2-photon laser scanning microscopy on native human cartilage
Abstract: Hyalin cartilage was investigated with 2-photon laser scanning microscope . NIR-2-photon-excitation and a specialized long distance objective lens allowed for autofluorescence and SHG measurements of the extracellular matrix up to 400 µm inside the sample.
Summary (2 min read)
1. INTRODUCTION & MOTIVATION
- Hyaline cartilage consists of chondrocytes that are embedded in an extracellular matrix tissue.
- The ECM, that was originally formed and is maintained by chondrocytes, contains mostly collagen II, proteogylcans like chondroitin sulfate and keratan sulfate, glucosamine, and water .
- Hence articular cartilage has a white optical appearance and is called hyalin.
- As the 2-photon excitation wavelength of the laser can be tuned, inducing autofluorescence of the sample (i.e. cartilage), staining protocols become obsolete.
- Two-photon excitation depends on extremely high photon densities that can only be realized by an adequate spatial and temporal focussing of light, such as in a diffraction-limited focus of a femtosecond laser beam.
2.1 Two-Photon Laser Scanning Microscope
- The authors 2-photon-laser-scanning-microscope (2PLSM) consists of a mode locked Tsunami Ti:Sa laser, pumped by a Millenia X solid-state laser (both Spectra-Physics) that generates 100 fs laser pulses between 760nm and 960nm , a TriM Scope scanning unit and an inverted microscope .
- Detection of the fluorescence signal is realized by a back illuminated EMCCD camera or a photomultiplier in a nondescanned manner.
- The scanning unit consists of an integrated pre-chirp section to compensate for laser pulse dispersion and two galvanometric mirror scanners to scan the laser foci in one optical x-y-plane (i.e. one depth) of the sample.
- This section consists of a set of ten 100% reflective mirrors and one 50% mirror.
- By introducing the 50% mirror between the set of 100% mirrors, as shown in figure 3, the laser beam can be split up into 1,2,4,..,64 beams resulting in an adjustable number of excitation foci in the sample .
2.2 Human Cartilage Tissue
- Human cartilage tissue has been received in healthy as well as in arthritic form directly from the surgical ward at a local hospital (Franziskus-Hospital, Bielefeld).
- Following biopsy, the tissue was kept at room temperature in Ringer solution and investigated within 6 hours after extraction.
- For sample preparation the tissue was cut with a scalpel and placed directly on a standard microscope glass slide and mounted onto the scanning stage of the inverted 2PLSM.
- During the transport, preparation and experiment the tissue was always kept in the solution .
- No additional staining or fluorescence labelling was applied.
- Figure 5 shows the 3D-autofluorescence reconstruction of unstained healthy human cartilage as measured with 2PLSM.
- The spectral discrimination between ECM and chondrocytic cells was achieved by recording two separate microscopy image stacks with fluorescence emission filtering for ECM (HQ 525/50) and for the chondrocytes (HQ 575/50).
- The superimposed image stack in figure 5, however, was printed in greyscale representation for technical reasons.
- The authors found chondrocyte densities that range from approximately 2·106 / cm3 to 20·106 / cm3 in the same cartilage sample.
- This change in morphology of arthritic tissue to a rough, fibrous surface is consistent with an increased friction and resulting wear between cartilage surfaces in contact.
4. SUMMARY & OUTLOOK
- Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols.
- Via a spectral autofluorescence separation these experiments allowed discrimination of the chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue.
- Furthermore, a comparison between healthy and arthritic cartilage tissue exhibited distinct differences in tissue morphology and, via relative autofluorescence intensity and penetration depth, distinct differences in ECM density.
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Q1. What are the contributions in "2-photon laser scanning microscopy on native human cartilage" ?
In this paper, a femtosecond, near-infrared ( NIR ) Ti: Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix ( ECM ) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 μm inside the sample.