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Third-Harmonic

Generation
Microscopy
Images Brain
Tumors

Ressa Muhrifah Novianti (140310140021)


1 LATAR BELAKANG

2 BRAIN TUMORS (GLIOBLASTOMA)

3 THIRD HARMONIC GENERATION

4 THG MICROSCOPY IMAGES BRAIN TUMORS


Kanker otak meliputi sekitar 85-90% dari
seluruh kanker susunan saraf pusat. Di Amerika
Serikat insidensi kanker otak ganas dan jinak
adalah 21.42 per 100.000 penduduk per tahun
(7.25 per 100.000 penduduk untuk kanker otak
ganas, 14.17 per 100.000 penduduk per tahun
untuk tumor otak jinak). Angka insidens untuk
kanker otak ganas di seluruh dunia
berdasarkan angka standar populasi dunia
adalah 3.4 per 100.000 penduduk.
Dari seluruh tumor primer susunan saraf
pusat, astrositoma anaplastik dan glioblastoma
multiforme (GBM) meliputi sekitar 38% dari
jumlah keseluruhan, dan meningioma dan
tumor mesenkim lainnya 27%. Sisanya terdiri
dari tumor otak primer yang bervariasi,
meliputi tumor hipofisis, schwannoma, limfoma
SSP, oligodendroglioma, ependimoma,
BRAIN TUMORS (GLIOBLASTOMA)

Glioblastoma multiforme (GBM) adalah tumor


otak primer ganas yang paling agresif dan
paling sering ditemui, melibatkan sel glia dan
bertanggung jawab untuk 52% kasus tumor
jaringan otak fungsional dan 20% tumor
intracranial. Tumor ini disebut sebagai satu dari
5 jenis kanker dengan harapan hidup terburuk
dibandingkan kanker lainnya.

Source : google image (glioblastoma)


THIRD HARMONIC GENERATION
SKEMA ALAT THIRD HARMONIC GENERATION
THIRD HARMONIC GENERATION MICROSCOPE

Efisiensi THG tergantung terutama pada suseptibilitas orde 3 (3) dan dari kondisi
medium dan fase-matching. Intensitas THG yang dihasilkan oleh sinar laser dengan Intensitas
I dan frekuensi sudut dalam medium diberikan oleh :

n = indeks bias medium


c = adalah kecepatan cahaya,

z = posisi sepanjang sumbu sinar


z1 dan z2 = batas medium
b = parameter sinar laser terfokus

Dalam medium homogen dengan fasa fase positif


k (phase matching) dalam persamaan akan bernilai nol dan menghasilkan THG terlepas dari
besarnya I dan (3)
KEUNGGULAN THIRD HARMONIC GENERATION

Menghasilkan pencitraan dengan resolusi


tinggi

Memberikan kontras yang baik (walaupun sampel


transparan)

Kemampuan memvisualisasikan morfologi jaringan


penuh

THG telah berhasil diterapkan pada citra sampel seperti embrio serangga, bibit
tanaman dan jaringan mamalia utuh, jaringan epitel, zebrafish embryos.

THG dapat memvisualisasikan sel, nukleus, kontur akson bagian dalam dan luar, sel
darah, dan pembuluh darah, sehingga menghasilkan visualisasi materi abu-abu dan
putih sampai depth kedalaman 350 m.
CONTOH VISUALISASI MICROSCOPE THIRD HARMONIC GENERATION

Figure 1 . Label-free live brain imaging using third-harmonic generation microscopy.

( A) Schematic of THG microscopy on brain tissue in an epidetection geometry.


( B) Focused laser beam at a dendrite. By setting the laserfocal volume several
times larger than the dendrite diameter, partial phasematching is achieved, and a
significant THG signal is produced.
( C) Focused laser beam in the cell body. Due to the poor structural phase-
matching conditions, no THG is produced.
( D) THG microscopy image of living neurons in mouse brain tissue. The neurons as
dark shadows.
PROSES MEMVISUALISASI JARINGAN OTAK

Sampel otak normal secara struktural dipotong dari korteks temporal dan materi putih subkortikal.

Sampel otak tumor dipotong dari daerah tumor margin (kasus glioma kelas rendah) dan dari inti tumor dan area
peritumoral (kasus glioma kelas tinggi).

sampel jaringan otak ditempatkan selama 30 detik pada cairan artificial cerebrospinal dingin (ACSF) pada suhu 4 C
yang mengandung NaCl, KCl, NaH2PO4, MgSO4, CaCl2, NaHCO3, Glukosa. Dengan osmolaritas 300 mosmol / kg.

