2.1.

The System DermaInspect

All components of the laser imaging system DermaInspect 110 are mounted on a transportable workstation (Fig. 1). The system, with a total dimension of 75×120×140 cm ³, is considered a class 1M device according the new European laser safety regulations. It consists of the following three major modules¹⁵:

Figure 1

Photograph of the multiphoton imaging tool DermaInspect 110 for 4-D autofluorescence and SHG imaging with intracellular spatial resolution and 250-ps temporal resolution.

The tissue phantoms for microscopy (TPM, Riemann Labortechnik, Jena, Germany) were placed in sterile microchambers with 0.17-μm-thick glass windows filled with 1 low-melting agarose. Intralipid 10 scattering particles and fluorescent microspheres of different sizes and different fluorescence spectra have been used to characterize the spatial and temporal resolution of the system.

2.2.

The Multiphoton Microscope

A confocal laser scanning microscope (LSM 410, Zeiss) was modified to create a multiphoton microscope in such a way that (1) the optics were changed to obtain a high transmittance of 0.7 (800 nm), (2) an interface (JenLab GmbH, Jena, Germany) for introduction of a femtosecond laser source was provided, and (3) a special baseport unit equipped with a fast PMT, CCD camera, and spectrometer was created.

2.3.

Biopsies and in Vivo Measurements

Biopsies were obtained from patients with a variety of diseases, including psoriasis, nevi, melanomas, and dermatomycosis. For transportation and safe measurement, the samples were placed in sterile biopsy chambers for microscopy (MiniCeM-biopsy, JenLab GmbH) consisting of silicon with 4-mm holes and 0.17-μm-thick glass windows on both sides for inverted and upright imaging. Isotonic sodium chloride (NaCl) solution was used to prevent drying. The measurements occurred within 1 h after excision. Optical sectioning of in vivo Caucasian human skin (type II) of a male volunteer 41 years old and a female volunteer 33 years old was carried out on the lower forearm, which was placed on an arm holder.

2.4.

Time-Resolved Autofluorescence Measurements

Using the TCSPC method, single fluorescence photons have been counted with a fast PMT (transient time spread 150 ps) equipped with short-pass Schott BG 39 filters. The system allows count rates of more than 10⁶ photons per second at 80 MHz. Typically, the two-photon excited autofluorescence of skin at moderate laser powers provided count rates of about 100,000 photons per second. The PMT signal was synchronized with the x,y,z beam position calculated from signals of the galvoscanner and the piezodriven objective. This information was used to provide spatially resolved autofluorescence decay curves per pixel; to fit these curves by a mono-, bi-, or three-exponential approach; and to calculate mean fluorescence lifetimes per pixel or per region of interest. The calculated mean fluorescence lifetimes were depicted as color-coded τ-images.

3.1.

Alignment

Tissue phantoms for microscopy have been used for proper beam alignment and to test the imaging system by the determination of point spread functions (PSF) dependent on the intratissue depth of the excitation volume and the fluorescence wavelength, and to probe the temporal resolution. We measured a lateral PSF width of 0.4 to 0.6 μm and an axial PSF of 1.2 to 2.0 μm during optical sectioning between 0 and 100 μm depth in spite of scattering material (0 to 1) and a temporal resolution of about 250 ps.

Alignment of the skin surface occurred typically during fast scanning (1 s per frame at 512×512=262,144 pixels) and detection of the strong autofluorescence of the outermost tissue layer, the stratum corneum. Typically, a mean power of 2 mW was chosen, which corresponds to a pulse energy of 25 pJ. In order to obtain high-contrast images, the scan speed was reduced to 8 s per frame, which corresponds to a mean beam dwell time per pixel of 30 μs. The z-position was varied in steps of 1, 5, or 10 μm to a deeper position. For in-depth imaging, the incident laser power was increased. When imaging at a tissue depth of 100 μm, typically the incident mean laser power was chosen to be 20 mW (750 nm).

3.2.

Optical Sectioning of an Area within the Human Forearm

Figure 2 demonstrates a high-resolution in vivo autofluorescence image of seven cells deep in the epidermis within the stratum spinosum. The intracellular dark areas reflect the nonfluorescent nuclei. Two-photon excitation at 760 nm resulted in NAD(P)H fluorescence. The reduced coenzymes NAD(P)H are mainly located in mitochondria. As is obvious from this figure, even single autofluorescent mitochondria can be imaged deep into the tissue. The right cell depicted in the image exhibits multiple nuclei that are most likely due to ongoing cell devision.

Figure 2

High-resolution in vivo multiphoton autofluorescence image of seven cells in the stratum spinosum at about a 45-μm depth in the left forearm of a female volunteer (laser excitation, 760 nm).

