Electron Microscope Observation of Collagen fibers

Xiaoxing Han

The Institute of Optics, University of Rochester

OPT407: SEM Practicum

Spring 2006 Final Project

Background

Collagen is the main protein of connective tissue in animals and the most abundant protein in mammals, making up about 40% of the total. It is one of the long, fibrous structural proteins whose structure and functions are quite different from those of globular proteins such as enzymes.

Fig 1. Collagen triple helix

Collagen is distinct from other proteins in that the molecule comprises three polypeptide chains which form a unique triple-helical structure (See figure 1). It is tough and inextensible, with great tensile strength, and is the main component of cartilage, ligaments and tendons, and the main protein component of bone and teeth. Along with soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. It strengthens blood vessels and plays a role in tissue development. It is present in the cornea and lens of the eye in crystalline form.

More than 20 types of collagen are known up to now, different types are determined by different polypeptide sequences. Their properties are different and they can be found in different places of animal body.

Type I
bone, tendon, fibrocartilage, dermis, cornea
Type II
nucleus pulposus, hyaline cartilage
Type III
intestinal and uterine wall
Type IV
endothelial, epithelial membranes
Type V
cornea, placenta, bone, heart valve

Samples

Samples used in this project including a rat cornea sample for SEM imaging collagen type I fibrils, a bovine collagen type III solution sample (Rockland Immunochemicals Inc. 1× PBS diluted to 0.1mg/ml) for TEM studying collagen type III fibrils and an insoluable bovine collagen type I fiber sample( Sigma Aldrich) for microscope observation.

Sample preparation

SEM sample peparation

The rat cornea sample used for SEM imaging was HMDS dehydrated, the fixing and dehydration process is as follows:

1) Immerse freshly cut tissue sample in physiological saline

2) Transfer the sample to a solution containing 1% Glutaraldehyde in 0.1M Cacodylate buffer, PH 7.

Allow 5 minutes for sample fixing process.

3) Wash in distilled water for 5 minutes

4) Dehydrate using a series of ethanol washes:

50% ethanol 5 minutes; 75% ethanol 5 minutes; 90% ethanol 5 minutes; 100% ethanol 5 minutes.

5) Immerse in HMDS for 15 minutes and air dried in room temperature in fume hood.

We then mounted the sample on the SEM sample stub and sputtered a layer of gold on it in a sputter coater.

After that the sample was well grounded with the conductive paint, and was ready for SEM imaging.

TEM sample preparation

To improve contrast of our TEM images, we used the negative staining technique.

For our collagen type III sample we used 1% phosphotungstic acid as our negative stain.

The stain process is as follow:

1) A drop of collagen solution is placed on a petri dish.

2) A carbon coated EM grid (Fig 2) is placed carbon side down on top of the collagen solution drop for approximately 1-3 minutes.

3) The grid is removed, blotted with filter paper and placed onto a drop of stain solution, for one minute.

4) Then remove the excess stain solution.



Fig 2. EM grid

 

Light microscope sample preparation

For light microscope samples, we don't need special sample preparation process. We simply put a piece of collagen fiber sample on a microscope slide, covered it with a coverslip and then put the sample under the microscope objective to observe.

Result

Different samples should be imaged by different imaging techniques. For bulk and nontransparent samples like the rat cornea sample we use SEM. For thin and transparent sample like the collagen type III solution sample we use TEM. For prior electron microscopy observation that doesn't need large magnifications, we use light microscope .

SEM imaging

Fig 3 is an SEM image we got from the rat cornea sample at 10000× magnification. We can see, in this picture, some big bundles of collagen fibrils lying around. Also, we can clearly see the fine fabrics of single collagen fibrils which make up the whole rat cornea .

Fig.3 Rat Cornea SEM image 10000×

We then took a closer look at a small area at 50000× magnification. Here we can clearly see single fibrils in the background, which are about 20nm in diameter in average. There is a big bundle of fibrils lying above the collagen fibril fabrics background. We can even almost see how single collagen fibrils twisted with each other to form the fibril bundle. The left image in figure is the original image and the right one is a colorized image.

SEM1

Fig.4 Rat Cornea SEM image 50000×

TEM imaging

Figure 5 and figure 6 are TEM images we got from the PBS buffered 0.1mg/ml bovine collagen type III solution sample. The contrast of these images are good because of the negative stain: 1% phosphotungstic acid we used in the experiment. The magnification is 125000×, and we can clearly see the big mess of the collagen type III fibrils. The fibrils are also about 20 nm in diameter. The left images in these figures are the original TEM images and the right ones are the colorized version. We can see the collagen type III fibrils in the solution form a gel and exhibit complicated 3D structure.

SEM2SEM2

Fig 5. Bovine collagen type III solution TEM image 125000×

SEM2SEM2

Fig 6. Bovine collagen type III solution TEM image 125000×

Light microscope imaging

Figure 7 is the light microscope image of the insoluable bovine collagen type I fiber sample.

SEM2

Fig 7. Bovine collagen type I fiber sample light microscope 100×

Conclusions and remarks

Conclusions

1) Techniques employed

Light microscopy, SEM, TEM, sample fixing and dehydration for SEM, sample sputter coating for SEM, negative staining for TEM, image colorization.

2) Results

Luckily the SEM and TEM images all looks good. We successfully observed different collagen types by different electron microscopy techniques and the images all look like what they are expected to be. The collagen type I fibrils in rat cornea form into a fine fabrics or twisted together into a big bundle. The collagen fibrils in the bovine collagen type III solution form into a collagen gel with complicated 3D structure. This project is very helpful for a better understanding on collagen structures and properties, and for a better knowledge on SEM, TEM and light microscope imaging techniques as well.

Remarks

Thanks Brian for his help and guidance. Thanks Ms. Karen for generous donation of 1% phosphotungstic acid negative stain. Thanks Dr. Edward Brown for cutting rat cornea and his guidence.

Reference

1) Kadler et al, Collagen fibril formation, Biochem. J. (1996) 316, 1-11