3. Need to develop a deeper understanding of the relationship
between microbial, enzymatic and physiochemical parameters
and their influence on cheese quality, consistency and ripening
patterns
main goals :
-To develop new methods to measure differences in micro
heterogeneity in cheese pH
-To apply advanced methods to visualize the cheese matrix
components: phospholipids, fats, …
-apply advanced microscopic techniques and different types of
fluorescent dyes to examine the cheese matrix composition
Cheese-natural cheese type
4. Current microscopic methods to examine
cheese
SEM: freeze-fracture, cryo-SEM
CLSM: Nile blue staining (fat-green , protein-red)
Bacterial colonies/bacteria (live /dead)
Studied properties:
Cheese matrix microstructure (SEM, ESEM)
Chemical composition (CLSM)
Mechanical properties (rheology, changes in structure
during heating and cooling)
TEM
Mamdouh El Bakry, Jeremiah Sheehan Journal of Food Engineering 2014 (125), 84-96
10 μm 10 μm 5 μm 5 μm
20 μm 20 μm
20 μm
5 μm
5. Cheese - natural cheese type
LIGHT MICROSCOPY TECHNIGUES
1.Two photon excitation microscopy (TPEM)
2.Second Harmonic Generation Microscopy (SHG)
3.Fluorescent Lifetime Imaging (FLIM)
4.Raman Confocal Microscopy
Treatment 50 °C, BS 50 °C, DS 40 °C, BS 40 °C, DS
Milk volume 454 kg 454 kg 454 kg 454 kg
Starter cultures 1% S. thermophilus
0.5% L. helveticus
1% S. thermophilus
0.5% L. helveticus
1% S. thermophilus
0.5% L. helveticus
1% S. thermophilus
0.5% L. helveticus
Cook 0.5 °C min-1
to 45 °C
1 °C min-1
to 50 °C
0.5 °C min-1
to 45 °C
1 °C min-1
to 50 °C
0.5 °C min-1
to 40 °C 0.5 °C min-1
to 40 °C
Drain pH pH 6.30 pH 6.30 pH 6.30 pH 6.30
Curd handling Pre-press and mould Whey drainage
Cheddar curd
Pre- press and mould Whey drainage
Cheddar curd
Salting method Brine
23% (w/w) NaCl, 0.2% (w/w)
Ca, pH 5.30
Dry salt at 1.45% (w/w) Brine
23% (w/w) NaCl, 0.2% (w/w)
Ca, pH 5.30
Dry salt at 1.45% (w/w)
Manufacture parameters used during cheese making trials. Cheese vats were cooked to two various max scalds either 50 °C or 40 °C. These
vats underwent salting via Brine salting (BS) or Dry salting (DS)
7. Diaspro, A. et al. (2002),'Functional imaging of
living Paramecium by means of confocal and two-
photon excitation fluorescence microscopy', Optical
Diagnostics of Living Cells V 4622(1), in Daniel L.
Farkas & Robert C. Leif, ed.,, SPIE, 24-31.
Second Harmonic Generation
non-linear optical process
Requirements: High power of
incoming radiation, breaking the
center of symmetry
1) The ability to produce SHG is
an intrinsic property of molecules
lacking a center of symmetry (e.g.,
collagen, crystals), therefore, it
does not require any staining and
there is no photobleaching
2) SHG emission is directional, i.e.
anisotropic
3) Signal intensity is proportional to
collagen concentration.
