacuole, and
(d) lipid droplets. Highest degree of co-localization can be seen for the lipid droplets (d).

for interesting studies on the lipid metabolism on a single-cell
level.
The impact of nutritional condition on lipid storage
To investigate whether nutritional state would affect the abun-
dance and size/morphology of lipid droplets, cells grown on
fermentative medium (glucose) or purely respiratory medium
752
shows examples of CARS images obtained from measurements
on the different types of yeast cells under study, illustrating
their different lipid storage morphology. Corresponding quan-
titative data obtained from the image analysis are summarized
in Table 1. With approximately 100 yeast cells in each group,
average droplet volumes of 0.21 and 0.18 µm3 were found for
cells grown on glucose and ethanol/glycerol, respectively. How-
ever, the average number of droplets per cell was found to be

(ethanol/glycerol) were compared by CARS microscopy. Figure 5 somewhat lower for cells grown on glucose. The evaluation re-

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c 2009 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2009, 40, 748–756
CARS microscopy of lipid stores in yeast

(a)

(b)

z
y

x
(c)
Figure 4. A three-dimensional reconstruction from a z-stack of two-
dimensional CARS microscopy images giving a volume image of the
lipid droplets in a yeast cell. The length of the x-axis arrow corresponds to
3 µm.

sulted in an average of 1.1 droplets (range: 0–4) per cell for cells
grown on glucose compared to 1.6 droplets (range: 0–4) per cell
for cells grown on ethanol/glycerol. Despite the slightly larger
volume per lipid droplet the total lipid volume per cell was still
(d)
lower (0.23 µm3 ) for glucose-grown cells than for ethanol/glycerol-
grown cells (0.28 µm3 ). This was confirmed by the biochemical
triglyceride determination, resulting in 1.5 ± 0.17 mg/g dry weight
on glucose-based medium and 4.9 ± 0.21 mg/g dry weight on
ethanol/glycerol-based medium.
The effect of long-term starvation was investigated by moni-
toring the lipid droplet morphology of commercial grade Baker’s
yeast. These cells prevail under starved conditions, and from
Fig. 5c we conclude that they show a large cell-to-cell variation in
(e)
their lipid contents. Beside the typical lipid droplet morphology
of well-fed cells, which is characterized by a few lipid droplets
per cell, there is a deviating population exhibiting a significantly
larger number of lipid droplets per cell. This is exemplified by
the uppermost cell in the image in Fig. 5c, where an entire clus-
ter of lipid droplets can be seen. The quantitative evaluation
confirmed that the number of lipid droplets per cell indeed is
larger for the starved cells compared to the well-fed cells. As
the size of the lipid droplets was similar, it follows that the to-
Figure 5. Examples of CARS microscopy images measured on yeast
tal lipid volume stored by the long-term starved cells is 3–4 cells of the different types investigated: (a) glucose-grown wild type,
times larger than the well-fed cells (see Table 1 for a summary of (b) ethanol/glycerol-grown wild type, (c) commercial grade Baker’s yeast,
data). (d) lipid-free mutant, and (e) bcy1-mutant.

The impact of gene deletion on lipid storage

Beside the nutritional impact on the regulation of the lipid
metabolism, we also studied the influence of specific genes central
for the cell metabolism. In the so-called lipid-free strain,[41] the
four genes controlling TGA and steryl ester synthesis (DGA1, LRO1,
ARE1 and ARE2) are lacking. These cells do not produce detectable
amounts of storage lipids,[41,47] and this was confirmed by us in
that no lipid droplets could be observed in the CARS microscopy
images, as exemplified in Fig. 5d. This is a further confirmation that
CARS microscopy probing the CH2 vibration specifically visualizes
For the bcy1 mutant, the complementary accumulation of
storage carbohydrates is instead deficient.[42] However, the impact
on lipid accumulation is not known, which motivated us to
investigate whether the bcy1-deletion strain also downregulates
neutral storage lipid levels. The bcy1 mutant exhibited lipid
droplets of similar size as the wild-type cells with an average
volume of 0.23 µm3 . However, the number of lipid droplets per
cell (on average 2.5) was higher, as shown in Fig. 5e and quantified
in Table 1. This means that the bcy1-deletion strain accumulates on
average twice as much storage lipids per cell as the corresponding
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the presence of TGA and steryl esters. wild-type strain.

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 C. Brackmann et al.

