Hyperspectral Deep Ultraviolet Autofluorescence of Muscle Fibers Is

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Hyperspectral deep UV autofluorescence of muscle fibers is affected by postmortem changes Caroline Chagnot, Annie Venien, Frédéric Jamme, Matthieu Refregiers, Mickaël Desvaux, and Thierry Astruc J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00668 • Publication Date (Web): 27 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Journal of Agricultural and Food Chemistry

Hyperspectral deep UV autofluorescence of muscle fibers is affected by postmortem changes

1 2 3 4 5 6 7 8

Caroline Chagnot1, 2, Annie Vénien1, Frédéric Jamme3, 4, Matthieu Réfrégiers4,

9

Mickaël Desvaux2 and Thierry Astruc1

10

1

11

France.

12

2

13

3

14

UR370 Qualité des Produits Animaux, INRA, F-63122 Saint-Genès Champanelle,

UR454 Microbiologie, INRA, F-63122 Saint-Genès Champanelle, France Synchrotron SOLEIL, BP48, L’Orme des Merisiers, F-91120 Gif-sur-Yvette, France

4

INRA, UAR1008 CEPIA, rue de la Géraudière, F-44316 Nantes, France

15 16 17 18

Corresponding author: Thierry Astuc

19

E-mail: [email protected]

20

Tel. +33 4 73 62 41 56

21

Fax +33 4 73 62 42 68

22 23 24

Short title: Postmortem muscle fibers fluorescence spectroscopy

25 26

1 ACS Paragon Plus Environment


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Abstract

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After slaughter, muscle cells undergo biochemical and physicochemical changes that

29

may affect their autofluorescence characteristics. The autofluorescent response of

30

different rat Extensor Digitorum Longus (EDL) and soleus muscle fiber types was

31

investigated by deep UV synchrotron microspectroscopy immediately after animal

32

sacrifice and after 24 hours storage in a moist chamber at 20 °C. The glycogen

33

content decreased from 23 to 18 micromoles / g fresh muscle in 24 hours

34

postmortem. Following a 275 nm excitation wavelength, the spectral muscle fiber

35

autofluorescence response showed discrimination depending on postmortem time (t0

36

versus t24h) on both muscles at 346 nm and 302 nm, and to a lesser extent at 408 nm

37

and 325 nm. Taken individually, all fiber types were discriminated, but with variable

38

accuracy, type IIA showing better separation of t0 / t24h than other fiber types.

39

These results suggest the usefulness of the autofluorescent response of muscle cells

40

for rapidly meat aging characterization.

41 42 43

Key words: rat muscle fibers; postmortem; meat; UV spectroscopy; histology ; fiber

44

types

45 46 47 48 49 50 51


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Introduction

53

Meat comes from skeletal muscle, which generally contains different proportions of

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the four pure myofiber types: Type I, Type IIA, Type IIX and Type IIB. These contain

55

respectively the myosin heavy chain isoforms (MyHC) I, IIa, IIx and IIb

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fibers intermediate between two pure types and containing two different myosin

57

isoforms (I-IIA, IIA-IIX, IIX-IIB) are also found. The muscle fibers are characterized by

58

their metabolism (oxidative or glycolytic) and their contraction speed (slow or fast).

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Type I are slow-twitch fibers and all Types II are fast-twitch fibers. Type I and IIA

60

fibers have an oxidative metabolism; IIX and IIB fibers have a glycolytic one 1, 2.

61

After slaughter of farm animals, muscle undergoes significant metabolic, physical,

62

structural and biochemical changes that determine the quality of meat and meat

63

products

64

cells will try to survive by degrading their glycogen stock through anaerobic

65

glycolysis. Lactate accumulation in cells is accompanied by a drop in pH. When the

66

energy reserves are depleted, muscle pH stabilizes at values generally between 5.7

67

and 6.2, termed the ultimate pH. The kinetics of change in pH is species-dependent,

68

and the time to reach the ultimate pH can range from 2 h in poultry to 24 h in cattle.

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After this first phase of postmortem changes, additional biochemical changes and

70

significant ultrastructural alterations are observed, related to the improvement of

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meat quality, especially meat tenderness

72

changes (also called meat maturation) is dependent on the species, and mostly on

73

the metabolic and contractile types of the muscles considered. Degradation of

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myofibrillar structure is faster in white than in red fibers 5, 7.

