Fluorescence LSCM to Assess the Penetration of Low Molecular Protein Hydrolyzates Into Hair

Nov 1, 2010 | Contact Author | By: Olga Freis and Dominique Gauché, Laboratoires Sérobiologiques division of Cognis; and Ute Griesbach and Hans-Martin Haake, Cognis
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Title: Fluorescence LSCM to Assess the Penetration of Low Molecular Protein Hydrolyzates Into Hair
penetrationx protein hydrolyzatex fluorescence laser scanning confocal microscopyx
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Keywords: penetration | protein hydrolyzate | fluorescence laser scanning confocal microscopy

Abstract: The present study uses confocal laser scanning fluorescence microscopy to assess the penetration of protein hydrolyzates into hair. While higher amounts of protein were found in the cuticle, still significant quantities were observed in the cortical parts of hair, and this penetration was enhanced by longer incubation times.

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O Freis, D Gauché, U Griesbach and H-M Haake, Fluorescence laser scanning confocal microscopy to assess the penetration of low molecular protein hydrolyzates into hair, Cosm & Toil 125(11) 30-35 (Nov 2010)

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Several studies have been published on the substantivity and penetration of fluorescein isothiocyanate (FITC)-labeled proteins and protein hydrolyzates into human hair using fluorescence microscopy or confocal laser scanning fluorescence microscopy. The latter method provides a new dimension of hair fiber observation and diagnostic perspective, as will be shown, due to its significantly higher resolution than classical microscopy, which locates penetrated labeled proteins more precisely.


Lab Practical: Using Fluorescence Laser Scanning Confocal Microscopy

  • A high performance fluorescence laser scanning confocal microscope and an experienced operator are required to perform comparable examinations.
  • The purification step after FITC labeling of substances to be analyzed is critical. The correct separation of unreacted label, i.e. FITC, from labeled molecules using e.g. chromatography is a prerequisite for further analysis.
  • Researchers are urged to take a rigorous approach to the treatment conditions and uniformity of treatment procedures.
  • There is significant background fluorescence in hair, thus it is important to always use the same conditions for a comparable series of image acquisitions.
  • An optimum equilibrium of laser power for correct imaging without bleaching the label is necessary.

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Figure 1. Schematic of fluorescence laser scanning confocal microscopy

Figure 1. Schematic of fluorescence laser scanning confocal microscopy

The key feature of confocal microscopy is its ability to acquire images that are in focus from selected depths, a process known as optical sectioning. Images are acquired point-by-point and reconstructed by a computer, which allows for three-dimensional reconstructions of topologically complex objects.

Figure 2. FITC-labeled protein penetration into control Caucasian hair

Figure 2. Example of FITC-labeled protein penetration

Example of FITC-labeled protein penetration into the control Caucasian hair: a) and d) without treatment; b) and e) on hair treated with 0.1% FITC-labeled protein for 30 min; and c) and f) on hair treated with 0.1% FITC-labeled protein for 24 hr

Figure 3. FITC-labeled protein penetration on bleached Caucasian hair

Figure 3. FITC-labeled protein penetration on bleached Caucasian hair

Example of FITC-labeled protein penetration on bleached Caucasian hair: a) and d) without treatment; b) and e) on hair treated with 0.1% FITC-labeled protein for 30 min; and c) and f) on hair treated with 0.1% FITC-labeled protein for 24 hr

Figure 4. FITC-labeled protein penetration on control African-American hair

Figure 4. FITC-labeled protein penetration on control African-American hair

Example of FITC-labeled protein penetration on control African-American hair: a) and d) without treatment; b) and e) on hair treated with 0.1% FITC-labeled protein for 30 min; and c) and f) on hair treated with 0.1% FITC-labeled protein for 24 hr

Figure 5. FITC-labeled protein penetration on relaxed African-American hair

Figure 5. FITC-labeled protein penetration on relaxed African-American hair

Example of FITC-labeled protein penetration on relaxed African-American hair: a) and d) without treatment; b) and e) on hair treated with 0.1% FITC-labeled protein for 30 min; and c) and f) on hair treated with 0.1% FITC-labeled protein for 24 hr

Figure 6. Hair treated with 0.5% labeled proteins

Figure 6. Hair treated with 0.5% labeled proteins

Hair treated with 0.5% labeled proteins; a) example of an optical longitudinal section (= one stack); b) maximal projection of three-dimensional reconstructed stacks of the whole hair fi ber; c) transparency effect; d) rotation of three-dimensional reconstructed stacks of the whole hair fi ber

Footnotes [Freis 125(11)]

a Caucasian hair was obtained from Fesnoux-Sécher, Paris.

b African-American hair was obtained from DeMeo Brothers, Inc., New York.

c Labeling and separation were conducted by Deutsches Wollforschungsinstitut (DWI), Aachen, Germany.

d The TCS-SP2 fluorescence laser scanning confocal microscope is manufactured by LEICA Microsystèmes, Nanterre, France.

e Gluadin WLM (INCI: Hydrolyzed Wheat Protein) is a product of Cognis.

f The Tissue-Tek brand automated slide stainer and coverslipper is a device of Sakura Tinetek Europe, Zoeterwoude, Netherlands.

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