In vivo confocal and multiphoton microendoscopy - PubMed (original) (raw)
In vivo confocal and multiphoton microendoscopy
Pilhan Kim et al. J Biomed Opt. 2008 Jan-Feb.
Abstract
The ability to conduct high-resolution fluorescence imaging in internal organs of small animal models in situ and over time can make a significant impact in biomedical research. Toward this goal, we developed a real-time confocal and multiphoton endoscopic imaging system. Using 1-mm-diameter endoscopes based on gradient index lenses, we demonstrate video-rate multicolor multimodal imaging with cellular resolution in live mice.
Figures
Fig. 1
(a) Schematic of the microendoscope imaging system and (b) spatial resolution and (c) signal strength in confocal microendoscopy measured with different pinhole sizes. Dotted lines in (b) represent theoretical diffraction-limited resolution of a 0.45-NA objective lens.
Fig. 2
Various images acquired with the imaging system. (a) to (c) Fluorescence images of a pollen grain sample. The images in (a) and (b) were obtained with a GRIN endoscope by two-photon excitation at 860 nm and by confocal excitation at 635 nm, respectively. The image in (c) was obtained by 860-nm two-photon excitation using a standard objective lens (40×, 0.6 NA), shown for comparison. (d) Endoscopic SHG image of starch in a sliced fresh potato. The polarization state of the excitation beam was linear along the horizontal axis. (e) and (f) Confocal endoscope images of a pine embryo [in (e), excitation; 491 nm, and emission; 520/35 nm; in (f) excitation; 635 nm, and emission; 692/40 nm]. (g) Merged image of (e) and (f). Image blurring near the FOV boundary is due to field curvature. Each image was averaged over 30 frames acquired in 1 s at the same sample position. Scale bars are 25 _μ_m. (Color online only.)
Fig. 3
Images of the intact ear skin in anesthetized mouse. (a) Confocal image showing Langerhans cells expressing GFP+ major histocmpatibility complex (MHC) class II molecules in a genetically engineered mouse [generously provided by Dr. Marianne Boes at Harvard Medical School (HMS)]. Excitation is 491 nm; emission is 520/35 nm. (b) Two-photon fluorescence image of blood vessels with free-flowing rhodamine-B-dextran conjugates after tail-vein injection (2,000,000 MW, 200 _μ_g/200 _μ_l). Excitation is 800 nm, 30 mW; emission is 590/80 nm. (c) Collagen fibrillar structure visualized by SHG. Excitation is 800 nm, 30 mW; emission is 417/60 nm. The images in (a) to (c) were averaged over 30 consecutive frames acquired in 1 s. (d) to (f) A sequence of frames showing ovarian cancer cells (OVCAR-1) in blood circulation, superimposed on a green fluorescence image of blood vessels in a GFP+ Tie2 mouse (generously provided by Dr. Scott Plotkin at HMS). The OVCAR-1 cells were labeled in vitro using DiD (Invitrogen) and injected at the tail vein [2 million cells in phosphate-buffered saline (PBS) 200 _μ_l]. Scale bar is 50 _μ_m. (Color online only.)
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