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Proceedings ArticleDOI

2-photon laser scanning microscopy on native human cartilage

12 Jun 2005-Vol. 5860, Iss: 29, pp 586003

AbstractHyalin 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.

Topics: Microscopy (56%), Autofluorescence (52%)

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.

3. RESULTS

  • 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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European Conference on Biomedical Optics (ECBO) - (Munich), June 12–17, 2005
Proceedings of SPIE, Vol. 5860, 16-21 (2005)
2-Photon Laser Scanning Microscopy on Native
Human Cartilage
Jörg Martini, Katja Tönsing, Michael Dickob*, Dario Anselmetti
Bielefeld University, Physics Faculty, Experimental BioPhysics & Applied NanoScience,
Universitätsstr. 25, 33615 Bielefeld, Germany
* Orthopedic Surgery, Bahnhofstrasse 30, 33602 Bielefeld, Germany
ABSTRACT
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 in a
nondestructive and sterile manner. Using 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. Via spectral autofluorescence separation these experiments allowed the
discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within
the cartilage tissue to approximately 0.2-2·10
7
/cm
3
. Furthermore, a comparison of the relative
autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in
tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our
measurement has the potential of being used in future diagnostic applications.
Keywords: three-dimensional microscopy; multiphoton processes, biomedical imaging, human cartilage
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 (Figure 1). Earlier experiments have exhibited that the chondrocytes are
located in tissue cavities (lacunae) and often group during their proliferation [1,2]. The three dimensional collocation of
the lacunae is therefore dependent on their relative position in the cartilage, i.e. distance to the bone. Degeneration of
Figure 1: (from left to right) Nano-, ultra- und microstructure of human knee joint cartilage with severe arthritic lesion on the right.

cartilage (arthritis) is due to a deterioration of ECM, water content and chondrocytes and its loss of intrinsic elasticity.
Due to the intrinsically dense structure of the ECM, cartilage is a tissue that strongly scatters but does not absorb much
light. Hence articular cartilage has a white optical appearance and is called hyalin. Consequently, penetration depth of
visible light is vastly reduced, making a characterization with conventional brightfield microscopy extremely difficult,
as they require staining and/or microtome cuts. A remedy is using NIR light that is less affected by light scattering and
is able to deeply penetrate such biological tissue. Combining this advantage with the benefit of NIR two-photon
excitation [3] (Figure 2) allows a proper 3-dimensional microscopic characterization of these tissues. As the 2-photon
excitation wavelength of the laser can be tuned, inducing autofluorescence of the sample (i.e. cartilage), staining
protocols become obsolete. However, 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.
Figure 2: (a) Conventional one-photon excitation (b) two-photon excitation
Therefore, in this paper, we investigate the application of two-photon laser scanning microscopy (2PLSM) for the
characterization of human cartilage tissue with respect to the following questions: 1) is an adequate and stain-free
imaging of the cartilage tissue possible with 2PLSM, 2) can chondrocytic cells be identified and discriminated from
ECM spectrally, and 3) is there a detectable difference that can be used in the future for optical diagnostic evaluation of
healthy and arthritic cartilage tissue.
2. EXPERIMENTAL SETUP
2.1 Two-Photon Laser Scanning Microscope
Our 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 (see
Figure 3, #1), a TriM Scope scanning unit (La Vision Biotec, see Figure 3, # 2, 3, 4) and an inverted microscope (IX 71,
Olympus, see Figure 3, #5). Detection of the fluorescence signal is realized by a back illuminated EMCCD camera
(IXON BV887ECS-UVB, Andor Technology; see Figure 3, #b) or a photomultiplier (H7422-40, Hamamatsu; see
Figure 3, #c) in a nondescanned manner. Therefore, the NIR laserbeams, used for excitation, are being directed with a
dichroic mirror (2P-Beamsplitter 680 DCSPXR, Chroma) onto the back aperture of the objective lens (see Figure 3, #9)
where stray light from the NIR in the detection path is blocked by a short pass filter (2P-Emitter E 700 SP, Chroma). In
order to perform spectrally resolved measurements, a filter wheel (La Vision Biotec) or a spectrograph (Triax 190,
HORIBA Jobin Yvon) can be introduced into the detection pathway (see Figure 3, #7). The scanning unit consists of an
integrated pre-chirp section (Figure 3, #2) to compensate for laser pulse dispersion and two galvanometric mirror
scanners (Figure 3, #4) to scan the laser foci in one optical x-y-plane (i.e. one depth) of the sample. The multiplexing