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Postmortem changes can be assessed by mechanical measurements and

76

biochemical and/or ultrastructural analysis, but these methods are usually destructive

1, 2

. Hybrid

3, 4

. After bleeding, the muscle is deprived of oxygen and nutrients. Muscle

5, 6

. The speed of these postmortem



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and time-consuming. Some authors have exploited the variation in the physical

78

properties of muscles to characterize differences in composition by fluorescence

79

spectroscopy 8-10. This highly sensitive method detects fluorophores naturally present

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in muscle whose properties are very sensitive to changes in their environment.

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Collagen,

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autofluorescent molecules in muscle 8. Several years ago, the autofluorescence of

83

tryptophan signal following meat sample excitation at 290 nm, allowed a correct

84

discrimination of non aged from aged meat samples

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performed on a macroscopic scale and did not address the metabolic and contractile

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properties of muscles. The analysis at the cell or even the ultrastructural scale is

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more difficult because of the low availability fluorescence microscopes that allow an

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excitation in the deep UV which is relevant for studying biomacromolecules that have

89

a strong absorption in this energy range. Indeed, although spectrometers can often go

90

down to 250 nm, microscopes are usually very limited in this range with either 350 nm

91

cut-off or access to only a limited number of excitation wavelengths. However, the

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combination of a microscope with a UV fluorescence spectrometer coupled to

93

synchrotron radiation allows an excitation range of 200 to 600 nm that has been used

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to characterize animal tissues at the ultrastructural level 12-15.

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Thanks to the availability of these tools, it becomes theoretically possible to detect

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small changes of the tissue and cells and therefore characterize the degree of

97

postmortem

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autofluorescence changes of individual muscle fibers from rat skeletal muscles

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stored under anoxia during 24 hours, regarding their metabolism and ultrastructure

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tryptophan,

change

tyrosine

in

muscle

or

NADH

cells.

are

Our

among the

11

most

abundant

. However, the analysis was

goal

was

to

investigate

the

changes.


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Materials and Methods

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Animals and samples

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The rat model was chosen because it allows a better control of rearing and slaughter

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conditions; it is easy to extract entire muscles, and obtain early samples in the

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laboratory by controlling the best kinetic sampling and temperature conditions.

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Twenty-four male Wistar rats aged 5 months and weighing about 500 g were bought

107

from Janvier (Saint-Berthevin, France) and housed at 22 °C under 12 h:12 h

108

dark:light cycles with water and food ad libitum at an INRA animal facility (Installation

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Expérimentale de Nutrition, Unité de Nutrition Humaine, INRA Theix, agreement

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No. C 63345.14). Rats were anaesthetized by isoflurane gas and decapitated with a

111

guillotine according to the recommendation of our Regional Ethics Committee

112

(C2E2A No. 2) and French national legislation (JO 87-848). As no act of pain was

113

performed, since the sampling took place after the death of animals, the experimental

114

project was outside the scope of the European Union Directive 2010/63 and therefore

115

did not require ethics review. However, the experimental project was followed by the

116

animal welfare structure integrated in the INRA animal facility installation according to

117

French law (Article JO R. 214-103).

118

Immediately after death, the lower limbs were severed and dissected under sterile

119

conditions. Extensor digitorum longus (EDL), tibialis anterior, gastrocnemius and

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soleus muscles from the right and left hindlegs were extracted from tendon to tendon

121

avoiding any lesions.

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Muscle postmortem kinetics

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Muscles from three rats were immediately cryofixed post-dissection (10 minutes after

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decapitation). For the other 21 rats, muscles were suspended at 20 °C in a sterile

128

moist chamber to prevent drying of the muscle and contamination from

129

microorganisms. Lead ballast was attached at the caudal tendon of each muscle

130

(1.5 g for soleus and EDL, 4.5 g for tibialis and gastrocnemius) to preserve the

131

anatomical muscle length (Figure 1).

132

After respectively 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h postmortem, muscles of

133

three rats were removed from their moist chambers and prepared for biochemical

134

and imaging analysis.

135 136

pH measurements

137

As EDL and soleus muscles are too small, pH measurements were made on tibialis

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anterior and gastrocnemius muscles localized around EDL and soleus muscle. pH

139

measurements were made according to Fernandez et al., (2002)

140

small quantity of muscle available. Briefly, muscles were weighed and ground in

141

5 mM idoacetate buffer (2.5 ml/g of muscle), and pH was measured in the muscle

142

suspension at room temperature.

