13 research outputs found
νλΆ μνκΈ° κΈ°κ΄μ λν μ΄κ΄μνλ―Έκ²½ μ°κ΅¬ λ° κ°λ°
DoctorTwo-photon microscopy (TPM) is a nonlinear fluorescence microscopic technique widely applied for various biological studies including neurobiology, cancer biology, and immunology since it provides three-dimensional (3D) cellular information within tissue based on endogenous and exogenous fluorophores. However, the lower gastrointestinal (GI) tract imaging still has been limited because of the location of the GI tissues which are deeply located inside the abdominal cavity. Also, these tissues are morpho-functionally complicated and sensitive tissue for maintaining immune homeostasis that makes the limitation of imaging. Moreover, the lower GI tract has numerous cells and tremendous amount of microorganism including bacteria and fungi, thus, it makes difficulty in controlling and assessing them for the longitudinal in vivo studies. Most previous studies for the lower GI tract imaging provided its superficial information by using transgenic mice or exogenous fluorescence markers in relatively short time window. Although autofluorescence (AF)-based imaging without using the fluorescence labeling could visualize all the cells within the tissue, it can be visualized either by having a sufficiently high excitation laser power or by having a slow imaging speed due to the weak AF level. Furthermore, TPM-based 3D cellular and structural information of GI tract have not been well elucidated in live condition. Hence, it is necessary to develop effective imaging method and tools for in vivo longitudinal studies of GI tract at cellular-level under expanded time window.
In this study, the aim is to develop a novel two-photon (TP) imaging method which overcomes several limitations of AF-based imaging and side-viewing rotational endomicroscopic imaging probe system for longitudinal studies of the lower GI tract with an outstanding image contrast at cellular-level, in vivo. First, I described the applications of moxifloxacin, an FDA-approved fluoroquinolone antibiotic, as a biocompatible cell-labeling agent for TPM to overcome the weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. By using TPM with moxifloxacin, various cell lines, and animal tissues including the lower