研究目的
To validate the potential of two-photon imaging for monitoring retinoid changes in human eyes by measuring the content of retinyl esters and all-trans-retinal condensation products in monkey and human eyes.
研究成果
These data show that human retina contains sufficient amounts of retinoids for two-photon excitation imaging. Greater amounts of 11-cis-retinyl esters relative to rodent retinas contribute to the fluorescence signal from both monkey and human eyes. These observations indicate that TPM imaging found effective in mice could detect early age- and disease-related changes in human retina.
研究不足
The potential risk of light-induced tissue damage requires minimization of the laser beam energy that could compromise the imaging signal.
1：Experimental Design and Method Selection:
Two-photon microscopy (TPM) was used to visualize excised retina from monkey eyes. Retinoid composition and content in human and monkey eyes were quantified by HPLC and mass spectrometry (MS).
2：Sample Selection and Data Sources:
Freshly enucleated eyes of cynomolgus monkey (Macaca fascicularis) were obtained from Ricerca Biosciences. Deidentified human eyes were obtained from the Cleveland Eye Bank.
3：List of Experimental Equipment and Materials:
Laser scanning confocal microscope (Leica TCS SP2 or Leica TCS SP5), fs laser (Chameleon), band-pass filter HQ 465/160, photomultiplier tube detector, HCX PL APO CS 40.0 × 1.25 oil objective, glass-bottom dish (MatTek Corp.), SpeedVac (Thermo Fisher Scientific, Inc.), LXQ linear ion trap mass spectrometer (Thermo Fisher Scientific, Inc.).
4：0 × 25 oil objective, glass-bottom dish (MatTek Corp.), SpeedVac (Thermo Fisher Scientific, Inc.), LXQ linear ion trap mass spectrometer (Thermo Fisher Scientific, Inc.). Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: Retinal tissue preparation for TPM imaging, 2PO imaging, analysis of retinoid composition in human and monkey eye.
5：Data Analysis Methods:
Fluorescence of the acquired raw images was quantified by statistical analyses with commercial confocal software (Leica Confocal Software version 2.6. SigmaPlot).
独家科研数据包，助您复现前沿成果，加速创新突破
获取完整内容-
Laser scanning confocal microscope
Leica TCS SP2 or Leica TCS SP5
Leica Microsystems
Used for two-photon microscopy imaging of retinal tissues.
-
fs laser
Chameleon
Coherent
Delivers pulses for two-photon excitation microscopy.
-
band-pass filter
HQ 465/160
Chroma Technology
Filters the fluorescence signal for detection.
-
photmultiplier tube detector
Detects the fluorescence signal induced by two-photon excitation.
-
HCX PL APO CS 40.0 × 1.25 oil objective
Leica Microsystems
Focuses the laser light on the sample and collects the induced fluorescence.
-
glass-bottom dish
MatTek Corp.
Holds the retinal sample for imaging.
-
SpeedVac
Thermo Fisher Scientific
Concentrates samples by evaporating solvents under vacuum.
-
LXQ linear ion trap mass spectrometer
Thermo Fisher Scientific
Quantifies levels of A2E in the analyzed samples.
-
登录查看剩余6件设备及参数对照表
查看全部