section (Figure 3, #3) of the TriM Scope can split up the incident laser beam into a variable number of beams of the
same average power. This section consists of a set of ten 100% reflective mirrors and one (adjustable) 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 (see Figure 3, #6). In addition, the
laser power can be adjusted in order to achieve short acquisition times without photodamage. A mechanical focus drive
(MFD, Märzhäuser) in combination with a NIR coated objective lens (XLUMPLFL20XW, Olympus) with a large
working distance (WD = 2 mm) allows for the acquisition of depth resolved fluorescence x-y-scans inside the sample.
Data acquisition and control is realized via a software package (Imspector, La Vision Biotec) controlling all necessary
experimental parameters such as laser attenuator, number of beams, detection filter settings, scanner settings and the
focus drive. 5-dimensional fluorescent data sets (including spectral and temporal data axis) are handled and processed
with Imspector, Image J [4] or Imaris 4.0 (Bitplane AG) software packages.
Figure 3: Scheme of two-photon laser scanning microscope
Figure 4: Experimental setup of two-photon laser scanning microscope

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 (Ringer). No additional staining or
fluorescence labelling was applied.
3. RESULTS
Figure 5 shows the 3D-autofluorescence reconstruction of unstained healthy human cartilage as measured with 2PLSM.
Measurements with a tissue penetration depth of up to 200 µm inside the sample (up to 460 µm are possible for arthritic
cartilage) at a spatial resolution of approximately 1 micron can be performed within minutes. The image was taken in
the 2PLSM mode at an excitation wavelength of 800 nm with 64 parallel foci at a total laser power of 260 mW, keeping
laser power at 4 mW per focus to prevent photodamage [5]. The spectral discrimination between ECM and chondrocytic
cells was achieved by recording two separate microscopy image stacks with fluorescence emission filtering for ECM
Figure 5: 3D-autofluorescence reconstruction of unstained healthy human cartilage tissue. The ECM and the chondrocytic cells,
originally represented in green and red colors, are shown in greyscale representation for printtechnical reasons. The presented plane
lies 50 microns below the tissue surface of the sample.

(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.
From the image in figure 5 it is evident that a spectral discrimination between ECM and chondrocytes is readily
possible. Furthermore, the complete mapping of the tissue allows a direct estimate of the corresponding chondrocyte
density in the tissue region displayed in figure 5, which was found to be approximately 20·10
6
/ cm
3
. This result
compares well with values for healthy cartilage found by other research groups [6]. It has to be mentioned thought that
the chondrocyte density varies vastly for different samples, even within the same sample, depending on the relative
position of the region under investigation in the cartilage. We found chondrocyte densities that range from
approximately 2·10
6
/ cm
3
to 20·10
6
/ cm
3
in the same cartilage sample. In addition, the lacunae in the ECM could be
identified by a reduced fluorescence signal in colocalization with the grouped chondrocytic cells.
We also investigated healthy and arthritic tissue samples (macroscopical diagnosis) from the same female test person
using 2PLSM in order to determine if any differences can be identified (Figure 6). Both tissue samples were
characterized with 2PLSM at 800 nm with a laser power of 240 mW in 64-foci parallel mode of operation. For mapping
the surface morphology here, only the green fluorescent emission filtering for the ECM (HQ 525/50) was recorded.
Figure 6: 3D-autofluorescence reconstruction of the surface of unstained healthy and arthritic human cartilage tissue from the same
proband. The ECM is shown in grayscale representation.
In figure 6 two things are evident: firstly, healthy cartilage tissue displays a much higher autofluorescence emission
from ECM than arthritic tissue, and secondly, the two outer surface structures significantly differ with respect of
smoothness and morphology. Whereas the surface of healthy tissue is smooth and isotropic, the arthritic surface is
fibrous and rough. 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. Furthermore the reduced fluorescence
emission intensity for arthritic tissue can be interpreted as an indication of lower tissue density.

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Frequently Asked Questions (1)
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.