GI tissues were imaged, analyzed and compared with endogenous fluorescence with second harmonic generation (SHG) signal within them. In addition, Paneth cells (PCs), key mediators for intestinal homeostasis by secreting antimicrobial factors, were studied in the intact mouse small intestine by TPM with moxifloxacin, in vivo. Finally, side-viewing rotational TP endomicroscopic system was developed and improved to study the lower GI tract, especially mouse colon, in vivo.
In conclusion, this dissertation suggests novel TP imaging method using moxifloxacin and side-viewing rotational endomicroscopic imaging probe system for longitudinal studies of the lower GI tissues at cellular-level, in vivo. In addition, TPM application to the lower GI tissues using moxifloxacin labeling could contribute to study the microenvironment of another internal organ via TP endomicroscopy.μ΄κ΄μνλ―Έκ²½ (two-photon microscopy, TPM) κΈ°μ μ 3μ°¨μ ν΄μλλ₯Ό κ°λ νκ΄ νλ―Έκ²½ κΈ°μ λ‘μ μΈν¬ λ° μ체쑰μ§μ ꡬ쑰μ μΈ μ 보λ₯Ό μ 곡ν μ μκΈ° λλ¬Έμ μλ¬Όν, μλͺ
곡ν λ° κΈ°μ΄μν μ°κ΅¬ λ±μμ λ€μνκ² μ¬μ©λκ³ μλ€. μ΅κ·Όμλ μ΄κ΄μνλ―Έκ²½ κΈ°μ μ κΈ°λ°μΌλ‘ ν λμ‘°μ§ λ΄ μ 경ꡬ쑰 κ΄μ°°, μ‘°μ§ λ΄ μ λ°μ/νμ±κ³Όμ λλ μΈν¬ κ±°λμ λν μ€μκ° κ΄μ°° λ±μ λν μΈν¬ μμ€μΌλ‘μ μ°κ΅¬ κ²°κ³Όλ€μ΄ μ£Όλͺ©μ λ°κ³ μλ€. μ΅κ·Όμλ μνκΈ° κΈ°κ΄ λ΄ λ©΄μμ²΄κ³ λ° κΈ°λ₯ μ°κ΅¬λ₯Ό μν΄μλ μ΄κ΄μνλ―Έκ²½ κΈ°μ μ΄ μ΄μ©λκ³ μλ€. κ·Έλ¬λ μμ₯μ‘°μ§ λ° λμ₯μ‘°μ§κ³Ό κ°μ νλΆ μνκΈ° κΈ°κ΄μ λν μμν λ° μ΄κ΄μνλ―Έκ²½ κΈ°μ μ κΈ°λ°μΌλ‘ νλ μ°κ΅¬λ μνκΈ° κΈ°κ΄ μ‘°μ§μ νΉμ± λλ¬Έμ μ΄μμλ μνμμ μ₯μκ°λμ μμννλλ° μ μ½μ΄ λ§μ μ΄λ €μμ΄ λ°λ₯Έλ€. μλνλ©΄, νλΆ μνκΈ° κΈ°κ΄μ λ³΅λΆ μμͺ½μ κΉμ΄ μμΉνκ³ μμ λΏλ§ μλλΌ μλ§μ μΈν¬λ€μ λ¬Όλ‘ λ°ν
리μ (bacteria), κ· μ’
(fungus)κ³Ό κ°μ μλ§μ λ―Έμλ¬Όλ€μ΄ 곡μνκ³ μμΌλ©° μν, ν‘μ λ° λ©΄μ체κ³μ μ μ§ λ±μ μν΄μ κΈ°λ₯μ μΌλ‘ λ―Όκ°νκ³ κ΅¬μ‘°μ μΌλ‘ λ§€μ° λ³΅μ‘ν μ‘°μ§μ΄κΈ° λλ¬Έμ΄λ€. λ°λΌμ νλΆ μνκΈ° κΈ°κ΄μ λν μ°κ΅¬λ€μ μ£Όλ‘ λ³΅μ‘νκ³ μ΄λ €μ΄ κ³Όμ λ€μ ν΅ν΄ μνλμ΄ μμΌλ©° μ΄κ΄μνλ―Έκ²½ κΈ°μ κΈ°λ° μ°κ΅¬λ€μ μ£Όλ‘ μ‘°μ§ νλ©΄μ μΌλΆμ λν΄ λΉκ΅μ 짧μ μκ° (~ μ΅λ 8μκ°) λ΄μμ κ΄μ°°λ μ 보λ§μ μ 곡νκ³ μλ€. λ°λΌμ νλΆ μνκΈ° κΈ°κ΄μ λν μλͺ
ν, λ©΄μν, μ λ°μν λ±μ μ μλ―Έν κΈ°μ΄μ°κ΅¬λ₯Ό μν΄μλ μ‘°μ§μ΄ μ΄μμλ μνμμ μΈν¬ μμ€μ μ 보λ₯Ό μ 곡νλ©° μ‘°μ§μ λν μ₯κΈ°κ°μ κ΄μ°°κ³Ό λΆμμ΄ κ°λ₯ν μ΄κ΄μνλ―Έκ²½ μμν κΈ°μ μ μ°κ΅¬ λ° κ°λ°μ΄ νμνλ€. λ³Έ μ°κ΅¬μμλ, νλΆ μνκΈ° κΈ°κ΄μ λν μ΄κ΄μνλ―Έκ²½ μμν κΈ°μ μ μ°κ΅¬ λ° κ°λ°μ λ€λ£¨κ³ μλ€.
