This study was approved by the Human Ethical committee at Göteborg University (approval number S166-1). All patients gave their informed consent.
Open surgical biopsies were obtained from human forearm muscles of five healthy patients (age 24–68 years) undergoing surgery of the forearm (fracture surgery, plate removal and tendon transfer surgery. The surgeon exposed the muscle of interest and the parallel orientation of the muscle fibres was defined by inspection. A small part (approximately 15 × 5 × 5 mm) of the muscle was freed by alternating sharp and blunt dissection taking care not to mechanically damage the central part of the biopsy. The biopsies were then carefully divided into smaller pieces by scissors in parallel with the fibre orientation and put in a test tube with relaxing solution (cf. below).
Samples were treated in two different ways. One part, defined as fresh (F), was taken from the relaxing solution (see below), embedded in OCT ("Optimal Cutting Temperature", a special low-temperature embedding medium for cryosectioning techniques. OCT; Miles Laboratories, Naperville, Il, USA) and frozen in isopentane (pre-cooled in liquid nitrogen).
The other part was stored in a storage solution, stored (T) in freezer at -20°C. After storage for 4 weeks the biopsies were washed in relaxing solution and then treated as described above.
Relaxing (or working) solution contained 7.5 mM EGTA ("Ethylene Glycol Tetraacetic Acid", a chelating agent with a high affinity for calcium and therefore useful for making buffer solutions that resemble the intracellular environment), 170 mM KPr, 2 mM MgAcetat, 5 mM Imidazole, 10 mM phosphocreatin, 4 mM Na2 ATP, 17 μg/ml leupeptin, 4 μg/ml E64 (E 64 is an inhibitor of the lysosomal proteinase Cathepsin B i.e., inhibitor of protein breakdown). Storage solution included the same constituents as the relaxing solution with an addition of NaN3 (to a concentration of 1 mM) and glycerol (to a concentration of 50%). This was obtained by adding 1 ml 0.5 M NaN3/500 ml storage solution and 250 glycerol/500 ml solution to the relaxing solution.
The biopsy and storage procedures were identical to that for the morphology part of this study. Stored (frozen) preparations were gently defrosted on ice-bed in relaxing solution. Single fibres were dissected under microscope (Leica MZ8, Heerbrugg, Switzerland) with epi-illumination (model DCR II, Fostec, Auburn, NY) using forceps (P-00019, S&T, Neuhausen, Switzerland) and scissors. The chosen fibre was then transferred to a glass-bottomed chamber containing relaxing solution, specially designed to fit to our microscope and laser set-up. The whole set-up was placed on a vibration isolation table (Newport Instruments, Irvine, CA, USA). The fibre was then mounted to titan-thread lever arms by 10-0 monofilament sutures under microscope (Leica model MZ95, Heerbrugg, Switzerland) while still in the relaxing solution. The lever arms were connected to a force transducer (Model 405A-10 V/gram, Aurora Scientific Inc, Ontario, Canada) and a manually regulated digital micromanipulator (Mitutoyo 0–1", Tokyo, Japan) respectively.
Fibre length (knot to knot) was measured indirectly on a video monitor (Sony Trinitron Color Video Monitor, PVM-14M2 MDE, Tokyo, Japan) by magnification via a camera (Ikegami CCD Color Camera Model ICD-810P, Tokyo, Japan) attached to the microscope. Fibre diameter was measured in the same way and fibre area was calculated assuming cylindrical shape. A laser beam from a HeNe-laser (Melles Griot Model U-1507, Carlsbad, CA, USA) was then directed through the chamber hitting the mounted fibre at a right angel and creating a diffraction pattern. Sarcomere length (SL) was calculated by measurement of distance between light peak maximum as described by Yeah .
To determine distance between peaks of light interference a digital calliper was used. Two observations of 0 th – 1st, 1 st – 1 st and 0th – 2nd diffraction order peak intensities were made after each stretch .
Initial sarcomere length was defined as SL with the fibre mounted and "uncoiled" but not stretched. Tension as response to stretch was registered on a voltmeter (Amprobe AM-15, Everett, WA, USA). The fibre was then stretched in a continuous protocol recording tension values after stress relaxation of 1 minute. The stretch steps were 250 μm up to a total stretch of 4 mm and in steps of 500 μm thereafter. Stretch was discontinued at a total stretch of 8 mm or at fibre rupture. Slope of stress-strain curve was determined for each sample by defining the linear portion of the curve in the range of SL between 1.7 and 4.8 μm. Stress-strain curves are presented with stress values, based on tension at 1 minute of stress relaxation, corrected for area change during stretch assuming linear deformation of a cylinder with a constant volume. Change in sarcomere length (SL) is expressed as relative SL. The initial SL was set to 1 (unit).
The OCT-embedded muscle biopsies were cut in a cryostat (Microm HM 500, Walldorf, Germany) in 10 μm thick sections and put on microscope slides and stained with Haematoxylin & Eosin (HE). Each slide was inspected by two independent and trained observers under light microscope (Nikon Eclipse E 600) to which a video camera (Sony Power HAD Video cam) was attached. Muscle cross sections were measured for single fibre diameter according to Dubowitz  using software for PC (Easy Image measure module 2000, Bergström Instrument AB, Stockholm, Sweden). Areas in the section were chosen with emphasis on finding polygonal or circular shape of the cut fibres and avoiding areas with semicircular or longitudinal cuts. At least 150 fibres were measured on each slide. Measured cells were counted. Overall morphology was based on homogeneity of cells, presence of inflammatory cells, and position and density/number of nuclei. Atypical findings were recorded. Fibre occupancy (FOC) was calculated as a quote of fibre area (FA) per total measured area including extra-cellular matrix (ECM).
Data regarding fibre diameter are presented for one of the observers (FE). Data from the other observer (ER) were used to calculate inter-observer error. The diameter of muscle fibres specific to each slide is presented with number of fibres (n), mean, SEM, and FOC. Two-sided Student's t-test for paired observations was used to detect differences in fibre size mean between the different preparations of the same biopsy. Mann-Whitney U-test was used to test for difference in mean FOC.
A probability of less than 0.05 at statistical analysis of the observed outcome was considered significant.
The elastic modulus was determined as tangent of a linear portion of the stress-strain curve located within a physiological range of the sarcomere length (up to 2.5 times initial SL). Data are presented for fresh and stored biopsies.