Sampel jaringan normal dibuat irisan koronal dengan tebal 300-350 m tebal.
Sampel jaringan tumor yang baru dipotong dibilas dengan ACSF untuk menghilangkan kontaminasi darah

Potongan sample otak normal kemudian dimasukkan ke dalam cawan Petri plastik (diameter 50 mm) dan ditutup
dengan penutup kaca tebal 0,17 mm.
Potongan sample otak glioma kemudian dimasukkan ke dalam cawan Petri plastik (diameter 25 mm) dan ditutup
dengan penutup kaca tebal 0,17 mm.

Divisualisasi dengan Third Harmonic Generation


THG MICROSCOPY IMAGES BRAIN TUMORS

THG/SHG microscopy for brain tissue imaging. (A) Energy level diagram of the second(SHG) and third (THG) harmonic
generation process. (B) Energy level diagram of the two-(2PF) and three-photon (3PF) excited auto-fluorescence process.
(C) Multiphoton microscopesetup: Laser producing 200 fs pulses at 1200 nm; GM X-Y galvo-scanner mirrors; SL scan
lens; TL tube lens; MO microscope objective; DM1 dichroic mirror reflecting backscattered THG/SHG photons to the
PMT detectors; DM2 dichroic mirror splitting SHG andTHG channels; F narrow-band SHG and THG interference filters;
L focusing lenses; PMT photomultiplier tube detectors. (D) Infrared photons (white arrow) are focused deep in the
brain tissue, converted to THG (green) and SHG (red) photons, scattered back (green/redarrows) and epi-detected.
The nonlinear optical processes result in label-free contrast imageswith sub-cellular resolution and intrinsic depth sectioning
THG MICROSCOPY IMAGES BRAIN TUMORS

Figure. (E) Jaringan tumor yang sudah dipotong (Glioblastoma tingkat rendah)
(F) Jaringan tumor yang sudah dipotong (Glioblastoma tingkat tinggi)
(G) Jaringan tumor yang divisualisasi dengan miksroskop multiphoton
THG MICROSCOPY IMAGES BRAIN TUMORS

Fig. 2. Structurally normal human brain tissue.


(A) THG (green) and SHG (red) structurally normal,
fresh human neocortex and subcortical white
matter
tissue.
(B) Myelin-stained histology image of a similar
region of the brain.
(C, D) large pyramidal neurons layer
(E,F) whitematter
(G, H) a capillary blood vessel with intraluminal
erythrocytes
(I, J). The stain in panels
(B, D,F, H) is luxol fast blue (LFB) to reveal myelin,
and hemotoxin-and-eosion (H&E) in (J).
Images C, E and G are composed of single 2-m-
thick optical sections, imaging time 1.2 s.
Image I is composed of 21 images taken with 2-m
steps over 40 m depth, imaging time 1.5
min. The inset of image I shows a cross-section of
the capillary along the white line, revealing
a round contour of this microvessel (SHG) and
corroborating the intraluminal position of the
erythrocytes (THG)
THG MICROSCOPY IMAGES BRAIN TUMORS

Fig. 3. Infiltrative low-grade glioma: transition zone. (AD) THG/SHG images of the low-to high cellularity transition zone in a tissue sample
diagnosed by the neuropathologist as diffuselow-grade oligodendroglioma on the basis of H&E-stained histological sections.
(A) Mosaicimage of the transition zone in the white matter. Image is composed of 3 2 = 6 tiles, 1.35 0.90 mm2, each tile 450 450 m2,
1000 1000 pixels2, total imaging time 52 sec.
(BD) Magnified low- (B), intermediate- (C) and high-cellularity (D) areas marked on the image (A)with white squares. Images (B, C) show 2-m-
thick optical sections taken at depths of 2030m with acquisition time of 0.6 s. (E to H) H&E images of the sample areas corresponding tothe
THG/SHG images: AE, BF, CG, DH.
THG MICROSCOPY IMAGES BRAIN TUMORS

Fig. 4. Various cytological and histological features observed with THG/SHG imagingmodalities in low- and high-grade glioma tissues. Label-free
images of 2-m-thick opticalsections taken at depths of 2030 m with acquisition times of 0.55 s and H&E images of corresponding tissue
areas. (A, B) Glial cell with a nearly round nucleus and large nuclear/cytoplasm ratio. (C, D) Glial cell with a round nucleus and smaller
nuclear/cytoplasm
ratio. (E, F) Glial cell with an indented nucleus and multiple nucleoli. (G, H) Highly cellulararea in high-grade glioma (glioblastoma) with multiple
pleomorphic tumor cell nuclei withdense chromatin and high nuclear/cytoplasmatic ratio. (I, J) A neuronal or glial cell withvacuolated cytoplasm in
the edematic peritumoral neocortex of the high-grade glioma tissue.Autofluorescent deposits in the neuropil appear as yellow dots. (K, L) Corpus
amylaceumsurrounded by neuropil. (M, N) Intense vascular proliferation in high-grade glioma focus.
TERIMAKASIH