16

, adapting to the

143 144

Glycogen determination

145

Glycogen content was determined on each right EDL and soleus rat muscle at each

146

time postmortem by enzymatic procedures according to Bergmeyer, (1974)17, with

147

slight modifications. About 0.15 g of muscle tissue was homogenized with 1.8 mL of

148

0.55 M perchloric acid. On two 0.5 ml aliquots, glycogen was hydrolysed in glucose

149

with amyloglycosidase. Glucose was converted into 6-phosphogluconolactone after


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hexokinase and G-6-P dehydrogenase action accompanied by NADH H+ production.

151

NADH, proportional to glucose content, was determined by spectrophotometry at 340

152

nm and compared with standards to calculate the glucose concentration. Results are

153

expressed in micromoles per gram of fresh tissue.

154 155

Histology

156

Cryofixation and histological sections: Parts of left EDL and soleus muscles at 0 (t0)

157

and 24 h (t24h) postmortem were positioned on a cork plate with embedding medium

158

(Tissue-Tek) and cryofixed by immersion at −160 °C in isopentane cooled with liquid

159

nitrogen (−196 °C). Serial cross-sections of entire muscles (10 µm thick) were cut

160

using a cryostat (Microm, HM 560) and collected on glass slides for histological

161

stains, and on quartz coverslips for DUV microspectroscopy fluorescence analyses.

162

Sections were stored at −20 °C under vacuum until use. Periodic acid Schiff (PAS)

163

staining was performed for glycogen characterization 18.

164 165

Muscle fiber type determination: Fiber types were identified by highlighting the

166

different myosin heavy-chain isoforms (MyHC) using specific mouse monoclonal

167

antibodies BA-D5, SC-71 and BFF3 (AGRO-BIO France). The different primary

168

MyHC antibodies were visualized by an Alexa Fluor 488 labelled goat anti-mouse

169

IgG secondary antibody (A 11001, Invitrogen). In addition, the ECM protein laminin,

170

surrounding the muscle fiber, was stained using an anti-laminin primary polyclonal

171

antibody (L9393 Sigma) and a Cyanine 3-labelled secondary antibody (111-165-008,

172

Jackson). The myofiber response to the different MyHC antibodies identified fiber

173

Types I, IIA, IIB and hybrid IIX-IIB. IIX fiber types corresponded to the remaining



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unmarked cells. Controls were performed with no primary antibody to validate the

175

results.

176 177

Image acquisitions: Observations and image acquisitions were performed using a

178

light microscope (Olympus BX 61) coupled to a high resolution digital camera

179

(Olympus DP 71) and the Cell F software. PAS-stained sections were examined, and

180

images were acquired in bright field mode. Immunohistochemistry images were

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acquired in fluorescence mode (Alexa Fluor 488: 495/519 nm, Cyanine 3: 550/570

182

nm).

183 184

Transmission electron microscopy: Analysis was conducted on EDL and soleus

185

muscles at 0 and 24 h postmortem from 6 rats (3 rats per postmortem time). Pieces

186

of EDL and soleus muscles of about 4 × 1 mm were fixed overnight at 4 °C by

187

immersion in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2, taking

188

into account muscle fiber direction. Small cubes of about 1 mm3 were post-fixed in

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1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at room temperature.

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These cubes were dehydrated through a graded ethanol series and embedded in

191

epoxy resin (TAAB, Eurobio France). Ultra-thin longitudinal sections (90 nm) (parallel

192

to the fiber direction) were stained with uranyl acetate and lead citrate, and examined

193

with a transmission electron microscope (Hitachi HM 7650) using a 80 kV

194

acceleration voltage at the CICS cellular imaging lab on the Clermont-Ferrand

195

University campus (France). Micrographs were acquired with a Hamamatsu AMT

196

digital camera system coupled with the microscope.


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Deep UV fluorescence microspectrocopy

198

Spotting of cell types on unstained muscle serial cross-section: From two rat soleus

199

and EDL muscles (0 and 24 h postmortem), an unstained serial section was

200

deposited

201

immunohistofluorescence on other serial sections, 20 Soleus muscle fibers (10 Type

202

I and 10 Type IIA) and 21 EDL muscle fibers (3 Type I, 2 Type IIA, 5 Type IIX, 8 Type

203

IIX-IIB and 3 Type IIB) were spotted for fluorescence spectra acquisition by deep UV

204

(DUV) microspectroscopy.

on

quartz

coverslips,

and

using

fiber

type

identification

by

205 206

Spectrum acquisition: Synchrotron DUV microspectroscopy was performed on the

207

DISCO beamline of the SOLEIL synchrotron radiation facility (Saint-Aubin, France) 19,

208

20

. DUV monochromatized light was used at 275 nm excitation wavelength to excite

209

tissue sections. The emission spectra were acquired from 290 to 540 nm. For each

210

excited pixel a fluorescence spectrum was recorded. On each spotted cell, 20

211

acquisitions were made in the intracellular space.

