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MEDE 4501 Biophotonics: The University of Hong Kong
MEDE 4501 Biophotonics: The University of Hong Kong
Bachelor of Engineering
MEDE 4501
Biophotonics
Examination
Only approved calculators as announced by the Examinations Secretary can be used in this
examination. It is candidates' responsibility to ensure that their calculator operates
satisfactorily, and candidates must record the name and type ofthe calculator used on the front
page of the examination script.
In many clinical dermatological applications, there are pressing needs for in-vivo 30 imaging
techniques. Two optical imaging modalities, namely multi photon microscopy (MPM) and optical
coherence tomography (OCT), are proven to be useful for in-vivo "optical biopsy". In this
examination paper, you will need to design an integrated imaging system which combines MPM
and OCT together. Such "multimodal" imaging platform would be able to visualize, with high
resolution, the 30 morphology of different skin layers and identify characteristic features of
various skin pathologies (mainly occurs in epidermis and dermis regions (Fig. 1)) using MPM and
OCT, without anv exogenous contrast agents/labels.
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Here in this imaging system, MPM obtains image signals via (i) two photon excited fluorescence
(TPEF) from endogenous fluorophores and (ii) second harmonic generation (SHG) from non-
centrosymmetric molecules. The main endogenous fluoropho res in skin are reduced
nicotinamide adenine dinucleotide (NADH), keratin, flavin adenine dinucleotide (FAD), melanin
and porphyrins. Their fluor escence emission spectra are shown in Fig. 2. In addition to TPEF,
collagen matrix in the dermis contributes to the SHG signal obtained from skin. The entire
volumetric MPM (including both SHG and TPEF) as well as the OCT images of the tissue are
acquired based on laser scanning techniques.
Melanin
Figure 2: Emission s pectra of the major autofluorescent compounds found in s kin tiss ue
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Figure 3: MPM/OCT images of a basal cell carcinoma (BCC) located on the forehead region. (a) A representative B-scan
obtained from the BCC. The yellow arrows indicates two signal free cavities, which are one of the characteristic features
of BCC identified using OCT. (b) 30 cut-through rendering of the BCC reconstructed using 512 OCT B-scans (c) En face
views of epidermis (EPJ), reticular dermis (RD) and deeper reticular dermis (ORD) obtained from the depth locations
indicated in the 30 rendering. (d) MPM images of stratum corneum (SC), stratum granulosum (SG), stratum spinosum
(SS), dermal-epidermal junction (DEJ), papillary dermis (PD) and reticular dermis (RD) respectively, obtained from the
BCC. Green signals are mainly from NADH emission where as the red signals (in false color) correspond to SHG from
collagen matrix (collagenized tumor cavities).
(a) (9%) Describe the origins of the image contrasts obtained by OCT, SHG and TPEF imaging
(hints: structural or molecular? And explain why.)
(b) (8%) The major challenge for translating optical imaging technologies to clinical applications
is its limited imaging penetration depth into the biological tissue. Such limitation is even more
obvious when compared to other established medical imaging technologies, such as MRI, CT, and
ultrasound imaging. So, explain why such integrated system (MPM plus OCT) can still attract
attenion in dermatological diagnosis, particularly for diagnosing BCC?
(c) (3%) What are the colors of fluorescence emission of NADH, FAD, and porphyrins'?
(d) (6%)If a water-immersion (n =1.33) objective lens with a numerical aperture (NA) = 1.3 is
used for TPEF imaging, estimate the diffraction-limited transevse and axial resolutions of TPEF
images of NADH? Explain why the measured resolutions in practice are often larger than your
estimated values.
(e) (6%) What does it mean by "optical sectioning" in optical microscopy? How do we achieve it
in one-photon laser scanning confocal microscopy as well as MPM?
(t) (5%) NADH can have fluorescence emission upon excitation directly by the light at -260 - 360
nm (i.e. through one-photon fluorescence excitation). So, what is the rationale behind the use of
two-photon excitation of NADH in this problem (i.e. for dermatological diagnosis)?
(g) (15%) In this system design, if you are only allowed to use ONE laser source for both SHG
imaging of collagen and TPEF imaging of NADH in skin, choose the best possible laser source from
Table 1? Explain your choice. Name the laser your choose.
Table 1 Laser sources for (g). Sources 3 and 5 are the continuous-wave laser sources. TBD: "to be determined" in the
following questions. *Sources 1, 2, and 4 are assumed to deliver transform-limited pulses.
(h) (8%) What is the wavelength of the SHG signal in this system? Thus, in order to detect both
the SHG signal of collagen and TPEF signal of NADH, a filter set (2 dichroic mirrors and 2 barrier
filters) should be used to spectrally separate these two signals, which are finally detected by two
PMTs (Fig. 4). Choose the best suitable filter set. (Note: specify the detected signal (either SHG or
TPEF signal) in each channel.)
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Figure 4: (Top) Transmission spectra of the choices of spectral filter set (barrier filters and dichroic
mirrors). (Bottom)The schematic of the two-channel signal (SHG and TPEF) detection.
(i) (5%) The laser power is accidentally tuned up such that the power incident onto the tissue is
500 mW. Explain what will happen.
(j) (8%) You are only allowed to use single laser source in this integrated system to perform both
MPM and OCT. Hence, what is the axial resolution of the OCT images oz1 captured by this system
defined by the bandwidth of the laser source you chose in (g)? If the OCT system employs the
same objective Jens in (d) (i.e. NA= 1.3), what is the depth offield of this OCT system oz2? Hence,
explain how to determine the actual axial resolution of this OCT system? oz1 or oz2? Thus explain
why another separate objective lens with lower NA should be used for OCT operation in this
system.
(k) (2%) You are given an optical spectrometer based on a CCD/CMOS sensor for OCT image
acquisition. Hence, what is this type of OCT?
(1) (7%) !fit is required to achieve real-time 3D imaging (512(x) x 512(y) x 1024(z) voxels) at a
speed of 1 frame per second (fps), i.e. 512 x 512 A-scans in 1 second, choose the best line camera
from the list shown in Table 2 and Fig. 5. Note that you should also make your choice based on
the source you choose in (g). Explain your choice. (**x and y are along the transverse directions
whereas z is along the axial direction.]
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Wavelength (µm)
(m) (18%) Based on all the above questions, sketch a complete schematic of the system and
explain how the MPM (including TPEF and SHG) and OCT image acquisition can be done (i.e. the
operation principles and procedures) in this single system, which can be configured in a
handheld probe based on the use of optical fiber for light delivery. as depicted in Fig. 6.
Hints:
{1) What are the light (illumination and detection) pathways for OCT and MPM,
respectively, in the system?
(2) Any special considerations taken for the pulse width of illumination light, as
well as fiber specification?
(3) For 3D OCT, what are the key components required? And where do we put
them in this system?
(4) Do the MPM and OCT modules use same objective lenses?
Optical fiber
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