λ
8. Fluorescence lifetime imaging Microscopy
(FLIM)
- Fluorescence based method
- analysis of the lifetime of the excited state of fluorescent molecule
- Combination of this analysis with imaging
- - spatially resolved distribution of fluorescent lifetimes
- - detect environmental parameters that affect lifetime
9. Raman scattering
- Instantaneous process (zero lifetime)
- very weak process (less than 1 in 1 million photons scattered)
- each vibrational level is represented by a peak in Raman spectrum
- - high information content (compared to fluorescence)
- - high chemical specificity
- Raman confocal = confocal microscope equipped with sensitive spectrometer
- - local chemical information in 3D
S0
S1
S2
virtual
state
(10-15
s)
proportional
to the energy
of vibration
10. CLSM - lipids
Nile blue – fat / protein Rhodamine-DOPE - phospholipids
Fluorescent
marker
Lifetime ( τ1)
[ns]
Colour in image
Nile Blue
2.5 ⁺/₋ 0.16 red
3.5 ⁺/₋ 0.16 green
Rhod- DOPE 2.2 ⁺/₋ 0.16 light green
Christelle Lopez, Valerie Briard-Bion, Eric Beaucher, Michel
Ollivon J. Agric. Food. Chem. 2008, 56, 2406-2414
20 μm 20 μm
10 μm 10 μm 10 μm
10 μm
FLIM
11. Non-linear microscopy
Two photon excitation microscopy
Second Harmonic Generation Imaging
CLSM 20-30 µm /TPEM 40 µm
SHG imaging - calcium lactate crystals - influence the cheese texture and
quality attributes
5 μm
100 μm
50 μm
12. Macro level
Standard methodology: pH meter
Micro level
A) pH-dependent fluorescent probes , CLSM
- Fluorescent probe - C-SNARF-4
- Non-fat cheese model cheese
- pH around Lactococcus colonies
- No pH microgradient occurred around
Lactococcus colonies
B) pH-dependent fluorescent probe, MP FLIM
- Fluorescent probe - BCECF-AM, FITC, acridine orange,
C-SNARF4
- Insufficient in standard natural cheese
- Sufficient fluorescent probe – oregon green 488
pH micro-heterogeneity in natural
cheese matrix
BCECF-AM
Sophie Jeanson, Juliena Floury, Al Amine Issulahi, Marie-NÖelle Madec,
Sylvie Lortal 2013 Applied and Environmental Microbiology 6516-6518
FITC
Acridine Orange
13. Oregon Green 488
MP-FLIM
- is more photostable than fluorescein
- havs a lower pKa (pKa = 4.7 versus 6.4 for fluorescein)
- pH sensitive in the weakly acidic range (pH 4 to 6)
in rev. Zuzana Burdikova, Zdenek Svindrych, Jan Pala, Cian Hickey, Martin G Wilkinson, Jiri Panek Mark A.E. Auty, Ammasi Periasamy, Jeremiah J.
Sheehan 2014 Measurement of pH micro-heterogenity in natural cheese matrices by fluorescent lifetime imaging
Left: FLIM images of cheese stained with Oregon Green 488 buffered to pH 3.0 to 6.0. The
pseudocolor represents lifetime (red – 1.5 ns, green – 2.5 ns, blue – 3.5 ns), intensity represents
photon count, captions denote the pH of the buffer solution (-.- denotes unbuffered sample).
Right: calibration data extracted from FLIM images.
14. Raman Confocal Microscopy in cheese
research
20 μm20 μm
10 μm
20 μm
Pseudocolor overlay
Red (Fat), Green (Protein), Blue (water)
15. Calcium lactate crystals
-can be seen with Second Harmonic Generation microscopy technique
Fats and phospholipids
- more precisely determine localisation of fats and phospholipids using
theTwo Photon Excitation Fluorescent Lifetime Microscopic
Techniques
Micro heterogeneity in pH
- can be measured with Oregon Green fluorescent probe and
two photon fluorescent excitation fluorescent lifetime microscopy
Detailed chemical composition of cheese (water, protein, fats)
- Raman confocal microscopy is very promising technique
Main conclusions
16. 1.Z. Burdiková, C. Hickey, M. A. E. Auty, J. Pala, Z. Švindrych, I. Steinmetz,V. Krzyzanek, K.
Hrubanova, J.J. Sheehan ( 2014). Cheese Matrix Microstructure Studied by Advanced
MicroscopicTechniques. Microscopy and Microanalysis: Vol.20 pp. 1336
2. Z. Burdikova, Z. Svindrych, J.Pala, C. Hickey,V. Cmiel, M.A.E. Auty, J.J. Sheehan Fluorescence
Lifetime pH Measurements in Cheese Matrix (2014). Microscopy and MicroanalysisVol. 20, pp
1358
3. in rev. Z. Burdikova, Z. Svindrych, J. Pala, C. Hickey, M. GWilkinson, J. Panek M. A.E. Auty, A.
