High resolution non-invasive 3D imaging devices are required to detect pathogenic microorganisms such as Anthrax spores, bacteria, viruses, fungi and chemical agents entering biological tissues such as the epidermis. Due to the low light penetration depth and the biodamage potential, ultraviolet light sources can not be employed to realize intratissue imaging of bio- and chemohazards. We report on the novel near infrared laser technology multiphoton tomography and the high resolution 4D imaging tool DermaInspect for non-invasive detection of intratissue agents and their influence on cellular metabolism based on multiphoton autofluorescence imaging (MAI) and second harmonic generation (SHG). Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence of both, skin tissues and microorganisms, originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Bacteria emit in the blue/green spectral range due to NAD(P)H and flavoproteins and, in certain cases, in the red spectral range due to the biosynthesis of Zn-porphyrins, coproporphyrin and protoporphyrin. Collagen and exogenous non-centrosymmetric molecules can be detected by SHG signals. The system DermaInspect consists of a wavelength-tunable compact 80/90 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezo-driven objective, fast photon detector and time-resolved single photon counting unit. It can be used to perform optical sectioning and 3D autofluorescence lifetime imaging (τ-mapping) with 1 μm spatial resolution and 270 ps temporal resolution. The parameter fluorescence lifetime depends on the type of fluorophore and its microenvironment and can be used to distinguish bio- and chemohazards from cellular background and to gain information for pathogen identification. The novel in vivo non-invasive imaging system offers the possibility to detect and to localize CB agents in tissues and to gain information on their impact on respiratory chain activity, cell division and metabolism. The system DermaInspect can also be used to detect food and water contamination.
[1]
Karsten König,et al.
Laser tweezers and multiphoton microscopes in life sciences
,
2000,
Histochemistry and Cell Biology.
[2]
B R Masters.
Three-dimensional confocal microscopy of the lens.
,
1996,
Ophthalmic research.
[3]
R. Webb,et al.
In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology.
,
1999,
The Journal of investigative dermatology.
[4]
P. So,et al.
Two-Photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.
,
1998,
Optics express.
[5]
B R Masters,et al.
Multiphoton Excitation Microscopy of In Vivo Human Skin: Functional and Morphological Optical Biopsy Based on Three‐Dimensional Imaging, Lifetime Measurements and Fluorescence Spectroscopy a
,
1998,
Annals of the New York Academy of Sciences.
[6]
R. Webb,et al.
Video-rate confocal scanning laser microscope for imaging human tissues in vivo.
,
1999,
Applied optics.
[7]
Axel Bergmann,et al.
Picosecond fluorescence lifetime microscopy by TCSPC imaging
,
2001,
SPIE BiOS.
[8]
Gerald W. Lucassen,et al.
Two-photon fluorescence microscopy of in-vivo human skin
,
2000,
European Conference on Biomedical Optics.