212 213

Spectrum processing and statistical analysis: Autofluorescence spectra were spike-

214

and noise-filtered using an in-house program written in MATLAB version 7.3 (The

215

MathWorks, Natick, MA). The Unscrambler software (v9.8, Camo Software AS,

216

Norway) was used to perform a baseline adjustment to zero, apply unit vector

217

normalization that normalizes sample-wise the spectral data to unit vectors, and

218

analyze the processed spectra by principal component analysis (PCA). After

219

analysis, the family label of each spectrum was revealed and the two first of the 10

220

components tested were plotted. The mean spectrum of each group was also plotted

221

for better referencing of the spectral discriminants. Score plots were used to show



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similarity maps for comparison of spectra regardless of sample categories. Loading

223

plots derived from the first (PC1) and second principal components (PC2) were used

224

to reveal and identify characteristic fluorescence peaks.

225

Numerical variables of fluorescence intensity for wavelengths of interest identified on

226

the PCA loadings were expressed as mean ± standard error of the mean (s.e.m.).

227

Variance analysis and mean comparisons were performed using one-way variance

228

analysis and the Student-Newman-Keuls test under the XLSTATsoftware 2010

229

(Microsoft office, Redmond, USA).


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Results and Discussion

231

Postmortem metabolism time course

232

The level of EDL and soleus muscles glycogen was about 23 µmol / g fresh muscle

233

at 10 minutes postmortem, and decreased slightly with time for both muscles (Figure

234

2). The decrease in contrast of PAS staining with time postmortem reflects a

235

decrease in intracellular glycogen, in agreement with our biochemical data and the

236

literature

237

on the fiber type. Glycolytic fibers (IIX and IIB) of the EDL were systematically richer

238

in glycogen than oxidative fibers (I and IIA), fully consistent with the literature on the

239

subject

240

a heterogeneous distribution. In this muscle, the IIA fibers contained less glycogen

241

than Type I fibers. This result is surprising at first sight because generally IIA fast-

242

twitch fibers have more glycogen reserves than Type I slow-twitch fibers to cope with

243

high energy consumption during vigorous exercise. This finding is explained by a

244

specific feature of rodent muscles (rats and mice), whose Type IIA fibers are richer in

245

mitochondria, and thus more oxidative than their Type I fibers

246

logical for it to be less richly supplied with glycogen. The initial level of glycogen

247

(around 23 µmol / g fresh muscle) was lower than reported by others who found 30–

248

40 µmol / g of fresh muscle in the rat muscle

249

the delay of 10 minutes necessary for muscle dissection when part of the glycogen

250

was depleted; 60% of muscle glycogen is degraded within 5 minutes after the death

251

of the animal

252

and gastrocnemius muscles), slightly lower than usual in vivo pH (7.2), as a

3, 4

3, 4

. However, soon after animal sacrifice, PAS staining intensity depended

. The soleus, which contains only oxidative fibers (I and IIA), also showed

23

23, 24

21, 22

. It is therefore

. This difference is probably due to

. This hypothesis is in accordance with the initial pH (6.9 in Tibialis

ACS Paragon Plus Environment

11


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consequence of glycogen degradation associated with a release of protons in the

254

tissue.

255

The rat muscle glycogen content appeared to be considerably lower than seen in

256

farm animal species where values of glycogen are about 60–70 µmol / g in rabbits 25,

257

85–100 µmol / g in pigs

258

29, 30

26, 27

, 45–50 µmol / g in turkeys

28

, 80–100 µmol / g in cattle

and about 70 µmol / g in sheep 31. Glycogen reserves remained relatively high in

259

rat muscle (Figure 2), unlike in farm animals whose meat glycogen reserves fall to

260

less than 10 µmol / g after rigor mortis, with a pH stabilization around 5.6–6.0

261

depending on the type of muscle 3, 4.

262

The initial pH of the gastrocnemius and tibialis muscle decreased and has reached

263

its minimum (6.06 ± 0.12 and 6.15 ± 0.03 respectively) after 2.5 hours postmortem,

264

which is consistent with the postmortem time of the stopped of glycogen consumption

265

in soleus and EDL muscles (Figure 2).