Periasamy, J. J. Sheehan (2015). Measurement of pH micro-heterogeneity in natural cheese
matrices by fluorescent lifetime imaging. Frontiers in Microbiology
4. in prep. Z. Burdikova, Z. Svindrych, C. Hickey, M.G.Wilkinson, M.A.E. Auty, J.J. Sheehan
(2015) Potential for application of advanced light microscopic techniques to gain deeper
insights into cheese matrix physico-chemistry. Dairy Science andTechnology
1.Microscopy and Microanalysis 2014 – poster presentation
2.18th
International Microscopy congress 2014 – poster presentation
3.9 th Cheese symposium 2014 – oral presentation
17. Teagasc:
Diarmuid J. J. Sheehan, Ph.D.
Bc. Cian Hickey
Mark A.E. Auty, Ph.D.
UCC
Msc. Kamil Drapala
Seamus O`Mahony, Ph.D.
Leica microscopy center Mannheim, Germany:
Dr. Jan Pala
Keck Center for Cellular Imaging, USA:
Prof. Ammasi Periasamy, Ph.D.
Zdenek Svindrych, Ph.D.
Acknowledgements
…. and you for your attention
19. E
p = χ2 E2
p (t) ≈ χ2 E0
2
cos(2ωt)
E(t)=E0cos(ωt)
Quadratic component of polarization
causes frequency doubling
Second Harmonic Generation
E
p = χ1 E
p (t)= χ1 E0 cos(ωt)
E(t)=E0cos(ωt)
Linear polarization leads to
refractive index 11 += χn
12 χχ < <
a non-linear optical process, in which an input (pump) wave generates
another wave with twice the optical frequency (i.e., half the wavelength).
20. C-SNARF-4 pH-sensitive fluorescent probe,
MP FLIM
The ratio of fluorescence
intensities Igreen/Ired of C-
SNARF-4 ratiometric pH
probe in pH range of 3.0 to
7.0, excited at 480 nm
(solid symbols) ad at
530 nm (open symbols).
Excitation-emission lambda scans of the C-
SNARF-4 solution buffered to pH 5.5 (A) without
timegate and (B) with timegate 1 ns (combination
of spectral information and lifetime information).
- seminaphthorhodaf-4F 5-(and 6) carboxylic acid ( C-SNARF-4)
-Long wavelength pH indicator
-In more acidic environments the pH sensitivity decreases rapidly
-In natural model cheese C-SNARF 4 indicates no pH sensitivity in the
range of 3.7- 5.0
The effect of pH on the C-
SNARF-4 fluorescence
lifetimes:, two-component
model, the longer lifetime
component τ2 shows
pronounced very weak pH
dependence.
Editor's Notes
Good afternoon Ladies and Gentlemen,
First of all I would like to thank the organisers for the opportunity to present my results on this exciting symposium.
My name is ZB and I work in Teagasc in dept. of Food Technology placed in Moorepark, Fermoy as a postdoc. ??? My supervisors are Diarmuid Sheehan and Mark Auty.
I would like to introduce few advanced light microscopy techniques and their applications in cheese research.
The title of my presentation is….
The processes during the cheese ripering are still not well known and to find out the relationship between the microbial, enzymatic, physiochemical parameters on the one side and cheese quality, consistency and ripening patterns on the other side is still challenging.
The main goals of our study were:
-
-
-
Let me now remind you the traditional microscopic techniques employed in cheese research.
These are namely scanning electron microscopy which allow us to see the detailed surface of the cheese matrix and cryo scanning electron microscopy.
With this technique we can study individual components, as well as details of bacteria or bacteria placement in protein matrix.
Researchers also widely apply the confocal laser scanning microscopy to cheese research.
The most common application in the cheese research is the visualisation of fat and protein components or separation of dead and live bacteria inside the cheese.
All my work was done on cheddar cheese.
The natural cheese type was prepared with a standard procedure described in the table ….
Diarmuid please add 2 sentences…
I will report my results using the following light microscopy techniques applied to the cheese matrix.
Namely it is …1-4.
The basic principe of flurescence microscopy is illustratet in the letf side of the image.
Standart fluorescence and confocal microscopy use the fluorescent lamp or laser. They are one photon excitation systems.
