There is an ever-increasing demand to do in vivo imaging, particularly live organ and live animal imaging for biomedical and clinical studies. Multiphoton microscopy provides a glimpse of real-time biology, making it ideal for application in almost all the fields of biomedical research and clinical study.
The Two-Photon Microscopy Core Facility at the Center for Biological Pathways is equipped with a Carl Zeiss multiphoton microscope LSM 7MP. All work is done on a fee-for-service basis to recover costs for equipment maintenance and upkeep. Priority use is given to MD Anderson investigators, but outside users also are accepted.
This core facility provides equipment and expertise to assist investigators in applying advanced multiphoton microscopy techniques to their research.
Multiphoton microscopy imaging: The LSM7MP will be used for advanced 3D image acquisition from live tissue, organ, embryo and animal, as well as time-lapse imaging.
Training: The core will provide training in usage of the multiphoton microscope. Training sessions are arranged in consultation with the facility manager.
Consultation: Consultation on experimental design and approach is provided by the facility manager and director.
Zeiss LSM 7MP: LSM 7 MP is a completely dedicated and optimized multiphoton laser scanning microscope system. This system is equipped with a powerful pulsed IR laser, IR laser delivery path, modulation optics, high sensitive non-descanned detectors (NDDs), and the Axio examiner microscope.
- High sensitive GaAsP detectors: 3 times more sensitive than traditional PMTs. This sensitivity increase will allow you to image deeper with less tissue damage than other multiphoton system.
- Ti:Sapphire near infrared laser system: High-quality femtosecond pulsed tunable near infrared laser with pulse energy of up to 3000 mW and repetition rates of 80
- NIR specific scanning system: Highest possible transmission over the range 600 nm to 1300 nm
- High flexibility of optics: The filters and dichroics are housed in ‘push and click’ filter cubes for quick and easy access and experimental changes
- AxioExaminer Microscope Stand: Specific design for two photon fluorescence and SHG imaging with the implementation of a custom stage
- ZEN software with Smart setup: The Smart setup tool automatically selects optimal settings of the LSM to suit the fluorophores in the sample
Powerful techniques of LSM7MP
- Significant increase of imaging depth into tissue thanks to its NIR wavelength deeper tissue penetration and efficient light detection.
- Less phototoxicity by using NIR excitation photons of energies lower than those of UV or visible wavelengths and by spatially confined fluorescence excitation to a minute sub-femtolitre focal volume
- Tremendous improvement of the optical localization and Z resolution with the smallest z-step of 25nm for 3D imaging
- Dramatically increase of signal-to-noise ratio by the large separation between the incident NIR excitation and the subsequent visible emission wavelengths, as well as eliminating fluorescence except at the focal point of the laser.
- The combination of 2PF and SHG brings to simultaneous imaging of cellular microcomponents and their hosting extracellular collagenous microstructure in bulk tissues, providing valuable information and offering new insights into the complex tissue structures.
Function of LSM7MP
Multiphoton Fluorescence Imaging
- Focused point excitation
- Excitation of only fluorophores in the focal center
- Minimal photo bleaching and photo damage
- Minimal or no background fluorescence
- No pinhole before the detector
- More efficient signal acquisition with NDD
- No need of UV excitation
- 3D optical sectioning imaging
- increase imaging depth to 750um
- intrinsic high Z resolution
- interactive volume rendering
- Multi-point imaging
- Multiple position imaging over time
- Live embryo and live tissue imaging over time
- Second harmonic generated signal (SHG) imaging
- Forward transmitted SHG imaging for thinner tissue
- Backward reflected SHG imaging for thicker tissue
- combination of fluorescence and SHG tomography
Targeted Research Applications
- Multiphoton microscopy in cancer research: Tumor pathophysiology, gene expression, angiogenesis, cell adhesion and migration, vascular, interstitial and lymphatic transport, metabolic microenvironment and drug delivery in deeper regions of tumor
- Detection of tumor in transgenic mouse models: Visualization and quantification of tumor stages of neoplastic progression, invasion, angiogenesis and metastasis methods, SHG to facilitate imaging of stromal collagen and tumor-stroma interactions, including the architecture and remodeling of the tumor microenvironment.
- Multiphoton microscopy in developmental biology: Study embryo development, track cell shape, nuclear position; localization of cytoskeleton molecules during formation of investigated organs in live wild-type and mutant embryos
- Detection of detailed information on tissue architecture and cellular morphology: Imaging of endogenous fluorescence and SHG eliminate the need to add often-toxic fluorescent labeling normally required to allow biomedical researchers to visualize sub-cellular and cellular structures in three dimensions and with minimum intervention; in situ even in vivo investigation of tissue microcomponent with high precision and contrast. NAD(P)H autofluorescence of mitochondria can be imaged and the cell morphology is reflected; cells can be identified through the autofluorescent cytoplasm and dark non-fluorescent nuclei and revealing of intact cellular network; the 3D tomography of cellular layers within tissue structure will be investigated.
- Bioenergetics and metabolism of live specimens in pathophysiology: Only the reduced form of NAD(P)/NAD(P)H are significantly fluorescent. The mitochondrial NAD(P)/NAD(P)H ration can be thereby determined as a function of mitochondrial metabolic states.