266 267

Postmortem ultrastructural changes

268

Representative images of ultrastructural analysis are shown in Figure 3. Shortly after

269

animal sacrifice, the myofibrils were well-preserved with well-defined sarcomeres and

270

aligned Z lines. However, the soleus showed contracted myofibrils that prevented the

271

I bands being distinguished. This contraction may be related to mechanical

272

stimulation during sampling, but also to cold shortening despite our precautions to

273

avoid this problem (fixative immersion at room temperature). The fact that only the

274

soleus muscle showed this state leads us to believe that it was due to cold

275

shortening as only red muscles are sensitive to cold shortening 3.

276

After 24 h postmortem, the myofibrils showed no significant change in their structure.

277

Soleus myofibrils were still contracted, but Z lines remained aligned, and


12

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mitochondria were well-preserved despite the storage temperature of 20 °C, which

279

promotes the action of endogenous proteases.

280

Appreciable changes were observed at 48 h postmortem on muscle sample prepared

281

exactly in the same conditions (see supplementary Figure S1).

282 283

Fluorescence response of muscle fibers according to time postmortem

284

Figure 4 shows the profile of fluorescence spectra acquired in soleus muscle fibers

285

at death (t0) and at 24 hours postmortem (t24h) (spectra acquired on the EDL muscle

286

show the same general pattern). The spectra show a peak at 332 nm characteristic

287

of tryptophan and a shoulder at 302 nm assigned to tyrosine

288

that emits fluorescence at 408 nm could not be identified with certainty. Collagen and

289

elastin fluoresce in this wavelengths range

290

extracellular matrix, which excludes the involvement of these compounds in the

291

observed fluorescence, as our spectral acquisitions were strictly intracellular. The

292

fluorescence peak at 408 nm could be attributed to NADH, since in living cells this

293

compound is suspected to fluoresce in this wavelength range 20.

294

The superposition of the spectra acquired at t0 and t24h shows some shifts which are

295

however difficult to see with the naked eye. However, these differences are

296

highlighted by the PCA and variance analysis that are presented in Figures 5–7.

297

PCA and loadings indicate a separation of the two postmortem times (t0 and t24h)

298

essentially in the PC2 on the fluorescence intensities at 302 and 346 nm, and to a

299

lesser extent on the PC1 at 325 nm and 408 nm, whichever the muscle considered

300

(Figures 5 and 6). In general, 302 nm and 408 nm fluorescence intensities were

301

higher, and 346 nm and 325 nm fluorescence intensities lower for t0 than for t24h

302

samples. Separation on the EDL PCA was less clearcut than in the soleus, mainly

14

12, 20, 32

. The compound

, but these proteins are located in the


13


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303

because the EDL is composed of four types of fiber plus IIX-IIB hybrid fibers, while

304

the soleus is composed of only I and IIA fibers.

305

Some cell types allow a better separation of postmortem times, such as Type IIA

306

fibers, which show excellent discrimination of postmortem times on both the EDL and

307

soleus muscles. IIX-IIB hybrid fibers and IIX fibers (absent in the soleus muscle) also

308

show a good separation as regards postmortem time. Postmortem time of soleus

309

muscle Type I fiber discrimination was somewhat less marked than for Type IIA, but

310

still very sharp. By contrast, postmortem time discrimination of EDL Type I and Type

311

IIB fibers was not as sharp on PC2. For these fiber types, the postmortem time was

312

discriminating on both PC2 at 302 nm and 346 nm and PC1 at 408 nm and 325 nm.

313

Discriminatory wavelengths of PC1 and PC2 were exactly the same for the two

314

muscles (EDL and soleus) and all types of fibers: 325 nm and 408 nm for PC1, 302

315

and 346 for PC2. Hence it seems that only few fluorophores are concerned in

316

postmortem time discrimination.

317

Significant differences in emission fluorescence intensities regarding the postmortem

318

time were amplified by the 346/302 and 408/325 ratios, which were systematically

319

lower and higher respectively for t0 than t24h muscle fibers (Figure 7). Soleus t0 fibers

320

I and IIA have a lower 408/325 ratio than EDL t0 I and IIA fibers (Figure 7). The

321

fluorescence intensity at 408 nm is significantly higher (p

Hyperspectral Deep Ultraviolet Autofluorescence of Muscle Fibers Is

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