1. During the normal fluorescence event a photon of sufficient energy is absorbed and the flourophore is excited to a higher electronic state. After a few nanoseconds the molecule goes back to the
ground state by emitting a photon of significantly lower energy (longer wavelength).
During two photon excitation event two photons of half the energy are absorbed simultaneously to achieve the excited state (the energy of a single photon is not sufficient to excite fluorescence of the fluorophore).
The main feature of TPEM is that the intensity of emmited light is not linearly proportional to the excitation intensity (as is the case for common fluorescence microscopy) but rather depend on the square of the excitation intensity. As a result the fluorescence originates mainly from the areas with maximum excitation intensity (the focus). So the out of focus fluorescence does not need to be blocked and the confocal pinhole is not necessary.
However, for this phenomenon to appear, the excitation intensity must be exceptionally high (megawatts per square micrometer) and this is only achievable with special pulsed lasers (about 100 femtosecond pulses).
V dvoufotonovem procesu je intenzita emise umerna druhe mocnine excitaxni intenzity, proto emise vychazi hlavne z mist s nejvyssi intenzitou exitacniho svetla – z ohniska.
Jsou potreba opravdu vysoke intenzity svetla, proto se pouzivaji pulsni lasery s delkou pulsu radove 100 fs (femtosekund) a spickovym vykonem radu jednotek az stovek MW (megawatt).
SHG is non-linear optical process with two requirements – high power of incoming radiation and non-centrosymmetric molecules in the sample
The SHG is based on the optical effects which are induced by specific inherent physical properties of a specimen.
The SHG signal is mostly emitted by collagen fibres or crystals.
This technique allows us to visualise specific structure inside the sample without labelling.
The fluorescence of organic molecules is not only characterized by their excitation and emission spectra, but also by their lifetimes.
When a fluorophore absorbs a photon it goes into the excited state and returns to the ground state by emitting a fluorescence
photon, converting the energy internally, or by transferring the energy to the environment, or a combination of those.
Molecular emission is stochastic event, but the lifetime of a population of molecules can be accumulated and plotted. If a large number of similar
molecules with similar local environments are excited by a short laser pulse, the time taken for fluorescence to decay can be plotted as a single exponential curve. Providing
that no energy is transferred to the environment, the lifetime described by this curve is called ‘natural’ fluorescence lifetime.
If energy is transferred to the environment, the actual fluorescence lifetime is shorter than the natural fluorescence lifetime. For almost all fluorophores, the rate of energy transfer
to the environment depends on the concentration of ions, oxygen, pH value or the binding of proteins in a cell. There is a direct relation between the concentrations of these ions,
called fluorescence quenchers, and the fluorescence lifetime of the fluorophore. Consequently, FLIM can not only be used to discriminate between different fluorophores
on the basis of their characteristic lifetimes (rather than their spectral properties) but also to distinguish among different environments within the cell based on changes in lifetime
of the same fluorophore if it is present in local environments containing varying concentrations of fluorescence quenchers.
Typical fluorescence lifetime is few (1-10) nanoseconds
Raman scattering is another process, that can be used in microscopy.
It is very weak process (typically less than 1 ppm of incoming photons is inelastically scattered).
It has zero lifetime, thus FLIM cannot be used to extract additional information. However the spectrum of the scattered photons (Raman spectrum) shows a high level of detail.
Every peak of the spectrum corresponds to some vibrational level of the scattering molecule, usually a specific chemical bond type.
the difference of energies of the incoming and scattered photon is equal to the energy of the vibrational level
databases of chemicals and their Raman spectra – decomposition (e.g. PCA) of the measured spectrum into individual chemical components
One of the main components of cheddar cheese are lipids, its not easy to separate them.
Standard microscopic methods to visualise lipids in cheese is to label the cheese by a lipophilic fluorescent marker and acquire the image by CLSM.
For the fats is usually applied Nile blue fluorescent dye. See the image 1 and 2
We apply the same fluorescent dye Nile blue and measure the emission signal with two photon excitation FLIM microscopy technique.
From the results is obvious, that the separation FLIM microscopic technique allow to us to more precise localisation fats in cheese.
The Nile blue in cheese matrix shows broad variability of lifetime, that apparently correlates with the size of fat droplets. This phenomenon deserves more testing, as it may suggest, that the composition of lipid droplets may depend on their size…
Based on the work of Christelle Lopes we also applied the fluorescent marker Rhodamine-DOPE for phospholipids.
