Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivo flow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivo at the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods.
Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 µs ex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED50,d) and cavitation (ED50,c) increased with pulse duration and were approximately equal for pulses of up to 10 µs. For 20 µs, the ED50,d was about 10% lower than the ED50,c; the difference increased with 40-µs pulses. Cells were killed predominantly by cavitation (up to 10-µs pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting.
Purpose: An in vivo flow cytometer was developed recently, providing quantification of fluorescently labeled cells in live animals without extracting blood samples. This non-invasive procedure allows continuously tracking a cell population of
interest over long periods of time to examine its dynamic changes in the circulation. However, it has not been shown
that counting signals arise from individual cells. Furthermore, cell morphology and cell-cell interaction in the blood
stream (e.g. aggregation) are not visualized. Here we describe an imaging in vivo flow cytometer.
Material and Methods: Fluorescence images are obtained simultaneously with quantitative information on a DiD-labeled cell population. As
fluorescent cells pass through the slit of light focused across a blood vessel, the excited fluorescence is detected
confocally. This cell counting signal triggers a strobe beam and an intensified CCD camera to capture a snapshot image
of the cell as it moves down-stream from the slit.
Results: Nearly all peaks counted as circulating T-cells originate from individual cells, while cell aggregates were rarely
observed (<2%). Counting signal amplitude variation is attributed to uneven dye-loading among cells. We identify
non-T-cells by their abnormal shape and size. Cell velocity was measured by determining the traveled distance from the
slit within the delay of the strobe pulse or by applying multiple strobe pulses during the integration time of the CCD
camera. Conclusions: An improved in vivo imaging flow cytometer can be a useful tool for studying cell populations in circulation.
Selective targeting of the Retinal Pigment Epithelium (RPE), by either applying trains of microsecond laser pulses or, in our approach, by repetitively scanning a tightly focused spot across the retina, achieves destruction of RPE cells while avoiding damage to the overlying photoreceptors. Both techniques have been demonstrated as attractive methods for the treatment of retinal diseases that are caused by a dysfunction of the RPE. Because the lesions are ophthalmoscopically invisible, an online control system that monitors cell death during irradiation is essential to ensure efficient and selective treatment in a clinical application. Bubble formation inside the RPE cells has been shown to be the cell damage mechanism for nano- and picosecond pulses. We built an optical system to investigate whether the same mechanism extends into the microsecond regime. The system detects changes in backscattered light of the irradiating beam during exposure. Samples of young bovine eyes were exposed in vitro using single pulses ranging from 3 μs to 50 μs. Using the viability assay calcein-AM the ED50 threshold for cell death was determined and compared to the threshold for bubble formation. We also set up a detection system on our slit lamp adapted scanning system in order to determine the feasibility of monitoring threshold RPE damage during selective laser treatment in vivo.
Intracellular cavitation was detected as a transient increase in backscattering signal, either of an external probe beam or of the irradiation beam itself. Monitoring with the irradiation beam is both simpler and more sensitive. We found the threshold for bubble formation to coincide with the threshold for cell damage for pulse durations up to 20 μs, suggesting that cavitation is the main mechanism of cell damage. For pulse widths longer than 20 μs, the cell damage mechanism appears to be increasingly thermal as the two thresholds diverge. We conclude that bubble detection can be used to monitor therapeutic endpoint for pulse durations up to 20 μs (or equivalent dwell time in a scanning approach). We have integrated a detection module into our slit lamp adapted laser scanner in order to determine threshold RPE damage during selective laser treatment in vivo.