Direct and instantaneous observation of intravenously injected substances using intravital confocal micro-videography

Abstract

We describe the development and application of intravital confocal micro-videography to visualize entrance, distribution, and clearance of drugs within various tissues and organs. We use a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The Nikon A1R allows simultaneous four channel acquisition and speed of 30 frames per second while maintaining high resolution of 512 × 512 scanned points. The key techniques of our intravital imaging are (1) to present a flat and perpendicular surface to the objective lens, and (2) to expose the subject with little or no bleeding to facilitate optical access to multiple tissues and organs, and (3) to isolate the subject from the body movement without compressing the blood vessels, and (4) to insert a tail vein catheter for timed injection without moving the subject. Ear lobe dermis tissue was accessible without surgery. Liver, kidney, and subcutaneous tumor were accessed following exteriorization through skin incision. In order to image initial extravasations of compounds into tissue following intravenous injection, movie acquisition was initialized prior to drug administration. Our technique can serve as a powerful tool for investigating biological mechanisms and functions of intravenously injected drugs, with both spatial and temporal resolution.

Label-free optical-resolution photoacoustic endomicroscopy in vivo

“…57 and 58 demonstrated 19.6 µm and 15.7 µm transverse resolutions, respectively), and an SNR of 29 dB from a 3-µm diameter microsphere illuminated by a pulse energy of 500-nJ in a 9.2 µm beam diameter. As shown in the in vivo and ex vivo image demonstrations, the major benefit of the OR-PAEM over existing IVM techniques [44][45][46][47][48][49][50][51][52][53][54][55][56] lies in its label-free angiographic imaging capability, which provides critical image in experimental biology and clinical medicine. Although other groups [57,58] developed endoscopic devices with optical focusing and achieved an even smaller probe diameter (i.e., ~1.1 mm in the case of ref.…”

“…OR-PAM realizes the key benefits of PAT in biological experimentation through its high-resolution imaging capability; further, its unique optical absorption-based contrast mechanism enables it to complement conventional highresolution microscopy tools, such as confocal microscopy [44][45][46][47][48][49], two-photon microscopy [44][45][46][50][51][52][53], and optical coherence tomography [44][45][46][53][54][55][56]. So far, considerable efforts have been made to improve the spatial resolution [15], scanning speed [7,12], and functional imaging capability [4][5][6][7][8][9] of OR-PAM.…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

“…IVM imaging of dynamic and fast events, such as blood flow or mobile immune cells, can be accomplished using resonant scanners, which allow imaging speeds up to 30 frames per second (Kirkpatrick et al, 2012;Matsumoto et al, 2010;Nguyen et al, 2001) or multibeam imaging, which can also reduce photodamage by spreading the excitation power over a number of focal spots (Niesner et al, 2007;Rinnenthal et al, 2013;Shimozawa et al, 2013).…”

Optical-resolution photoacoustic endomicroscopy in vivo

“…Intravital microscopy (IVM) techniques [1][2][3][4][5][6][7][8][9][10][11][12][13] have been opening new research and clinical avenues by providing unprecedented image information on biological processes in vivo. In addition to earlier approaches that imaged the body surfaces of small animals [1][2][3][4][7][8][9], technical advances in miniaturized imaging devices have now enabled extending IVM to endomicroscopy [1,5,6,10,11,13], which enables the imaging of internal organs.…”

“…In addition to earlier approaches that imaged the body surfaces of small animals [1][2][3][4][7][8][9], technical advances in miniaturized imaging devices have now enabled extending IVM to endomicroscopy [1,5,6,10,11,13], which enables the imaging of internal organs. So far, two-photon microscopy [1][2][3][7][8][9][10] and confocal microscopy [1][2][3][11][12][13] have been the major technical platforms for this approach. However, in most cases, these techniques are based on complex labeling with fluorescent molecules, which could disturb delicate biological processes.…”