Quantification of lipid droplet volumes (a)

The evaluated lipid droplet volumes depend on the spatial

resolution achieved in the experiment. The size of the probe
volume for the two objectives used was determined from
experimental data on polystyrene beads and polymer films, and
values of 0.19 µm3 (axial resolution ∼1.5 µm) and 0.13 µm3 (axial
resolution ∼1 µm) were obtained for the 60 × objective and 100
× objective, respectively. The tight focusing of the laser beams
also strongly influences the signal generation from a small object
and confines smaller lipid droplets to only appear, with contrast
sufficient for evaluation, in a single plane of an z-stack with a
step-size in the z-direction of 0.5 µm. Hence, the data evaluation (b)
resulted in distributions, shown in histograms in Figs. 6a–d, of lipid
volumes including values smaller than that of the probe volume.
Even with an uncertainty in the real volume of these small droplets,
they were clearly distinguishable in the evaluation and constitute a
part of the total evaluated lipid content from the CARS microscopy
images. Average lipid droplet volumes have also been calculated
neglecting the small droplets restricted to appearing in a single
plane, and these values are also presented in Table 1 (indicated
by (∗ )). This evaluation naturally resulted in higher average lipid
droplet volumes, but nevertheless the sequence with the lowest
value for ethanol-metabolizing cells and the highest for Baker’s
(c)
yeast remains the same.

Discussion
We have developed CARS microscopy for noninvasive, high-
resolution, three-dimensional imaging of unlabeled yeast, one
of the most prominent model organisms for the fundamental
understanding of cellular processes. To our knowledge, this is
the first study of S. cerevisiae by means of CARS microscopy.
Proof-of-principle measurements on fission yeast (Schizosaccha-
romyces pombe) have been presented by Kano and Hamaguchi,[48] (d)
hypothesizing that the strong CARS signal at the CH2 stretching vi-
bration would be derived from mitochondria and the endoplasmic
reticulum (ER). Based on thorough comparison with two-photon
fluorescence microscopy on a selection of relevant GFP-labeled
organelles, the present study instead suggests that the images in
the work of Kano and Hamaguchi show lipid droplets, which in
turn prompts for thorough co-localization studies of fission yeast.
That CARS microscopy is able to specifically visualize intracellu-
lar lipid stores in the genetically tractable model organism yeast
highlights the potential of the technology within lipid research.
The high-resolution CARS microscopy images allowed us to
compare the number of stationary lipid droplets, their size, and Figure 6. Histograms of the evaluated lipid droplet size distributions
for the investigated types of yeast cells: (a) glucose-grown wild type,
the total lipid volume per cell. This kind of data has previously been (b) ethanol/glycerol-grown wild type, (c) commercial grade Baker’s yeast,
presented, though averaged over full populations based on either and (d) bcy1-mutant.
large-scale analysis of cellular extracts[49,50] or on fluorescently
labeled lipid droplets,[22,41,51] without considering the large cell-
to-cell variability. This work shows that the specific capabilities of of the large individual variability of lipid accumulation in higher
CARS microscopy give us access to unique single-cell data, from eukaryotes. We consider two possible explanations, not mutually
which we conclude that there is a clear individual variability in exclusive, for the heterogeneity: (1) one or several regulatory
lipid droplet content within a population. We have been unable factors involved in determining expression of lipid biosynthetic
to correlate this with cell-cycle phase, cell size, or replicative age or degradative proteins is subject to major stochastic noise or
in terms of bud scars (data not shown). Instead, the variation (2) high cell-to-cell variation in the number of organelles (e.g.
might be a result of stochastic processes (‘biological noise’), which ER, mitochondria, and peroxisomes) central for the synthesis or
is known to contribute to cell population heterogeneity. This degradation of storage lipids.[54] In order to conclude whether the
is well studied on the level of gene expression,[52,53] but the cellular heterogeneity in lipid stores is matched by a similar
existence and implications on lipid storage has not previously heterogeneity on the level of lipid-biosynthetic and/or lipid-
754

been discussed – a topic of interest also for the understanding degrading enzymes, we suggest combining CARS microscopy