λ³Έ λ
Όλ¬Έμμλ μ΄κ΄μνλ―Έκ²½ μμν κΈ°μ μ λν΄μ, λ³Έ μ°κ΅¬μμ μ μν λͺ©μνλ‘μ¬μ (moxifloxacin)μ μ‘°μ§ λ΄ μΈν¬ μΌμμ λ‘μ νμ©μ κ²μ¦νμκ³ μ‘°μ§ λ΄ μΈν¬ μμνμ λν νκΈ°μ μΈ μ±λ₯ν₯μμ 보μμΌλ©° μΈμ²΄ λ΄ μΈν¬ κ΄μ°°μ κ°λ₯μ±κΉμ§ μ μνμλ€. ννΈ, λͺ©μνλ‘μ¬μ μ μ΄μ©ν μ΄κ΄μνλ―Έκ²½ μμνλ₯Ό ν΅ν΄ νλΆ μνκΈ° κΈ°κ΄ μ€ νλμΈ μμ₯μ‘°μ§μ λν΄μ μ λ§ λ©΄μ λ° νμμ± μ μ§μ ν΅μ¬μ μΈ μν μ νλ νλ€μ€ μΈν¬ (Paneth cell)λ₯Ό μ΄μμλ λ§μ°μ€μμ μμννλ λ°©λ²μ κ°λ°νμλ€. λ³Έ μ°κ΅¬μμ κ°λ°ν μ체 λ΄ μμ₯μ‘°μ§μ νλ€μ€ μΈν¬μ λν μμν κΈ°μ μ ν₯ν νλ€μ€ μΈν¬μ κΈ°λ₯λΏλ§ μλλΌ κ΄λ ¨ μ§λ³μ°κ΅¬, λ©΄μμ°κ΅¬ λ±μλ μμ©λ μ μλ€. λμκ° νλΆ μνκΈ° κΈ°κ΄ μ€ νλμΈ λμ₯μ‘°μ§μ λν μ체 λ΄ μμν λ° μ₯κΈ°κ°μ μ°κ΅¬λ₯Ό μνμ¬ κ΅΄μ λ₯ λΆν¬ν (gradient index, GRIN) λ μ¦λ₯Ό κΈ°λ°μΌλ‘ νλ μν μ΄κ΄μ λ΄μνλ―Έκ²½ (endomicroscopy) νλ‘λΈ μμ€ν
μ κ°λ°νμλ€. κ·Έλ¦¬κ³ μ΄κ΄μ λ΄μνλ―Έκ²½μ μμμ ν₯μνκΈ° μν΄μ λ΄μκ²½ νλ‘λΈλ‘ μ¬μ©λ GRIN λ μ¦μ μμ°¨λ¬Έμ λ₯Ό κ°μ νμμΌλ©° μ΄λ₯Ό ν΅ν΄ μ΄μμλ λ§μ°μ€μ μ체 λ΄ λμ₯μ‘°μ§μ ννΌ (epithelium)λΆν° μλμ (gland; crypt) ꡬ쑰κΉμ§ λ΄μκ²½μ μΈ λ°©λ²μ ν΅ν΄ μ²μμΌλ‘ μμννλλ° μ±κ³΅νμλ€. λ³Έ μ°κ΅¬μμ κ°λ°ν GRIN λ μ¦ κΈ°λ°μ μ΄κ΄μ λ΄μνλ―Έκ²½ νλ‘λΈ μμ€ν
μ ν₯ν λμ₯μ‘°μ§μ ꡬ쑰 κ΄μ°°λΏλ§ μλλΌ λμ₯μ‘°μ§ κ΄λ ¨ μ§λ³λͺ¨λΈμ μ₯κΈ°μ μΈ κ΄μ°° λ° κΈ°μ΄μ°κ΅¬μ νμ©λ μ μμ κ²μ΄λ€. λ³Έ λ
Όλ¬Έμμ λ€λ£¨κ³ μλ κ° νμμ°κ΅¬λ€μ λν λ΄μ©μ λ€μμ μμ½μ κ°λ΅ν κΈ°μ λμ΄ μλ€.
κ²°λ‘ μ μΌλ‘, λ³Έ μ°κ΅¬μμλ νλΆ μνκΈ° κΈ°κ΄μ λν μ΄κ΄μνλ―Έκ²½ μ°κ΅¬μ μμν κΈ°μ κ³Ό μλ‘μ΄ μμ€ν
κ°λ° λ΄μ©μ λ€λ£¨κ³ μμΌλ©° νλΆ μνκΈ° κΈ°κ΄μ λν μ체 λ΄ μμν λ° μ°κ΅¬κ° κ°λ₯νλ€λ κ²μ κ²μ¦νμλ€. λμκ° λ³Έ μ°κ΅¬μμ κ°λ°ν κΈ°μ λ° μμ€ν
μ΄ νλΆ μνκΈ° κΈ°κ΄λΏλ§ μλλΌ λ€λ₯Έ μ‘°μ§λ€μ μ체 λ΄ μ°κ΅¬μλ μμ©λ κ²μΌλ‘ μ λ§νλ©° μ΄κ΄μνλ―Έκ²½ μμκΈ°μ μ λν μμμΌλ‘μ νμ©κ°λ₯μ±μ μ μνλ€λ μ μμλ κΈ°μ¬νμλ€κ³ λ³Ό μ μλ€