Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 µm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.
The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses non- invasively. This paper describes the development of a micro manipulation workstation integrating two-photon, 3-D imaging with a high-force, uniform-gradient, magnetic manipulator. The uniform-gradient magnetic field applies nearly equal forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5 micrometers diameter paramagnetic particles and over 800 pN on 5.0 micrometers ferromagnetic particles. These forces vary less than 10% over an area 200 x 200 micrometers 2. The compatibility with the use of high numerical aperture (approximately equals 1.0) objectives is an integral part of the workstation design allowing sub- micron resolution 3-D two-photon imaging. Three dimensional maps of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses is not always a local deformation.
We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers 2. Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order of 1 micrometers . Elastic, viscoelastic, and creeping responses of cell surfaces were observed. Our results are consistent with previous observations using similar magnetic techniques.