Figure 3 shows images at different tissue depths separated by 5 μm out of a stack of optical sections of a male volunteer using 750-nm femtosecond laser pulses. Single cells, cellular components, extracellular structures, and transitions between tissue layers could be clearly recognized by nonlinear NIR-excited autofluorescence. In particular, the autofluorescence image of connective tissue from a depth of 80 μm demonstrated excellent in-depth resolution. The stratum corneum, with its fluorescent component keratin, exhibited a strong autofluorescence when excited within the tuning range of 750 to 850 nm. Within the broad excitation band, maxima at 750 nm and at around 820 nm have been detected in a small skin area of one volunteer. However, excitation spectra varied among different locations within this cornified tissue layer. The cell borders of the hexagonal-shaped corneocytes with a mean cell diameter of 36±15 μm often induced the strongest fluorescence of the tissue. The fluorescence pattern on the skin surface was interrupted by nonfluorescent areas with a thickness of up to 100 μm, owing to epidermal ridges. However, in the lower epidermal layers, the fluorescent borders (corneocytes) of these incisions were responsible for highly luminescent areas down to 50 μm or more. Hairs appeared as highly fluorescent structures. The thickness of the stratum corneum in the area of interest (lower forearm) was found to be 10 to 15 μm (female) and 15 to 20 μm (male).

Figure 3

In vivo optical tomography of the forearm of a male volunteer. The image at 5 μm shows fluorescent structures of the stratum corneum whereas the images at 30, 55, and 60 μm reflect fluorescent epidermal organelles. The images at 80 and 100 μm show connective structures in the dermis.

The transition to the stratum granulosum was detected by the occurrence of living cells with a different cell shape, a fluorescent cytoplasm, and nonfluorescent nuclei. In general, the autofluorescence of this tissue layer appeared of low contrast and more diffuse, owing to the coexistence of living and dead fluorescent cells. A typical thickness of about 10 μm for this tissue layer was found.

Within the stratum spinosum, the single cells and cellular compartments could be clearly seen by strong-fluorescent granula in the cytoplasm and the borders of the large, round, nonfluorescent nuclei. The cell diameter decreases with increasing tissue depth and was found in the upper part of this layer to be 26±3 μm and in the deepest cell layer 16±4 μm. Also, the ratio between the volume of the cytoplasm and the volume of the nuclei decreased. Sometimes cells with two nuclei were found.

The single innermost cell layer of the epidermis, the stratum basale, was determined by cuboidal cells with higher fluorescence intensity than living cells in the upper layers. Very likely these are the basal cells that can be differentiated and that migrate toward the skin surface. Within these cells, strong fluorescent granula in the upper part of the perinuclear region have been detected. The basal cells had the lowest diameter of the epidermal cells, with 9±1 μm, and the lowest ratio of cytoplasm to nuclei volume. During optical sectioning, the basal cells occurred as a circular cluster around the tip of a papilla.

The transition between epidermis and corium (cutis) was determined by the distribution of the papillae. Typically this junction was found at a depth of 55 to 70 μm for the female and 70 to 100 μm for the male volunteers. Connective tissue, in particular, single fluorescent long fibers with a thickness of less than 1 μm, could be clearly visualized. These highly fluorescent structures are properly single elastin fibers.

The images in Fig. 4 reflect the dependence on excitation wavelength. The figure shows some in vivo images at 770 and 820 nm out of a wavelength stack. The laser was tuned from 750 to 850 nm in steps of 10 nm. In general, the fluorescence intensity increased when the laser was tuned to short wavelengths. This is in accordance with the absorption behavior of the coenzymes NAD(P)H and flavins. However, optical sectioning with excitation wavelengths larger than 780 nm through the junction of the epidermis and dermis and within the dermis resulted in an additional source of luminescence and its detection. This luminescence is based on a second harmonic generation (SHG) of collagen. Therefore, the signal obtained from the dermis was found to be a combination of two-photon excited autofluorescence and of SHG formation at half the excitation wavelength. The SHG part was detected by determination of the emission wavelength using a variety of narrow bandpass filters and by tuning the excitation wavelength. It was clearly found that the signal possesses a short spectral bandwidth in contrast to fluorescence. In addition, a component with a short (picosecond) decay time was detected in the dermal region. The reason the SHG signal could be measured only at excitation wavelengths above 780 nm is due to the reduced transmission of the short-pass filters in the ultraviolet. In order to further verify that SHG signals are generated in the dermis, 10-μm-thick cryosections of a biopsy of Caucasian skin were investigated at 820 nm. No SHG signal was detected in the layers of the epidermis. However, a strong blue signal between 400 and 420 nm was detected in dermal collagen.