On the image from CLSM we see the placement of phospholipids on the edge between the fats droplets and protein matrix.
Again the corresponding FLIM image shows additional details….
Whit this microscopy technique we try to visualise the crystals inside the cheese matrix.
First we applied the standard methodology: here is the example: calcium lactate crystal visualised by cryo SEM.
The arrow points to calcium lactate crystal in the cheese.
On the left side are images of calcium lactate crystals visualised by SHG microscopy techniques.
The SHG signal also depends on the polarisation of excitation light with respect to crystallographic orientation of calcium salt crystal. To demonstrate this we rotated a polarizer and acquire the image at angles 0 (green) 30 (red) and 60 (blue) degrees. This final image is a merge of all tree angles.
We can overlay this SHG image with a fluorescent signal which originates from proteins (red), fat (green). Nile blue was used in this case.
The white signal is SHG signal which originate from calcium lactate crystals.
One of the main challenging question was how to measure the pH at microscale.
Traditionally we can measure the pH at macrolevel with a pH meter (probe), but there is significant knowledge gap relating to the degree of microheterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency etc.
On the micro level Sophie Jeanson measured the pH gradient around Lactococus colonies in non-fat model cheese. To measure pH gradient around bacterial colonies she applied the C-Snarf-4 pH sensitive
fluorescent dye and confocal laser scanning microscopy. She found no pH gradient in non-fat model cheese around the colonies.
In our case we labeled the natural type of cheese with pH sensitive pH fluorescent dyes: namely BCECF-AM, FITC, Acridine Orange, C-snarf-4 and Oregon Green 488. Then we measure the lifetimes of these fluorescent dyes in different pH’s using time domain single photon counting FLIM.
From our result we learned that BCECF-AM, FITC and Acridine Orange are not pH sensitive in natural type of cheese.
Finally we found that Oregon Green 488 (dextran) displays good pH sensitivity even when applied to the cheese matrix.
We have verified this by buffering the stained cheese sample to different pH values.
Left: FLIM images of cheese stained with Oregon Green 488 buffered to pH 3.0 to 6.0. The pseudocolor represents lifetime (red – 1.5 ns, green – 2.5 ns, blue – 3.5 ns), intensity represents photon count, captions denote the pH of the buffer solution (-.- denotes unbuffered sample (just water)). Right: solid symbols – the pH dependence of mean lifetime calculated from the FLIM images, open triangles – mean lifetime of stained cheese without pH buffer (determined from image denoted -.-). Error bars represent the standard deviation, mainly caused by sample inhomogeneity.
We also used Confocal Raman Imaging to study the composition of the cheese.
The main problem here is that the Raman signal is extremely weak and can easily get lost in sample autofluorescence.
However, we succesfully separated the main cheese components, namely fat (in red), protein (in green) and water (in blue).
I would like to thank …
A you for your attenttion….
detailed principle of SHG
it is clear from signal theory that any nonlinearity generates higher harmonics of the excitation field
even though these nonlinearities are extremely weak, they can become apparent in extremely strong field
used extensively in laser technology – 532nm (green) lasers
useful in microscopy – only limited number of asymmetric systems generates second harmonic (various interfaces, crystals without inversion symmetry, biology: collagen, actin/myosin, …)
C-SNARF-4 dye shows rather good sensitivity towards pH when the ratio of fluorescence intensity in two spectral channels is measured. However,
at pH less than 5.0 the sensitivity drops quickly.
We performed excitation-emission spectral scans to confirm, that the spectral properties of the dye does not change at acidic pH’s.
But we observed quite strong effect when we used timegating, i.e. the photons arriving within 1 ns after the extitaion pulse are discarded.
Indeed, with true single photon FLIM measurements we were able to extend the useful pH range of the dye down to about 3.5.
But due to the strong affinity of the dye towards the protein matrix this dye is not usable in practical situations (both the spectral properties and lifetime change significantly when applied to cheddar cheese matrix).
spectral scan: Vertical axis shows excitation wavelengths of White Light Laser (470-670 nm), horizontal axis displays emission spectra (480-730 nm).