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c 2009 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2009, 40, 748–756
CARS microscopy of lipid stores in yeast
studies with gene expression analysis, as well as with fluorescence
microscopy on GFP-labeled ER, mitochondria, and peroxisomes.
Beside the access to single-cell data, the major advantage of
CARS microscopy is the ‘true’ visualization of the distribution
of lipid droplets in the sense that the technique must not
rely on the labeling/expression of a reporter molecule. Due
to the small size of the lipids and their characteristic physical
properties, there are presently few fluorophores that provide
reliable labeling of this important category of biomolecules. It
is associated with, e.g. uncertainties in the labeling/expression
efficacy, photo-degradation of the reporter molecule, influence
of pH, temperature, and other environmental parameters on the
fluorescence generation, and altered physical properties of the
lipids.[23 – 27] Furthermore, in CARS microscopy there is no need to
induce overexpression of the target genes in order to display the
lipid droplets, i.e. the influence of genetic regulation mechanisms
can be studied under natural conditions. This is of particular
importance when studying the impact of genetic alterations on
lipid synthesis and degradation. Considering the discrepancy in
the results of genetic screening studies with respect to lipid
synthesis/degradation in S. cerevisiae employing fluorescence
microscopy, it would be interesting to repeat these studies with
the noninvasive CARS microscopy technique. While Szymanski
et al.[22] identified 59 genes affecting lipid storage, Fei et al.[51]
report 133 genes with an overlap between the two studies of
merely approximately 20 genes. This limited overlap indicates a
need for a more stringent approach, providing consistent results
and possibly also being able to identify further genes.
The usefulness of CARS microscopy within lipid research was
here further supported by addressing the highly relevant question
as to what extent the nutritional state (starvation, respiratory,
or fermentative growth) affects lipid storage mechanisms. In
addition to the interesting cell-to-cell variability, a slight difference
was still observed between the glucose- and ethanol/glycerol-
metabolizing cells when averaging the data, which was also
confirmed by biochemical analysis. Whereas the CARS microscopy
data suggest a factor of 1.3 larger lipid droplet volume per cell
at respiratory growth, the biochemical analysis results in a factor
of approximately 3. CARS microscopy probes both TGA and steryl
esters, and our data therefore suggests that it is primarily the
amount of TGA which is larger under respiratory growth conditions
whereas the difference in total lipid volume is minor. This notion is
supported by gene expression data in which DGA1, but not LRO1,
ARE1, and ARE2, was clearly induced following the diauxic shift (i.e.
after a shift from fermentative to purely respiratory growth).[55]
Dga1p is the major enzyme involved in acylation of diglycerides,
whereas Lro1p and Are1/2p are mainly involved in acylation of
sterols. The increased level of triglycerides has also been found
from a biochemical analysis of ‘stationary phase’ cells,[56] which is
probably equivalent to oxygen-limited cells on ethanol growth.
The most significant consequence of nutritional state on lipid
storage was observed for a subpopulation of the starved yeast cells,
exhibiting clusters of multiple lipid droplets. This may be a result of
genomic rearrangements reported for starved populations as an
adaptation to a nutrient depleted environment.[57] After 1 month
of starvation, genomic adaptation was observed for ∼70% of
the population compared to a control group. This prompts for
further investigations in which the lipid storage morphology of
a population exposed to long-term starvation is characterized
during the adaptation period by means of CARS microscopy and is
correlated with genetic analysis in order to identify the underlying
chromosomal rearrangements.
J. Raman Spectrosc. 2009, 40, 748–756 Copyright

c 2009 John Wiley & Sons, Ltd.
With the prospects of a genomewide screening, we studied
two different mutants: the so-called lipid-free strain (H1246) and
the bcy1-deletion strain. In the former, all four genes controlling
TGA and steryl ester synthesis (DGA1, LRO1, ARE1, and ARE2)
are simultaneously deleted,[41] and indeed no lipid droplets can
be observed in the CARS microscopy images (Fig. 5d). In a full
screening study, this must be complemented by the collection
and analysis of CARS microscopy images of mutants with single-
gene disruption, the results of which will be highly interesting
to compare with fluorescence microscopy studies.[22,41,51] As
an example of a single-gene deletion, the implication of the
bcy1-deletion on lipid storage, which has not been studied
previously, was investigated. The bcy1-mutant, which lacks the
regulatory subunit of protein kinase A (PKA) and therefore exhibits
constitutively high PKA activity, is known to be defective in the
accumulation of storage carbohydrates and also to be stress
sensitive.[42] Interestingly, the quantitative analysis of the CARS
microscopy images indicates that the deficiency in accumulating
glycogen and trehalose is to some degree compensated by
increased flow of carbon to storage lipids (see Fig. 5e), since
the average lipid volume per cell for the bcy1-mutant was
approximately twice that of the wild type.
Conclusion
In conclusion, we have paved the ground for label-free and
noninvasive imaging of lipid storage mechanisms in one of
the most important model organisms within cell and molecular
biology – yeast – by means of CARS microscopy. It allows us to
visualize and quantify lipid stores with molecular specificity and
sub-micrometer resolution. The access to a complete library of
viable deletion mutants together with the ability of label-free
microscopy opens up for a new era within genetic screening in the
field of lipid research, avoiding all uncertainties associated with
the addition of exogenous fluorophores. As initial examples of
this, the consequence of a multiple deletion of genes regulating
the synthesis of storage lipids (DGA1, LRO1, ARE1/2) was studied,
as well as a single-gene deletion of BCY1 regulating the synthesis
of storage carbohydrates. Finally, the intriguing question – also on
a higher eukaryotic level – of how lipid accumulation is affected
by the nutritional condition was addressed. Interestingly, we find
that lipid storage exhibits biological noise in the same manner as
previously reported for gene expression. Beyond this individual
variability, a fourfold increase in accumulated lipids could be
observed for a subpopulation of cells in conjunction with long-
term starvation. We also report the novel finding that the deficient
carbohydrate storage of the bcy1-mutant was compensated by
a twofold increase in neutral lipid storage. With the ability to
monitor lipid storage in yeast cells, a plethora of interesting studies
is made possible, with the prospects of improved fundamental
understanding of the impact from nutritional state and genetic
alterations on the lipid metabolism on a single-cell level.
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