Figure 4

In vivo optical tomography in dependence on excitation wavelength. Signals in the dermis at 820-nm excitation are mainly due to SHG radiation (a,c,e: 770 nm; b,d,f: 820 nm, female volunteer).

In addition to the imaging of autofluorescence intensity, in vivo fluorescence decay kinetics per pixel have been measured. From these large amounts of data two major fluorescence lifetime components have been determined. The shorter one has a time within the response time of the detector and is most likely due to the SHG signal in the UV and blue spectral range and a small amount of scattered light that was able to transmit the filters. The long component is due to autofluorescence. Its lifetime has been calculated and depicted as a false-color fluorescence lifetime image. Typically the fluorescence lifetime of an intratissue cell was found to be on the order of 1.8 to 2.4 ns. Figure 5 shows two optical sections out of a stack of 3-D τ-maps. One image shows the autofluorescence lifetime image of the stratum corneum. The histogram of the fluorescence lifetime distribution (right upper corner of the image) shows a maximum at about 1.9 ns. The lifetime image is dominated by yellow pixels because of the false color table chosen in which yellow corresponds to 1.78 to 1.94 ns. A particular decay curve of a part of the cell border is shown in the lower part. Deconvulation resulted in the calculation of a long-lived component with a mean fluorescence lifetime of 1.85 ns. In contrast, the lower image taken at a depth of 50 μm within the stratum spinosum shows a histogram maximum of about 2.4 ns and a single fluorescence decay curve of a pixel in the perinuclear region within an organelle that might be mitochondria. The biexponential deconvulation shows a component with a relatively long fluorescence lifetime of 2.7 ns. Mitochondrial protein-bound NAD(P)H has typical lifetimes in the range of 2 to 3 ns, in contrast to NAD(P)H in solution, which has picosecond lifetimes (Table 1).

Figure 5

In vivo autofluorescence lifetime images of stratum-corneum (upper panel, 5 μm) and stratum spinosum (lower panel, 50-μm tissue depth).

These in vivo studies on normal human skin demonstrate the capability of four-dimensional autofluorescence imaging in deep tissue with a subcellular resolution in the range of 1 μm and picosecond temporal resolution. Time-correlated single-photon counting allows the measurement of intratissue fluorescence decay kinetics and the calculation of mean fluorescence lifetimes. In addition, it supports the detection of second harmonic generation in the tissue depth by the detection of a short luminescence component limited by the temporal resolution of the detector (in this experimental setup, 250 ps). The process of SHG occurs immediately, in contrast to fluorescence with its typical lifetime in the nanosecond range.

3.3.

Imaging of Dermatological Disorders

Sixty biopsies of patients with different dermatological disorders that included psoriasis, warts, fungal infections, nevi, and benign and malignant skin cancers have been investigated by multiphoton-excited autofluorescence imaging. The pattern and intensity of the autofluorescence differed clearly from surrounding normal tissue.

In the images of the patient with trichophytosis (dermatomycosis) hexagonal-shaped strong fluorescent corneocytes with a mean diameter of 90±20 μm have been detected in depths down to 80 μm. It is interesting that these corneocytes revealed strong fluorescent nuclei compartments. Such nuclei compartments can arise during hyperkeratosis. Within this 90-μm-thick tissue layer, the fluorescence intensity increased with increasing tissue depth (Fig. 6). NIR femtosecond laser pulses can be also used to excite intense fluorescence of the melanin pigment (Fig. 7). Pigmented and nonpigmented areas could be clearly differentiated. In the case of a melanocytic compound nevi, the intensity of this autofluorescence was significantly increased. This was based on strongly fluorescent cells in all tissue layers, from the stratum granulosum down to the dermal–epidermal junction. The fluorescence from these cells arose from fluorescent granula.

Figure 6

Autofluorescence images of skin with fungal infection (trichophytosis).

Figure 7

Multiphoton autofluorescence image and transmission image of a pigmented cryosection demonstrating multiphoton-excited luminescence of melanin.

In the case of a malignant melanoma, no clear transitions between the different tissue layers were recognized (Fig. 8). Clusters of highly fluorescent cells appeared. In tissue depths of 40 μm and more that were measured, the fluorescence intensity was so large that the fluorescence excitation power could be reduced by 30 to 50. As indicated by the autofluorescence images and transmission images of cryosections of pigmented skin from patients with melanocytic nevi and superficially spreading melanomas, the pigmented dark compartments that contain melanin revealed the strongest fluorescence compared with nonpigmented areas.

Figure 8

Ex vivo optical sectioning through a superficial spreading melanoma. Five images at 3, 21, 33, 42, and 62 μm depth out of a stack are characterized by bright fluorescent granula.